OA21025A - Engineered hepatitis B virus neutralizing antibodies and uses thereof. - Google Patents
Engineered hepatitis B virus neutralizing antibodies and uses thereof. Download PDFInfo
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- OA21025A OA21025A OA1202200535 OA21025A OA 21025 A OA21025 A OA 21025A OA 1202200535 OA1202200535 OA 1202200535 OA 21025 A OA21025 A OA 21025A
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Abstract
The present disclosure relates, in part, to antibodies, and antigen-binding fragments thereof, that can bind to the antigenic loop region of hepatitis B surface antigen (HBsAg) and,optionally, can neutralize infection hepatitis B virus (HBV), and further optionally, of hepatitis delta virus (HDV). Presently disclosed antibodies and antigen-binding fragments have advantageous production characteristics, such as reduced formation of aggregates and/or improved production titer in transformed host cells, as compared to a reference antibody or antigenbinding fragment. The present disclosure also relates to to fusion proteins that comprise an antigen-binding fragment, and to nucleic acids that encode and cells that produce such antibodies, antigen-binding fragments, and fusion proteins. In addition, the present disclosure relates to the use of the antibodies, antigenbinding fragments, fusion proteins, and related polynucleotides, vectors, host cells, and compositions of the present disclosure in the diagnosis, prophylaxis and treatment of hepatitis B and hepatitis D. Also provided are combination therapies comprising (i) an antibody or antigenbinding fragment and (ii) an agent that is an inhibitor of HBV gene expression and/or that reduces HBV antigenic load.
Description
DETAILED DESCRIPTION
The présent disclosure relates to the field of immunotherapy for hepatitis B virus (HBV) and hepatitis delta virus (HDV). Disclosed bînding proteins, e.g., antibodies, antigen-binding fragments, and fusion proteins, are capable of binding to an epitope located in the antigenic loop région of the S domain of the HBV envelope protein (HBsAg), are capable of neutralizing a HBV infection and, in some embodiments, a HDV infection.
Presently disclosed binding proteins possess advantageous production properties (e.g., reduced formation of antibody dimers and/or increased production in a host cell), 10 such as compared to a reference anti-H BV antibody comprising the CDRs and, optionally, the VH and VL of HBC34-v35, disclosed in PCT Publication No. WO 2020/132091 ). Briefly, HBC34-v35 antibody has favorable binding and neutralization properties, but, as disclosed herein, can form antibody dimers through inter-light chain interactions during antibody production/purification. HBC34-v35 dimers hâve reduced ability to bind to HBsAg as compared to HBC34-v35 antibody monomers. Reducing dimer formation may tmprove, e.g., efflciency of antibody (or antigen-binding fragment) production and potency of a dose of the antibody (or antigen-binding fragment).
In certain embodiments, presently disclosed binding proteins can bind to any or 20 all of the known HBsAg génotypes, as well as HBsAg variants, and can neutralize HBV infection, as well as HDV infection. In certain embodiments, a presently disclosed binding protein can bind to and/or can neutralize HBV and/or HDV with similar or even increased potency as compared to HBC34-v35.
Nucleic acids that encode, and host cells that express, such binding proteins are also provided herein. In addition, the présent disclosure provides methods of using the binding proteins described herein in the diagnosis, prophylaxis, and treatment of diseases, as well as in methods of screening.
For example, embodiments of the antibodies, antigen-binding fragments, and fusion proteins according to the présent description may be used in methods of preventing, treating, or attenuating, or diagnosing HBV and HDV. In particular embodiments, the antibodies, antigen-binding fragments, and fusion proteins described herein bind to two or more different génotypes of hepatitis B virus surface antigen and to two or more different înfectious mutants of hepatitis B virus surface antigen. In spécifie embodiments, the antibodies, antigen-binding fragments, and fusion proteins 5 described herein bînd to ail known génotypes of hepatitis B virus surface antigen and to ail known înfectious mutants of hepatitis B virus surface antigen.
The présent disclosure also provides a method of treating chronic HBV infection in a subject in need thereof, comprising: administering to the subject an anti-HBV antibody or antigen-binding fragment in combination with an agent that reduces HBV
I0 antigenic load. The présent disclosure also provides a method of treating chronic HBV infection in a subject in need thereof, comprising: administering to the subject an antiHBV antibody or antigen-binding fragment in combination wîth an inhibitor of HBV gene expression.
In some methods, compositions for use, or uses described herein, the agent that i 5 reduces HBV antigenic load or the inhibitor of HBV gene expression is an RNAi agent (e.g., an siRNA, such as HBV00I or HBV002 or HBV003).
Prior to setting forth this disclosure in more detail, it may be helpfu) to an understanding thereof to provide définitions of certain terms to be used herein. Additional définitions are set forth throughout this disclosure.
Unless defined otherwise, ail technical and scientifie terms used herein hâve the same meaning as commonly understood by one of ordinary skill in the art.
Throughout this disclosure, unless the context requires otherwise, the term comprise, and variations thereof, such as comprises, and comprising, is used synonymously with, e.g, having, has, including, includes, or the like, and will 25 be understood to imply the inclusion of a stated member, ratio, integer (including, where appropriate, a fraction thereof; e.g., one tenth and one hundredth of an integer), concentration, or step but not the exclusion of any other non-stated member, ratio, integer, concentration, or step. Any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited 30 range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an înteger), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated.
The term consisting essentially of' is not équivalent to comprising and refers 5 to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, région, or module (e.g., a binding domain) or a protein consists essentially of ' a particular amino acid sequence when the amino acid sequence of a domain, région, module, or protein includes extensions, délétions, mutations, or a combination thereof 10 (e.g., amino acids at the amino- or carboxy-terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1 %) of the length of a domain, région, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domaîn(s), region(s), moduie(s), or protein (e.g., the target binding afflnity of a binding protein).
In addition, it shouid be understood that the individual compounds, or groups of compounds, derived from the various combinations of the structures and substituents described herein, are disclosed by the présent application to the same extent as if each compound or group of compounds was set forth individually. Thus, sélection of particular structures or particular substituents is within the scope of the présent disclosure.
The ternis a and an and the and sîmilar reference used in the context of describing the disclosure (including in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the alternative (e.g., or) shouid be understood to mean either one, both, or any combination of the alternatives. Recitation of ranges of values herein is intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the disclosure as if it were individually recited
herein. No language in the spécification should be construed as indicating any nonci aimed element as essential to the practice of the subject matter disclosed herein.
The word substantially does not exclude completely; e.g., a composition which is substantially free from Y may be completely free from Y. In certain embodiments, substantially refers to a gîven amount, effect, or activity of a composition, method, or use of the présent disclosure as compared to that of a reference composition, method, or use, and describes a réduction in the amount, effect, or activity of no more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%, or less, ofthe amount, effect, or activity of the reference composition, method, or use.
As used herein, the term about means ± 20% of the indicated range, value, or structure, unless otherwise indicated. In certain embodiments, about includes ± 15%, ± 10%, or ±5%.
Optional or optionally means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which they do not.
As used herein, amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modifted, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that hâve the same basic Chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, méthionine sulfoxide, méthionine methyl sulfonium. Such analogs hâve modified R groups (e.g., norleucine) or modîfied peptide backbones, but retain the same basic Chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to Chemical compounds that hâve a structure that is different from the general Chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
As used herein, the terms peptide, polypeptide, and protein, and variations of these terms, refer to a molécule that comprises at least two amîno acidsjoined to each other by a (normal or modified) peptide bond. Accordingly, a protein or polypeptide comprises a polymer of amîno acid residues. For example, a peptide, 5 polypeptide or protein may comprise or be composed of a plurality of amîno acids selected from the 20 amino acids defined by the genetic code or an amîno acid analog or mimetic, each being linked to at least one other by a peptide bond. A peptide, polypeptide or protein can comprise or be composed of L-amino acids and/or D-amino acids (or analogs or mimetics thereof). The terms peptide, polypeptide, protein also include peptidomimetics w'hich are defined as peptide analogs contaîning nonpeptidic structural éléments, which peptides are capable of mimicking or antagonîzing the biological action(s) of a natural parent peptide. In certain embodiments, a peptidomimetic lacks characteristics such as enzymatically scissile peptide bonds.
A peptide, polypeptide or protein may comprise amino acids other than the 20 15 amino acids defined by the genetic code in addition to these amino acids, or it can be composed of amino acids other than the 20 amino acids defined by the genetic code. In certain embodiments, a peptide, polypeptide or protein in the context of the présent disclosure can comprise amino acids that are modified by natural processes, such as post-translational maturation processes, or by Chemical processes (e.g., synthetic processes), which are known in the art and include those described herein. Such modifications can appear anywhere in the polypeptide; e,g., in the peptide skeleton; in the amino acid chain; or at the carboxy- or amino-terminal ends. A peptide or polypeptide can be branched, such as following an ubiquitination, or may be cyclic, with or without branching. The terms peptide, polypeptide, and protein also include modified peptides, polypeptides and proteins. For example, peptide, polypeptide or protein modifications can include acétylation, acylation, ADPnbosylatîon, amidation, covalent fixation of a nucléotide or of a nucléotide dérivative, covalent fixation of a lipid or of a lipidic dérivative, the covalent fixation of a phosphatidylinositol, covalent or non-covalent cross-linking, cyclization, disulfide bond 30 formation, déméthylation, glycosylation including pegylation, hydroxylation,
iodization, méthylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prénylation, racemization, seneloylation, sulfatation, amino acid addition such as arginylation or ubiquîtination. Such modifications hâve been described in the literature (see Proteins Structure and Molecular Properties (1993) 2nd Ed., T. E.
Creighton, New York; Post-translational Covalent Modifications of Proteins (1983) B. C, Johnson, Ed., Academie Press, New York; Seifter et al. ( 1990) Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182; 626-646 and Rattan et al., (1992) Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663: 48-62). Accordingly, the terms peptide, polypeptide, protein can include ] 0 for example lipopeptides, lipoproteîns, glycopeptîdes, glycoproteins and the like.
Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated. În certain embodiments, variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein. As used herein, (poly)peptide and protein may be used interchangeably in reference to a polymer of amino acid residues, such as a pluralîty of amino acid monomers linked by peptide bonds.
Nucleic acid molécule or polynucleotide or nucleic acid refers to a polymeric compound including covalently linked nucléotides, which can be made up of natural subunîts (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracîl, thymine, and cytosine. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodîthioate, phosphoroselenoate, phosphorodiselenoate, phosphoroaniiothioate, phosphoranilidate, phosphoramidate, or the like.
Nucleic acid molécules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic
DNA, any of which may be single or double-stranded. If single-stranded, the nucleic acid molécule may be the coding strand or non-coding (anti-sense strand). Also contemplated are microRNA, siRNA, viral genomic RNA, and synthetic RNA. Polynucleotides (including oligonucleotides), and fragments thereof may be generated, for example, by polymerase chaîn reaction (PCR) or by in vitro translation, or generated 5 by any of ligation, scission, endonuclease action, or exonuclease action.
A nucleic acid molécule encoding an amino acid sequence inciudes all nucléotide sequences that encode the same amino acid sequence. Some versions of the nucléotide sequences may also include intron(s) to the extent that the intron(s) may be removed through co- or post-transcriptional mechanisms. Different nucléotide sequences may encode the same amino acid sequence as the resuit of the redundancy or degeneracy of the genetic code, or by splicing, or both.
Variants of nucleic acid molécules of this disclosure are also contemplated. Variant nucleic acid molécules are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molécule of a defined or reference 15 polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molécule variants retain the capacîty to encode a fusion protein or a binding domain thereof having a functionality described herein, such as 20 specifically binding a target molécule.
As used herein, the term sequence variant refers to any sequence having one or ntore alterations in comparison to a reference sequence, whereby a reference sequence is any published sequence and/or of the sequences listed in the Table of Sequences and SEQ ID Numbers (sequence listing) herein. Thus, the term sequence 25 variant inciudes nucléotide sequence variants and amino acid sequence variants. In certain embodiments, a sequence variant in the context of a nucléotide sequence, the reference sequence is also a nucléotide sequence, whereas in certain embodiments for a sequence variant in the context ofan amino acid sequence, the reference sequence is also an amino acid sequence. A sequence variant as used herein can be at least 50%, 30 at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference sequence.
Percent sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences. Methods to détermine sequence identity can be designed to give the best match between the sequences being compared. For example, the sequences may be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to détermine sequence identity and similarity can be found in publîcly available computer programs. Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or
BLASTX). The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Within the context ofthîs disclosure, it will be understood that where sequence analysis software îs used for analysis, the results of the analysis are based on the default values of the program referenced. Default values mean any set of values or parameters which originally load with the software when first initialized.
A sequence variant in the context of a nucleic acid (nucléotide) sequence has an altered sequence in which one or more of the nucléotides in the reference sequence is deleted, or substituted, or one or more nucléotides are inserted into the sequence of the reference nucléotide sequence. Nucléotides are referred to herein by the standard one- letter désignation (A, C, G, or T). Due to the degeneracy of the genetic code, a sequence variant of a nucléotide sequence can either resuit in a change in the respective reference amino acid sequence, i.e. in an amino acid sequence variant or not. In certain embodiments, a nucléotide sequence variant does not resuit in an amino acid sequence variant (e.g., a silent mutation). In some embodiments, a nucléotide sequence variant that results in one or more non-silent mutation is contemplated. In some embodiments, a nucléotide sequence variant of the présent disclosure encodes an amino acid sequence that is at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference amino acid sequence. Nucléotide and 5 amino sequences as disclosed herein refer also to codon-optimized versions of a reference or wild-type nucléotide or amino acid sequence. In any of the embodiments described herein, a polynucleotide of the présent disclosure may be codon-optimized for a host cell containing the polynucleotide. Codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimumGene™ tool, or the 10 GeneArt Gene Synthesis Tool (Thermo Fisher Scientific). Codon-optimized sequences include sequences that are partially codon-optimized {i.e., at least one codon is optimized for expression in the host cell) and those that are fully codon-optimized.
A sequence variant in the context of an amino acid sequence has an altered sequence in which one or more of the amino acids is deleted, substituted, or inserted in 15 comparison to a reference amino acid sequence. As a resuit of the alterations, such a sequence variant has an amino acid sequence which is at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference amino acid sequence. For example, per 100 amino acids of the reference sequence a variant sequence that has no more than 10 alterations, i.e. any combination of délétions, insertions or substitutions, is at least 90% identical to the reference sequence.
A conservative substitution refers to amino acid substitutions that do not signîflcantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced 25 with an amino acid residue having a similar sîde chaîn. Conservative substitutions include a substitution found in one of the following groups: Group l : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histîdine (Hîs or H); Group 5:
Isoleucine (lie or I), Leucine (Leu or L), Méthionine (Met or M), Valine (Val or V); and
Group 6: Phenylalanîne (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by sîmilar function, Chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile. Other conservative substitutions groups include: sulfur-containing; Met and Cysteîne (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: Hîs, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, lie, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.
Amino acid sequence insertions can include amino- and/or carboxyl-tenninal fusions ranging in length from one residue to polypeptides containing a hundred or 15 more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include the fusion to the N- or C-terminus of an amino acid sequence to a reporter molécule or an enzyme.
In general, alterations in the sequence variants do nol abolish or significantly reduce a desired functionality of the respective reference sequence. For example, it is 20 preferred that a variant sequence of the présent disclosure does not significantly reduce or abrogate the functionality of a sequence of an antibody, or antigen-binding fragment thereof, to bind to the same epitope, to sufficiently neutralize infection of HBV and FIDV, and/or does not cause or increase formation of antibody dimer, and/or is not produced at a lower titer in a host cell, as compared to antibody or antigen binding fragment having (or encoded by) the reference sequence.
As used herein, a nucleic acid sequence or an amino acid sequence derived from a specified nucleic acid, peptide, polypeptide or protein refers to the orîgîn of the nucleic acid, peptide, polypeptide or protein. A nucleic acid sequence or amino acid sequence which is derived from a particular sequence may hâve an amino acid sequence 30 that is essentially identical to that sequence or a portion thereof, from which it is
derived, whereby essentially identifiai includes sequence variants as defined above. A nucleic acid sequence or amino acid sequence which is derived from a particular peptide or protein may be derived from the corresponding domain in the particular peptide or protein. In this context, corresponding refers to possession of a same functionality or characterîstic of interest. For example, an extracellular domain corresponds to another extracellular domain (of another protein), or a transmembrane domain corresponds to another “transmembrane domain” (of another protein). Corresponding parts of peptides, proteins and nucleic acids are thus easily identifiable to one of ordinary skill in the art. Lîkewise, a sequence derived from another (e.g., source) sequence can be identifïed by one of ordinary skill in the art as having its origin in the source sequence,
A nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be îdentical to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived). However, a nucleic [ 5 acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may also hâve one or more mutations relative to the starting nucleic acid, peptide, polypeptide or protein (from which it is derived), in particular a nucleic acid sequence or an amino acid sequence derived from another nucleic acid, peptide, polypeptide or protein may be a functional sequence variant as described above 20 of the starting nucleic acid, peptide, polypeptide or protein (from which it is derived).
For example, in a peptide/protein, one or more amino acid residues may be substituted with other amino acid residues, or one or more amino acid residue insertions or délétions may occur.
As used herein, the term mutation relates to a change in a nucleic acid 25 sequence and/or in an amino acid sequence in comparison to a reference sequence, e.g. a corresponding genomic, wild type, or reference sequence. A mutation, e.g. in comparison to a reference genomic sequence, may be, for example, a (naturally occurring) somatic mutation, a spontaneous mutation, an induced mutation, e.g. induced by enzymes, Chemicals or radiation, or a mutation obtained by site-directed mutagenesis 30 (molecular biology methods for making spécifie and intentional changes in the nucleic acid sequence and/or in the amino acid sequence). Thus, the terms mutation or mutatîng shall be understood to also include physically making or inducing a mutation, e.g. in a nucleic acid sequence or in an amino acid sequence. A mutation includes substitution, délétion and/or insertion of one or more nucléotides or amino acids, as well as inversion of several successive nucléotides or amino acids. To achieve a mutation in an amino acid sequence, a mutation may be introduced into the nucléotide sequence encoding said amino acid sequence in order to express a (recombinant) mutated polypeptide. A mutation may be achieved, for example, by altering (e.g., by site-directed mutagenesis) a codon (e.g., by alterning one, two, or three nucléotide bases 10 therein) of a nucleic acid molécule encoding one amino acid to provide a codon that encodes a different amino acid, or that encodes a same amino acid, or by synthesizing a sequence variant.
A functional variant refers to a polypeptide or polynucléotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide.
In other words, a functional variant of a polypeptide or encoded polypeptide of this disclosure has similar binding, similar affmity or similar activity when the functional variant displays no more than a 50% réduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant).
As used herein, a functional portion or functional fragment refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment ofthe parent or 30 reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99%, 99.9%, or 100% level of activity ofthe parent polypeptide, or provides a biological benefit (e.g., effector function). A functional portion or functional fragment of a polypeptide or encoded polypeptide of this disclosure has similar binding or similar activity when the functional portion or fragment displays 5 no more than a 50% réduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log différence as compared to the parent or reference with regard to affinity).
The term isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a 10 naturally occurring nucleic acid or polypeptide présent in a livîng animal is not isolated, but the same nucleic acid or polypeptide, separated from some or ail of the co-existing materials in the natural system, is isolated. Such nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part ofthe natural environment for the nucleic acid or polypeptide. Isolated can, in some embodiments, describe an antibody, antigen-binding fragment, fusion protein, polynucleotîde, vector, host cell, or composition that is outside of a human body.
The term gene means the segment of DNA or RNA involved in producing a polypeptide chain; in certain contexts, it includes régions preceding and following the 20 coding région (e.g., 5’ untranslated région (UTR) and 3’ UTR) as well as intervening sequences (introns) between individual coding segments (exons).
The term introduced in the context of inserting a nucleic acid molécule into a cell, means transfection, or transformation or transduction and includes reference to the incorporation of a nucleic acid molécule into a eukaryotic or prokaryotic cell wherein the nucleic acid molécule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
The term recombinant, as used herein (e.g. a recombinant antibody, a recombinant protein, a recombinant nucleic acid, or the like), refers to any molécule (antibody, protein, nucleic acid, or the like) which is prepared, expressed, created or
isolated by recombinant means, and which is not naturally occurring. Recombinant can be used synonymously with engineered or non-natural and can refer to to an organism, microorganism, cell, nucleic acid molécule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic 5 acid molécule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molécules encoding proteins, fusion proteins or enzymes, or other nucleic acid molécule additions, délétions, substitutions or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory régions in which the modifications aller expression of a polynucleotide, gene or operon.
As used herein, heterologous or non-endogenous or exogenous refers to any gene, protein, compound, nucleic acid molécule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molécule, or activity 15 native to a host cell or a subject that has been altered. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molécules that hâve been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molécules. In certain embodiments, heterologous, non-endogenous, or exogenous genes, proieins, 20 or nucleic acid molécules may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molécules may hâve been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molécule may intégrale into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self25 replicating vector). The term homologous or homolog refers to a gene, protein, compound. nucleic acid molécule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may hâve an altered structure, sequence, expression level, or any combination thereof.
A. non-endogenous poiynucleotide or gene, as well as the encoded polypeptide or activity, may be from the same species, a different species, or a combination thereof. As used herein, the term endogenous or native refers to a poiynucleotide, gene, protein, compound, molécule, or activity that is normally présent in a host cell or a subject.
The term expression, as used herein, refers to the process by which a polypeptide is produced based on the encodîng sequence of a nucleic acid molécule, such as a gene. The process may include transcription, post-transcriptîonal control, posl-transcriptional modification, translation, post-translational control, posttranslational modification, or any combination thereof. An expressed nucleic acid molécule is typically operably lînked to an expression control sequence (e.g., a promoter).
The term operably linked refers to the association of two or more nucleic acid molécules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when il is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Unlinked means that the associated genetic éléments are not closely associated with one another and the function of one does not affect the other.
As described herein, more than one heterologous nucleic acid molécule can be introduced into a host cell as separate nucleic acid molécules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molécule, as a single nucleic acid molécule encodîng a protein (e.g., a heavy chain of an antibody), or any combination thereof. When two or more heterologous nucleic acid molécules are introduced into a host cell, it is understood that the two or more heterologous nucleic acid molécules can be introduced as a single nucleic acid molécule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of referenced heterologous nucleic acid molécules or protein activities refers to the number of encodîng nucleic acid molécules or the number ot protein activities, not the number of separate nucleic acid molécules introduced into a host celL
As used herein, the terms cell, cell line, and cell culture are used interchangeably and ail such désignations include progeny. Thus, the terms transformants and transformed cells and host cells include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that ail progeny may not be precisely identical in DNA content, due to delîberate or înadvertent mutations. Variant progeny that hâve the same or substantially the same function, phenotype, or biological activity as screened for in the originally transformed cell are included. Where distinct désignations are intended, it will be clear from the context.
The term construct refers to any polynucleotide thaï contains a recombinant nucleic acid molécule (or, if the context clearly indicates, a fusion protein of the présent disclosure).
In certain embodiments, polynucleotides of the présent disclosure may be operatively linked to certain éléments of a vector. For example, polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signais such as splicing and polyadenylation signais; sequences that stabilize cytoplasmîc mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibiy sequences that enhance protein sécrétion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
Antibodies and Antigen-Bindîng Fragments
Presently disclosed embodiments include antibodies, and antigen-binding fragments thereof, that are capable of binding to the antigenic loop région of HBsAg (HBsAg and the antigenic loop région are described in further detail here) and, optionally, neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof; i.e., any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, or all ten of these génotypes. As 5 discussed further herein, presently disclosed antibodies and antigen-binding fragments possess other advantages, including, for example and not limited to, characterîstics that favor production in a host cell, and reduced propensity to form undesirable aggregates, such as dimers.
As used herein, and unless the context clearly indicates otherwise, antibody refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chai ns inter-connected by disulfîde bonds (though it will be understood that heavy chain antibodies, which lack light chains, are still generally encompassed by the term antibody, though preferred embodiments of the présent disclosure comprise both of a VH and a VL, and in some embodiments, both of a heavy chain and a light chain), as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molécule recognized by the intact antibody, such as, for example, a scFv, Fab, or F(ab')2 fragment. Thus, the term antibody herein is used in the broadest sense and inciudes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g·, sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fuily human antibodies, humanized antibodies, and heteroconj ugate antibodies, multispecific, e.g., bispecific, antibodies, diabodîes, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv, and other antibody formats known in the art. Unless otherwise stated, the term antibody should be understood to encompass functional antibody fragments thereof. The term antibody also encompasses intact or full-length antibodies, including antibodies of any class or sub class thereof, including IgG and sub-classes thereof (IgGl, IgG2, IgG2, IgG4), IgM, IgE, IgA, and IgD.
As used herein, in the context of an antibody, the ternis antigen-bînding fragment, fragment, and antibody fragment are used interchangeably to refer to any fragment of an antibody of the disclosure that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, a single chain antibody, Fab, Fab’, F(ab’)2, Fv or scFv.
Human antibodies are known (e.g., van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001 ) 368-374). Human antibodies can be produced în transgenic animais (e.g., mice) that are capable, upon immunization, of producing a full répertoire or a sélection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will resuit in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 ( 1993) 2551 -2555; Jakobovits, A., et al., Nature 362 (l 993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (l 992) 381388; Marks, J. D., étal., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for the préparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Lîss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). Human monoclonal antibodies may be prepared by using improved EBV-B cell immortal îzation as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871-5. The term human antibody as used herein also comprises such antibodies which are modified, e.g., in the variable région or the constant région, to generate properties according to the antibodies and antibody fragments of the présent disclosure.
Antibodies according to the présent disclosure can be of any isotype (e.g., IgA, IgG, IgM, IgE, IgD; i.e., comprising an α, γ, μ, ε, or Ô heavy chain). Within the IgG isotype, for example, antibodies may be IgGl, IgG2, IgG3 or IgG4 subclass. In spécifie embodiments, an antibody of the présent disclosure is an IgGl antibody. Antibodies or 5 antigen binding fragments provided herein may include a κ or a λ light chain.
Preferably, an antibody or antigen-binding fragment can comprise a λ light chain. In certain embodiments, HBsAg-specific antibodies described herein are of the IgG isotype (e.g., IgGlM,l7 l allotype) and may block the release of HBV and HBsAg from infected cells. Accordingly, in certain embodiments, an antibody according to the 10 présent description can bind intracellularly and thereby block the release of HBV virions and HBsAg.
The terms Vl or VL” and Vh or VH refer to the variable région (also called variable domain) from an antibody light chain and an antibody heavy chain, respectively; typically, these régions are involved directly in the binding of an antibody 15 or antigen-binding fragment to an antigen. A VL (as well as a CL or a light chain) can be a kappa (κ) class (also VK” herein) or a lambda (λ) class. The variable binding régions comprise discrète sub-regions known as complementarity determining régions” (CDRs) and framework régions (FRs). The terms complementarity determining région, and CDR, are synonymous with hypervariable région or HVR, and refer 20 to sequences of amino acids within antibody variable régions, which, in general, together confer the antigen specîficity and/or binding affinity of the antibody, wherein consecutive CDRs (i.e., CDRl and CDR2, CDR2 and CDR3) are separated from one another in primary amino acid sequence by a framework région. There are three CDRs in each variable région (HCDRl, HCDR2, HCDR3; LCDRl, LCDR2, LCDR3; also referred to as CDRHs and CDRLs, respectively). In certain embodiments, an antibody VH comprises four FRs and three CDRs as follows: FRI -HCDRl -FR2-HCDR2-FR3HCDR3-FR4; and an antibody VL comprises four FRs and three CDRs as follows: FRI -LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. In general, the VH and the VL together form the antigen-binding site through their respective CDRs, though it will be understood that in some cases, a binding site can be formed by or can comprise one, two, three, four, or five of the CDRs which CDR(s) may disposed in the VH, in the VL, or in both.
In certain embodiments, antibody CDRs and amino acid numbering of variable régions are according to the System deveîoped by the Chemical Computing Group (CCG); e.g., using Molecular Operating Environment (MOE) software (www.chemcomp.com).
In certain embodiments, antibody CDRs and amino acid numbering of variable régions are according to the IMGT numbering scheme (see, e.g., Lefranc et al., Dev. Camp. Immunol. 27:55, 2003).
Equivalent residue positions can be annotated and for different molécules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300).
As used herein, a variant of a CDR refers to a functional variant (as provided herein) of a CDR sequence having up to 1-3 amino acid substitutions, délétions, or 15 combinations thereof.
In certain embodiments, the présent disclosure provides an antibody, or an antigen-binding fragment thereof, comprising (i) a heavy chain variable région (VH) that comprises therein the amino acid sequence of SEQ ID NO.:34, the amino acid sequence of SEQ ID NO.:35 or SEQ ID NO.:36, and the amino acid sequence of SEQ
ID NO.:37; and (ii) a light chain variable région (VL) that comprises therein the amino acid sequence any one of SEQ ID NOs.:41,40, 42, and 43, the amino acid sequence according to any one of SEQ ID NOs:49, 44-48, and 50-53, and the amîno sequence according to SEQ ID NO.:55 or 56, wherein, optionally, the VL comprises a R60N substitution mutation, a R60A substitution mutation, a R60K substitution mutation, a S64A substitution mutation, a I74A substitution mutation, or any combination thereof, relative to SEQ ID NO.:58 and wherein the amino acid numbering of the substitution mutation(s) is according to SEQ ID NO.:58, and still further optionally wherein the VL does not comprise any further mutation(s) relative to SEQ IDNO.:58, and wherein the antibody or antigen-binding fragment thereof is capable of binding to the antigenic loop région of HBsAg and, optionally, neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof.
In some embodiments, the antibody or antigen-binding fragment comprises: (î) in the VH, the amino acid sequences according to SEQ 1D NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:4l, 45, and 55, respectively; (ii) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, and in the VL, the amino acid sequences according to SEQ ID NOs.: 41, 46, and 55, respectively; (iii) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.: 41,47, and 55, respectively; (îv) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:4l, 48, and 55, respectively; (v) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:4l, 49, and 55, respectively; (vi) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:4l, 50, and 55, respectively; (vii) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:4l, 51, and 55, respectively; (viii) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:4l, 52, and 55, respectively; or (ix) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:4l, 53, and 55, respectively.
In certain embodiments, the présent disclosure provides an antibodj, or an antigen-binding fragment thereof, comprising (i) a heavy chain variable région (VH) comprising a CDRHl amino acid sequence according to SEQ ID NO.:34, a CDRH2 amino acid sequence according to SEQ ID NO.:35 or 36, and a CDRH3 amino acid
sequence according to SEQ ID NO.:37; and (ii) a light chain variable région (VL) comprising a CDRL1 amino acid sequence set forth in any one of SEQ ID NOs.:40-43, a CDRL2 amino acid sequence according to any one of SEQ ID NOs:45-53, and a CDRL3 amino acid sequence according to SEQ ID NO.:55 or 56, wherein the CDRs are according to CCG, wherein the antibody or antigen-binding fragment thereof is capable of binding to the antîgenic loop région of HBsAg and neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof.
In certain embodiments, the CDRHl, CDRH2, CDRH3, CDRLl, CDRL2, and
CDRL3 amino acid sequences are according to SEQ ID NOs.: (i) 34, 35, 37, 41, 45, and 55, respectively; (ii) 34, 35, 37, 41, 46, and 55, respectively; (iii) 34, 35, 37, 41, 47, and 55, respectively; (iv) 34, 35, 37, 41, 48, and 55, respectively; (v) 34, 35, 37, 41, 49, and 55, respectively; (vi) 34, 35, 37, 41,50, and 55, respectively; (vîi) 34, 35, 37, 41,51, and 55, respectively; (viii) 34, 35, 37, 41, 52, and 55, respectively; or (ix) 34, 35, 37,
41,53, and 55, respectively, wherein CDRs are according to CCG.
Table I provides the CDR amino acid SEQ ID NOs. of certain antibodies, wherein CDRs are defined according to CCG.
Table 1. CDR (CCG numbering) amino acid SEQ ID NOs. of Certain Antibodies
| Antibody | CDRHl | CDRH2 | CDRH3 | CDRLl | CDRLl | CDRL3 |
| HBC34-v35 | 34 | 35 | 37 | 41 | 44 | 55 |
| HBC34-v36 | 34 | 35 | 37 | 41 | 45 | 55 |
| HBC34-v37 | 34 | 35 | 37 | 41 | 46 | 55 |
| HBC34-v38 | 34 | 35 | 37 | 41 | 47 | 55 |
| HBC34-v39 | 34 | 35 | 37 | 41 | 48 | 55 |
| HBC34-v40 | 34 | 35 | 37 | 41 | 49 | 55 |
| HBC34-v41 | 34 | 35 | 37 | 41 | 50 | 55 |
| HBC34-v42 | 34 | 35 | 37 | 41 | 51 | 55 |
| HBC34-v43 | 34 | 35 | 37 | 41 | 52 | 55 |
| HBC34-v44 | 34 | 35 | 37 | 41 | 53 | 55 |
| HBC34-v45 | 34 | 35 | 37 | 41 | 44 | 55 |
| HBC34-v46 | 34 | 35 | 37 | 41 | 44 | 55 |
| EIBC34-v47 | 34 | 35 | 37 | 41 | 51 | 55 |
| EIBC34-v48 | 34 | 35 | 37 | 41 | 44 | 55 |
| [-IBC34-V49 | 34 | 35 | 37 | 41 | 51 | 55 |
| HBC34-v50 | 34 | 35 | 37 | 41 | 44 | 55 |
In certain embodiments, an antibody or antigen-binding fragment comprises CDRHl, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of: HBC34-v36; HBC34v37; HBC34-v38; HBC34-v39; HBC34-v40; HBC34-v4l; HBC34-v42; HBC34-v43;
HBC34-v44; HBC34-v45; HBC34-v46; HBC34-v47; HBC34-v48; HBC34-v49; or HBC34-v50, wherein the CDRs are according to CCG, optionally wherein the VL further comprises a R60N substitution mutation, a R60A substitution mutation, a R60K substitution mutation, a S64A substitution mutation, a I74A substitution mutation, or any combination thereof, relative to SEQ ID NO.:58 and wherein the amino acid numbering of the substitution mutation(s) is according to SEQ ID NO.:58, and further optionally wherein the VL does not comprise any other mutation(s) relative to SEQ ID NO.:58.
Table 2 provides the CDR amino acid SEQ ID NOs. of certain antibodies, wherein CDRs are defined according to IMGT (short and long versions of CDRH2 and 15 CDRL2 are disclosed).
Table 2. CDR (IMGT numbering) amino acid SEQ ID NOs. of Certain Antibodies
| Antibody | CDRHl | CDRH2 (short/long) | CDRH3 | CDRL1 | CDRL2 (short/long) | CDRL3 |
| HBC34-v35 | 150 | 151/152 | 153 | 154 | 155/163 | 173 |
| HBC34-v36 | 150 | 151/152 | 153 | 154 | 156/164 | 173 |
| HBC34-v37 | 150 | 151/152 | 153 | 154 | 157/165 | 173 |
| HBC34-v38 | 150 | 151/152 | 153 | 154 | 158/166 | 173 |
| HBC34-v39 | 150 | 151/152 | 153 | 154 | 159/167 | 173 |
| HBC34-v40 | 150 | 151/152 | 153 | 154 | 156/168 | 173 |
| HBC34-v4I | 150 | 151/152 | 153 | 154 | 158/169 | 173 |
| HBC34-v42 | 150 | 151/152 | 153 | 154 | 160/170 | 173 |
| HBC34-v43 | 150 | 151/152 | 153 | 154 | 161/171 | 173 |
| HBC34-v44 | 150 | 151/152 | 153 | 154 | 162/172 | 173 |
| HBC34-v45 | 150 | 151/152 | 153 | 154 | 155/163 | 173 |
| HBC34-v46 | 150 | 151/152 | 153 | 154 | 155/163 | 173 |
| HBC34-v47 | 150 | 151/152 | 153 | 154 | 160/170 | 173 |
| HBC34-v48 | 150 | 151/152 | 153 | 154 | 155/163 | 173 |
| HBC34-v49 | 150 | 151/152 | 153 | 154 | 160/170 | 173 |
| HBC34-v50 | 150 | 151/152 | 153 | 154 | 155/163 | 173 |
In certain embodiments, an antibody or antigen-binding fragment comprises CDRHl, CDRH2, CDRH3, CDRLl, CDRL2, and CDRL3 of: HBC34-v36; HBC34v37; HBC34-v38; HBC34-v39; HBC34-v40; HBC34-v4] ; HBC34-v42; HBC34-v43; HBC34-v44; HBC34-v45; HBC34-v46; HBC34-v47; HBC34-v48; HBC34-v49; or
HBC34-v50, wherein the CDRs are according to IMGT, optionaliy wherein the VL further comprises a R60N substitution mutation, a R60A substitution mutation, a R60K substitution mutation, a S64A substitution mutation, a I74A substitution mutation, or any combination thereof, relative to SEQ ID NO.:58 and wherein the amino acid numbering of the substitution mutâtion(s) is according to SEQ ID N0.:58, and further optionaliy wherein the VL does not comprise any other mutation(s) relative to SEQ ID NO.:58.
Labié 3 provides the VH and VL amîno acid SEQ ID NOs, of certain antibodies.
Table 3. VH and VL amino acid SEQ ID NOs. of Certain Antibodies
| Antibody | VH | VL |
| HBC34-v35 | 38 | 57 |
| HBC34-v36 | 38 | 58 |
| HBC34-v37 | 38 | 59 |
| HBC34-v38 | 38 | 60 |
| HBC34-v39 | 38 | 6I |
| HBC34-v40 | 38 | 62 |
| HBC34-v4l | 38 | 63 |
| HBC34-v42 | 38 | 64 |
| HBC34-v43 | 38 | 65 |
| HBC34-v44 | 38 | 66 |
| HBC34-v45 | 38 | 67 |
| HBC34-v46 | 38 | 68 |
| HBC34-V47 | 38 | 69 |
| HBC34-v48 | 38 | 70 |
| HBC34-v49 | 38 | 71 |
| HBC34-v50 | 38 | 72 |
In certain embodiments, an antibody or antigen-binding fragment comprises the 15 VH and VL amino acid sequences of: HBC34-v36; HBC34-v37; HBC34-v38; HBC34v39; HBC34-v40; HBC34-v4l; HBC34-v42; HBC34-v43; HBC34-v44; HBC34-v45; HBC34-v46; HBC34-v47; HBC34-v48; HBC34-v49; or HBC34-v50.
In certain embodiments, antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein: (i) the VH comprises or consists of an amino acid sequence having at least 90% (Le., 90%, 9I%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any noninteger value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 58-66, 69, 71, or 72. In particular embodiments, the VH and the VL comprise or consist of amino acid sequences having at least 90% (i.e., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any non-integer value therebetween) identity to the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 58, respectively; (ii) 38 and 59, respectively; (iii) 38 and 60, respectively; (iv) 38 and 61, respectively; (v) 38 and 62, respectively; (vi) 38 and 63, respectively; (vii) 38 and 64, respectively; (viii) 38 and 65, respectively; (ix) 38 and 66, respectively; (x) 38 and 71, respectively; or (xi) 38 and 72, respectively. As a non-limiting example, in certain embodiments, the VH comprises an amino acid sequence having at least 90% identity to SEQ ID NO.:38 and the VL comprises an amino acid sequence having at least 90% identity to SEQ ID NO.:62.
In some embodiments, the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO.; 38 or 39; and/or the VL comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs.: 58-66, 69, 71, or 72. In particular embodiments, the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.; (i) 38 and 58, respectively; (ii) 38 and 59, respectively; (iii) 38 and 60, respectively; (iv) 38 and 61, respectively; (v) 38 and 62, respectively; (vî) 38 and 63, respectively; (vii) 38 and 64, respectively; (viii) 38 and 65, respectively; (ix) 38 and 66, respectively; (x) 38 and 71, respectively; or (xi) 38 and 72, respectively.
In certain embodiments, an antibody or antigen-binding fragment comprises a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:38 and a VL comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.:58-72.
In certain embodiments, an antibody or antigen-binding fragment comprises a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:38 and a VL comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.:59-72.
In another aspect, the présent disclosure provides an antibody or anligen-binding fragment, comprising: a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 67, respectively; or (ii) 38 and 68, respectively, wherein the antibody or antigen-binding fragment thereof is capable of binding to the antigenic loop région of HBsAg and neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof.
Also provided is an antibody or antigen-binding fragment comprising a VH according to SEQ ID NO.:38 or 39 and a VL variant of any one of SEQ ID NOs.:57-72 that comprises any one or more of the following mutations (in framework région 3, as determined by CCG numbering): R60A, R60N, R60K, S64A, I74A. In certain further embodiments, the VL variant does not comprise any further mutations as compared to SEQ ID NO.:57-72 (respectively))
Also provided is an antibody or antigen-binding fragment comprising a VH according to SEQ ID NO.:38 and a VL variant of any one of SEQ ID NOs.:57-72 that comprises a substitution mutation (such as, for example, a conservative amino acid substitution, or a mutation to a germline-encoded amino acid) at Q78, D8l, or both (CCG numbering).
Also provided is an antibody or antigen-binding fragment comprising a VH according to SEQ ID NO.:39 and a VL variant of any one of SEQ ID NOs.:57-72 that comprises a substitution mutation (such as, for example, a conservative amino acid substitution, or a mutation to a germline-encoded amino acid) at Q78, D81, or both (CCG numbering).
As discussed further herein in, presently disclosed antibodies and antîgenbinding fragments hâve a reduced propensity to form aggregates (e.g., dimers), and/or hâve improved productivity (e.g., higher titer) in a host cell, and/or hâve similar or substantially identical or even improved: binding to HBsAg; HBV neutralization;
and/or thermostability, as compared to a reference antibody disclosed herein, It will be understood that a reference antîbody or antigen-binding fragment refers to an antibody or antigen-binding fragment that is identical to the subject antibody or antigen-binding fragment, respectively, with the exception of the identified or enumerated features (e.g., différences in CDR and/or variable région framework sequence(s)). In some embodiments, the reference antibody comprises the CDRHl, CDRH2, CDRH3, CDRLl, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 41,44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57,
As a non-limiting example, an antibody or antigen-binding fragment of the présent disclosure can be an IgGl isotype and comprise a wild-type IgG I Fc moiety, and a reference antibody or antigen-binding fragment comprises CDRHl, CDRH2, CDRH3, CDRLl, CDRL2, and CDRL3 amino acid sequences according to the amino acîd sequences set forth in SEQ ID NOs,:34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57, and is the IgGl isotype and comprises a wild-type IgGl Fc moiety. It will further be understood that when comparing a presently disclosed antibody or antigen-binding fragment with a reference antibody or antigen-binding fragment under certain conditions, the conditions (e.g., amount of starting material, température, buffer, identity of host cell line, culture conditions, duration of a relevant time period, codon-optîmization of an encoding polynucleotide, or the like) will, unless explicitly stated otherwise, be identical as between the presently disclosed molecule(s) and the reference molecule(s), or as close to identical as the conditions permit (e.g., two antibodies may differ in their amino acid sequences by one or a number of amino acids, but will be otherwise identical, and will
be encoded by a comparable polynucleotide (e.g., each antibody can be encoded by a respective codon-optîmized polynucleotide).
As a non-limiting example, presently disclosed antibodies and antigen-binding fragments produce fewer aggregates (e.g., in the form of antibody:antibody dimers, 5 antibody:antigen-binding fragment dimers, or antigen-binding fragment:antigen-bînding fragment dimers), and/or hâve a higher production tîter in a host cell, as compared to a reference antibody or antigen-binding fragment, respectîvely.
In this context, a dimer is a complex or aggregate comprising two antibody or antigen-binding fragment molécules (e.g., an antibody:antibody dimer, a Fab:Fab dimer, or an antibody:Fab dimer). As discussed further herein, dimerization in this context is distinct from typical associations between antibody heavy chain and light chain components, or between two antibody heavy chain polypeptides, that occur in the formation of an intact tetrameric antibody, Fv, or Fab and may in volve associations between two monomers. Accordingly, it will be understood that in the présent context, ] 5 a dimer or does not refer to the association of an antibody heavy chain with an antibody light chain to provide a half-antibody comprising a functional Fab, and also does not include association of two heavy chains of an antibody (e.g., hinge-hinge and Fc-Fc) or VH-VL associations (e.g. that occur via disulfide bonds), such as in a Fv or in a Fab.
In certain embodiments, a dimer is formed by association between the VLs of two discrète antibody or antigen-binding fragment molécules. An illustration of a dimer formed by association of two VLs of discrète antibody molécules is shown in present Figure 7. Such dimerization can, for example, reduce binding valency and/or binding affinity and/or avidity and/or neutralization potency ofone or both ofthe antibody or antigen-binding fragment molécules comprised therein. In general, an increased presence of such dimers in a composition comprising a plurality of antibodies or antigen-binding fragments reduces the overall binding and/or neutralizing potency of the composition.
Antibody or antigen-binding fragment dimers can be identified using, known 30 techniques, such as, for example, size-exchision chromatography. A dimer will hâve a molecular weight that is higher than the molecular weight of each individual (monomer) subunit thereof and will typically equal or approximate the sum ofthe molecular weights of each individual subunit thereof. For example, a homo-dimer (i.e., which comprises two antibody molécules that are identical or substantially identical in their amino acid sequences) will generally hâve a molecular weight that is about twice the molecular weight of each monomeric subunit thereof. For example, a typical human IgG l immunoglobulin molécule has a molecular weight of around 150 kilodaltons (for example, with each of the two heavy chains weighing around 50 kilodaltons, and each of the two lîght chains weighing around 25 kilodaltons), and a dimer comprising two such immunoglobulin molécules will hâve a molecular weight of around 300 kilodaltons. Of course, it will be understood that one antibody may hâve a slightly or somewhat different molecular weight than a different antibody of the same general structure and isotype, e.g., due at least in-part to any différences in the respective amino acid sequences.
As another, non-limiting, example, an antibody molécule may hâve a molecular weight of between 140 kilodaltons and 160 kilodaltons, and an antibody dirner comprising two antibody molécules may hâve a molecular weight of between 280 kilodaltons and 320 kilodaltons. Dimers may be referred-to as high-molecular weight species or HMWS.
The presence of dimers in a composition or sample comprising a plurality of antibody (and/or antigen-binding fragment) molécules can be evaluated using, for example, absolute size exclusion chromatography (aSEC). The amount of dimer in a composition or sample can be expressed as the percentage of total antibody or antigenbinding fragment molécules in the composition or sample that are comprised in a dimer. By way of illustration, for an antibody composition comprising 12% dimers, 88% ofthe total antibody molécules in the sample are présent as monomers.
In any of the presently disclosed embodiments, in a sample comprising a plurality of the antibody or antigen-binding fragment (i.e., a plurality of antibody or antigen-binding fragment molécules), less than 12%, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less
of the plurality is comprised in a dimer when the sample has been incubated for about 120 to about 168 hours at about 40°C, wherein, optionally, the presence of dimer is determined by absolute size-exclusion chromatography.
In any of the presently disclosed embodiments, incubation of a plurality of presently disclosed antibody or antigen-binding fragment molécules results in reduced formation of dimers as compared to incubation of a plurality of a reference antibody or antigen-binding fragment molécules, wherein the reference antibody or antigen-binding fragment comprises CDRHl, CDRH2, CDRH3, CDRLI, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.;34, 35, 10 37, 41,44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57, and wherein, optionally, the presence of antibody dimer is determined by absolute size-exclusion chromatography. Such a reference antibody or antigen-binding fragment (e.g., Fv, Fab) can form dimers that, in some embodiments, collectîvely comprise more than 2%, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, or up to I2% ofthe antibody or antigen-binding fragment molécules in a sample (e.g., when incubated for about 120 to about 168 hours at about 40°C). In other words, in some embodiments, up to 12% or more of reference antibody or antigen-binding fragment molécules are comprised in a dimer, while a lesser percentage, preferably 2% or less, of presently disclosed antibody or antigen-binding fragment molécules are comprised in a dimer.
In some embodiments, a presently disclosed antibody or antigen-binding fragment forms a lower amount of dimer, and/or forms dimers at a reduced frequency and/or as a lower percentage of total antibody or antigen-binding fragment molécules in 25 a sample or composition, (e.g., as determined using Size Exclusion Chromatography) as compared to a reference antibody: (i) in a 5-day, a 15-day, and/or a 32-day incubation at 4°C; (ii) in a 5-day, a 15-day, and/or a 32-day incubation at 25“C; and/or (iii) in a 5-day, a 15-day, and/or a 32-day incubation at 40°C, wherein the reference antibody or antigen-binding fragment comprises CDRHl, CDRH2, CDRH3, CDRLI, CDRE2, and 30 CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ
ID NOs.:34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57.
in some embodiments, the percentage of presently disclosed antibody or antigen-binding fragment molécules in a composition that are comprised in a dimer îs less than 4/5, less than 3/4, less than 1/2, less than 1/3, less than 1/4, less than 1/5, less than 1/6, less than 1/7, less than 1/8, less than 1/9, or less than 1/10 the percentage of the reference antibody molécules in a composition that are présent in a dimer, respectively. As a non-limiting example, following a 32-day (768-hour) incubation at 10 40°C, 22% or more of the reference antibody molécules in a composition can be comprised in a dimer, while 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less of presently disclosed antibody or antigen-binding fragment molécules in a composition are comprised in a dimer, respectively.
In some embodiments, a host cell (e.g., a CHO cell such as an ExpîCHO cell) transfected with a polynucleotide encoding a presently disclosed antibody or antigenbinding fragment provides 1.5x or more, 2x or more, 3x or more, or 4x or more the amount of antibody or antigen-binding fragment, respectively, (e.g., measured as a 20 concentration in mg/mL) than a reference host cell transfected with a polynucleotide encoding a reference antibody or antigen-binding fragment, wherein the reference antibody or antigen-binding fragment comprises CDRH 1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 41, 44, and 55, respectively, and optionally comprises 25 the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57.
In some embodiments, a presently disclosed antibody or antigen-binding fragment thereof is produced in transfected cells at a hîgher tîter as compared to a reference antibody or antigen-binding fragment is produced in reference transfected 30 cells, wherein the reference antibody or antigen-binding fragment comprises CDRH1,
CDRLI2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 41,44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57.
In some embodiments, a presently disclosed antibody or antigen-binding fragment thereof is produced in transfected cells at titers of at least 1.5-fold, at least 2fold, at least 3-fold, or at least 4-fold, higher than the titer at which a reference antibody or antigen-binding fragment is produced, wherein the reference antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth în SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57.
In any of the presently disclosed embodiments, the antibody or antigen-binding fragment is capable of binding to a HBsAg (e.g., of subtype adw) with an EC50 (ng/ml) of 3.5 or less, about 3.2 or less, less than 3.0, less than 2.5, less than 2.0, less than 1.5, or less than l .0. In some embodiments, the antibody or antigen-binding fragment is capable of binding to a HBsAg (e.g., of subtype adw) with an EC50 (ng/ml i of less than 3.5, less than 3.4, less than 3.3, less than 3.2, less than 3.1, less than 3.0, less than 2.9, less than 2.8, less than 2.7, less than 2.6, less than 2.5, less than 2.4, less than 2.3, less than 2.1, less than 2.0, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1,2, less than 1.1, or less than 1.0. In some embodiments, the antibody or antigen-binding fragment is capable of binding to a HBsAg (e.g., of subtype adw) with an EC50 (ng/ml) of between 0.9 and 2.0, or of between 0.9 and 1.9, or of between 0.9 and 1.8, or of between 0.9 and 1.7, or of between 0.9 and 1.6, or of between 0.9 and 1.5, or of between 0.9 and 1.4, or of between 0.9 and 1.3, or of between 0.9 and 1.2, or of between 0.9 and 1.1, or of between 0.9 and 1.0, or of between 1.0 and 2.0. In certain embodiments, the antibody or antigen-binding fragment is capable of binding to a HBsAg (e.g., of subtype adw) with an EC50 (ng/ml) of 2.0 or less. In some embodiments, a binding EC50 is determined by ELISA (e.g., direct antigen-binding ELISA assay, with binding curves determined by fitting the curves using Graphpad prism).
In any ofthe presently disclosed embodiments, the antibody or antigen-binding fragment thereof is capable of neutralizing hepatitis B virus infection with a neutralization of infection EC50 of less than 20 ng/ml, preferably 15 ng/ml or less, more preferably 10 ng/mL or less. In some embodiments, the antibody or antigenbinding fragment thereof is capable of neutralizing hepatitis B virus infection with a neutralization of infection EC50 of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 ng/mL. In some embodiments, the antibody or antigen-binding fragment thereof is capable of neutralizing hepatitis B virus infection with a neutralization of infection EC50 that is lower than the neutralization of infection EC50 (using the same assay) of a reference antibody or antigen-binding fragment that comprises CDRH!, CDRFI2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ IDNOs.:34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57. In some embodiments, a neutralization of infection EC50 is determined following incubation of cultnred cells, e.g., difièrent!ated HepaRG cells, with a fixed amount of HBV in the presence or absence of the antibody to be tested. In such an embodiment, incubation may be carried out, for example, for 16 hours at 37°C. That incubation may be performed in a medium (e.g. supplemented with 4% PEG 8000). After incubation, cells may be washed and further cultivated. To measure virus înfectivity, the levels of hepatitis B surface antigen (HBsAg) and/or hepatitis B e antigen (HBeAg) secreted into the culture supernatant, e.g. from day 7 to day 11 post-infection, may be determined by enzyme-linked immunosorbent assay (ELISA). Levels of HBsAg and/or HBeAg from treated cells can be compared to those of untreated cells to détermine the presence and extent of neutralization.
Fv is a small antibody fragment that contains a complété antigen-recognition and antigen-binding site. This fragment generally consists of a dimer of one heavy- and one light-chain variable région domain in tight, non-covalent association. However,
even a single variable domain (or half of an Fv comprising only three CDRs spécifie for an antigen) can hâve the ability to recognize and bind antigen, although typically at a lower affinity than the entîre binding site.
Single-chain Fv also abbreviated as sFv or scFv, are antibody fragments 5 that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide comprises a polypeptide linker disposed between and linking the VH and VL domains that enables the scFv to retain or form the desired structure for antigen binding. Such a peptide linker can be încorporated into a fusion polypeptide using standard techniques well known in the art. Additionally 10 or alternatvely, Fv can hâve a disulfide bond formed between and stabilizing the VH and the VL. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269315 (1994); Borrebaeck 1995, infra. In certain embodiments, the antibody or antigenbinding fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain. In particular embodiments, a scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH-linker-VL orientation or in a VL-lînker-VH orientation. Any scFv of the présent disclosure may be engineered so that the C-terminal end of the VL domain is linked by a short peptide sequence to the N-terminal end of the VH domain, or vice versa (i.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-iinker-(N)VL(C). Alternatively, in some embodiments, a linker may be linked to an N-terminal portion or end of the VH domain, the VL domain, or both.
Peptide linker sequences may be chosen, for example, based on: (1) their ability to adopt a flexible extended conformation; (2) their inability or lack of ability to adopt a 25 secondary structure that could interact with functional epitopes on the first and second polypeptides and/or on a target molécule; and/or (3) the lack or relative lack of hydrophobie or charged residues that might react with the polypeptides and/or target molécule. Other considérations regarding linker design (e.g., length) can include the conformation or range of conformations in which the VH and VL can form a functional 30 antigen-binding site. In certain embodiments, peptide linker sequences contain, for example, Gly, Asn and Ser residues. Other near neutraî amino acids, such as Thr and A la, may also be included in a lînker sequence. Other amino acid sequences which may be usefully employed as linker include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy étal., Proc. Natl. Acad. Sci. USA 83:8258 8262 (I986); U.S. Pat. No. 4,935,233, and U.S. Pat. No. 4,751,180. Other illustrative and non-limiting examples of linkers may include, for example, those disclosed by Chaudhary et al., Proc. Natl. Acad. Sci. USA 87:1066-1070 (1990), and Bird et al., Science 242:423-426 (1988)) and a pentamer of four glycine residues linked in sériés, the C-terminal glycine oi the sériés being linked to a single serine, when présent in a single itération or repeated 1 to 5 or more times, or more. Any suitable lînker may be used, and in general can be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20,21, 22, 15 23,24, 25, 26, 27, 28, 29, 30,40, 50, 60, 70, 80, 90, or 100 amino acids in length, or less than about 200 amino acids in length, and will preferably comprise a flexible structure (can provide flexibility and room for conformâtional movement between two régions, domains, motifs, fragments, or modules connected by the linker), and will preferably be biologically inert and/or hâve a low risk of immunogenicity in a human.
scFv can be eonstructed using any combination of the VH and VL sequences or any combination ofthe CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences disclosed herein. In some embodiments, linker sequences are not required; for example, when the first and second polypeptides hâve non-essential N-terminal amino acid régions that can be used to separate the functional domains and prevent steric interférence.
In some embodiments, an antibody or antigen-binding fragment comprises a light chain constant région (or a portion or fragment thereof), a heavy chain constant région (or a portion or fragment thereof), or both. The term CL refers to an immunoglobulin light chain constant région or a light chain constant région, i.e., a constant région from an antibody light chain. The term CH refers to an immunoglobulin heavy chain constant région or a heavy chain constant région, which is further divisible, depending on the antibody isotype into CH 1, CH2, and CH3
4l (IgA, IgD, IgG), or CHl, CH2, CH3, and CH4 domains (IgE, IgM). The Fc région of an antibody heavy chain is described further herein. In any of the presently disclosed embodiments, an antibody or antigen-binding fragment of the présent disclosure comprises any one or more of CL, a CHl, a CFE, and a CH3. In certain embodiments, a CL comprises an amino acid sequence having at least 90%, 9l%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.:79. In certain embodiments, a CH1-CH2-CH3 comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.:73, or a variant thereof that comprises one or more of the following amino acid substitutions (EU numberîng): G236A; A330L; I332E; M428L; N434S. Fc moietîes are described elsewhere herein.
It will be understood that, for example, production in a mammalîan cell line can remove one or more C-terminal lysine of an antibody heavy chain (see, e.g., Liu et al. mAbs 6(5):1145-1154 (2014)). Accordingly, an antibody or antigen-binding fragment ofthe présent disclosure can comprise a heavy chain, a CH1-CH3, a CH3, or an Fc polypeptide wherein a C-terminal residue is présent or is absent; in other words, encompassed are embodiments wherein the C-terminal residue of a heavy chain, a CHl -CH3, or an Fc moiety is not a lysine, and embodiments where a lysine is the Cterminal residue. In certain embodiments, a composition comprises a plurality of an antibody and/or an antigen-binding fragment ofthe présent disclosure, wherein one or more antibody or antigen-binding fragment does not comprise a lysine residue at the Cterminal end of the heavy chain, CH 1-CH3, or Fc moiety, and wherein one or more antibody or antigen-binding fragment comprises a lysine residue at the C-terminal end of the heavy chain, CH1-CH3, or Fc moiety.
A Fab (fragment antîgen binding) is the part of an antibody that binds to antigens and includes the variable région and CHl of the heavy chain linked to the light chain via an inter-chain disulfide bond. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still
capable of cross-linking antigen. Both the Fab and F(ab’)2 are examples of antigenbinding fragments. Fab' fragments differfrom Fab fragments by having additional few residues at the carboxy terminus of the CH l domain including one or more cysteines from the antibody hînge région. Fab'-SH is the désignation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab’)2 antibody fragments originally were produced as pairs of Fab' fragments that hâve hinge cysteines between them. Other Chemical couplings of antibody fragments are also known. Fab fragments may bejoined, e.g., by a peptide linker, to form a single chain Fab, also referred to herein as scFab. In these embodiments, an inter-chain disulfide bond that is présent în a native Fab may not be présent, and the linker serves in full or in part to link or connect the Fab fragments in a single polypeptide chain. A heavy chain-derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VH + CHl, or Fd) and a light chain-derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VL + CL) may be linked in any arrangement to form a scFab.
For example, a scFab may be arranged, in N-terminal to C-terminal direction, according to (heavy chain Fab fragment - linker - light chain Fab fragment) or (light chain Fab fragment - linker - heavy chain Fab fragment). Peptide linkers and exemplary linker sequences for use in scFabs are discussed in further detail herein.
In any of the presently disclosed embodiments, the antibody, or the antigen20 binding fragment thereof, comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, a Fab, a Fab’, a F(ab’)2, a Fv, or a scFv.
Fragments of the antibodies described herein can be obtained from the antibodies by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by Chemical réduction. Alternatively, fragments 25 ofthe antibodies can be obtained by cloning and expression ofpart of the sequences of the heavy or light chains. The présent disclosure encompasses single-chain Fv fragments (scFv) derived from the heavy and light chains of an antibody as described herein, including, for example, an scFv comprising the CDRs (and, optionally, the variable régions) from an antibody according to the présent description, heavy or light chain monomers and dimers (i.e., VH-VL dimer, HC-LC dimer, HC-HC dimer), single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, in which the heavy and light chain variable domains or régions are joined by a peptide lînker.
In certain embodiments, an antibody according to the présent disclosure, or an antigen binding fragment thereof, comprises a purified antibody, a monoclonal antibody, a single chain antibody, Fab, Fab’, F(ab')2, Fv or scFv.
Antibodies and antigen binding fragments of the présent disclosure may, in embodiments, be multispecific (e.g., bispecific, trispecific, tetraspecific, or the like), and may be provided in any multispecific format, as disclosed herein. In certain embodiments, an antibody or antigen-binding fragment of the présent disclosure is a multispecific antibody, such as a bispecific or trispecific antibody. Formats for bispecific antibodies are disclosed in, for example, Spiess et al., Mol. Immunol. 67(2):95 (2015), and in Brinkmann and Kontermann, mAbs 9(2):182-212 (2017), which bispecific formats and methods of making the same are incorporated herein by reference and include, for example, Bispecific T cell Engagera (BiTEs), DARTs, Knobs-lnto-FIoles (KIH) assemblîes, scFv-CH3-KIH assembiies, K.IH Common LightCham antibodies, TandAbs, Triple Bodîes, TriBi Minibodies, Fab-scFv, scFv-CH-CLscFv, F(ab')2-scFv2, tetravalent HCabs, Intrabodies, CrossMabs, Dual Action Fabs (DAFs) (two-in-one or four-in-one), DutaMabs, DT-IgG, Charge Pairs, Fab-arm Excbange, SEEDbodies, Triomabs, LUZ-Y assembiies, Fcabs, κλ-bodies, orthogonal Fabs, DVD-IgGs, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, lgG(H)-V, V(H)-IgG, lgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, lgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (four-in-one). A bispecific or multispecific antibody may comprise a HBV- and/or FIDV-specific binding domain of the instant disclosure in combination with another HBV- and/or HDV-specific binding domain of the instant disclosure, or in combination with a different binding domain that specifically binds to HBV and/or HDV (e.g., at a same or a different epitope), or with a binding domain that specifically binds to a different antigen.
Antibody fragments of the disclosure may impart monovalent or multivalent interactions and be contaîned in a variety of structures as described above. For instance, scFv molécules may be synthesîzed to create a trivalent tnabody or a tetravalent tetrabody. The scFv molécules may include a domain of the Fc région resulting in bivalent minibodies. In addition, the sequences of the disclosure may be a component of multispecific molécules in which the sequences ofthe disclosure target the epitopes ofthe disclosure and other régions of the molécule bind to other targets. Exemplary molécules include, but are not limited to, bispecific Fab2, trispecîfic Fab3, bispecific scFv, and diabodies (Holliger and Hudson, 2005, Nature Bïotechnology 9: I126-II36).
Antibodies or antigen-binding fragments thereof such as those described herein, including but not limited to scFv, may, in certain embodiments, be comprised in a fusion protein that is capable of specifically binding to an antigen as described herein.
As used herein, fusion protein refers to a protein that, in a single chain, has at least two distinct domains or motifs, wherein the domains or motifs are not natural!y found together, or in the given arrangement, in a protein. A poiynucleotide encodîng a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesîzed.
In some embodiments, a fusion protein is capable of expression at a surface of a host cell, e.g., a T cell, NK cell, or NK-T cell. In certain embodiments, a fusion protein comprises (i) an extracellular component comprising the antibody or antigen binding fragment thereof (e.g., a scFv); (ii) a transmembrane component (e.g., a transmembrane domain from CD4, CD8, CD27, CD28, or a functional variant or portion thereof, or any combination thereof); and (iii) an intracellular component comprising a signaling domain from a costimulatory protein, or a functional variant or portion thereof (e.g., a signaling domain from from CD27, CD28, 4-IBB (CD137), OX40 (CDI34), CD2, CD5, ICAM-l (CD54), LFA-l (CD l la/CDI 8), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, MKG2C, SLAMF7, NKp80, CD160, B7-H3, a ligand that specifically binds with CD83, or a functional variant thereof, or any combination thereof), and/or an effector domain (e.g., from CD3e, CD38, CD3Ç, CD25, CD79A, CD79B, CARDl l, DAPIO, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCHl, N0TCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk,
SLAMFl, Slp76, ρΤα, TCRa, TCRp, TRJM, Zap70, PTCH2, or any combination thereof).
In certain embodiments, a fusion protein comprising an antibody or antigen binding fragment comprises a chimeric antigen receptor molécule (CAR), which may be expressed on a cell surface of a host cell such as a T cell, a NK cell, or a NK-T cell for use in a cellular immunotherapy. CAR molécules and principles of design are described in, for example: Sade la in et al., Cancer Discov., 3(4):388 (2013); Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016); Stone et al., Cancer Immunol. Immunother., 63(l 1 ):ï 163 (2014); Xu étal., 2018 Oncotarget 9:13991; Androulla et al., 10 2018 Curr. Pharm. Biotechnol. Volume 19 (April 2018); Wu étal., 2016 Expert Opin.
Biol. Ther. 16:1469; and Renet aL, 2017 Protein Cell 8:634, which CAR molécules, CAR designs, and CAR design principles are herein incorporated by reference in their entirety.
Throughout this disclosure, antibodies, antigen binding fragments thereof, and 15 fusion proteins may individually or collectively (e.g., in any combination) be referred to as binding proteins.
Binding proteins according to the présent disclosure may be provided in purified form. For example, an antibody may be présent in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% 20 and more usually less than 50% of the composition is made up of other polypeptides.
Binding proteins according to the présent disclosure may be immunogenic in human and/or in non-human (or heterologous) hosts; e.g., in mice. For exaniple, an antibody may hâve an idiotope that is immunogenic in non-human hosts, but not in a human host. Antibodies of the disclosure for human use include those that are not ty pically isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, or the like, and in some instances are not obtained by humanization or from xeno-mice. Also contemplated herein are variant forms of the disclosed antibodies, which are engineered so as to reduce known or potentîal immunogenicity and/or other potentia] liabilities, or to confer a desired structure and/or functionality of the antibody în a non30 human animal, such as a mouse (e.g., a murinized antibody wherein one or more human amino acid residue, sequence, or motif is replaced by a residue, sequence, or motif that has reduced or abrogated immunogenicity or other liability, or has a desired structure and/or function, in a mouse; e.g., for model studies using a mouse).
As used herein, a neutralizing antibody (or antigen binding fragment, or fusion protein) is one that can neutralize, i.e., prevent, inhibit, reduce, impede or interfère with, the ability of a pathogen to initiate and/or perpetuate an infection in a host (e.g, host organisai or host cell). The ternis neutralizing antibody and an antibody that neutralizes or antibodies that neutralize are used interchangeably herein. These antibodies can be used alone, or in combination (e.g., two or more of the presently disclosed antibodies in a combination, or an antibody ofthe présent disclosure in combination with another agent, which may or may not be an antibody agent, including an antibody that is capable of neutralizing an HBV B and/or HBV D infection), as prophylactic or therapeutic agents upon appropriate formulation, in association with active vaccination, as a diagnostic tool, or as a production tool as described herein. Accordingly, presently disclosed antibodies or antigen-binding fragments are capable of neutralizing infection by a HBV, a HDV, or both.
As used herein, specifically binds or spécifie for refers to an association or union of a binding protein (e.g., an antibody or antigen binding fragment thereof) or a binding domain to a target molécule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of l/M) equal to or greaterthan l0s M-1 (which equals the ratio of the on-rate [Kon] to the offrate [Koff] for this association reaction), while not significantly associating or unitîng with any other molécules or components in a sample. Binding proteins or binding domains may be classified as high-affinity binding proteins or binding domains or as low-affinity binding proteins or binding domains. High-affinity binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of at least 107 M’1, at least 108 M-1, at least 109 M'1, at least 1010 M'1, at least 10 M’’,at least IO12 M or at least 1013 M'1. Low-affinity binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of up to 107 M1, up to 106 M'1, or up to HP M-i. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., ΙΟ'5 M to 10’1 M). The terms binding” and specifically binding and similar references do not encompass non-specific sticking.
Binding of a binding protein can be determined or assessed using an appropriate assay, such as, for example, Surface Plasmon Résonance (SPR) methods, e.g., a Biacore™ system; kînetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet platform); isothermal titration calorimetry (ITC), or the like, an antigen-binding ELISA (e.g., direct or indirect) with imaging by, e.g., optical density at 450nm, or by flow cytometry, or the like.
In certain embodiments, binding proteins according to the present disclosure can bind to the antigenic loop région of HBsAg. The envelope of the hepatitis B virus generally contains three HBV envelope proteins (also known as HBsAg, hepatitis B surface antigen): S protein (for small, also referred to as S-HBsAg), M protein (for middle, also referred to as Μ-HBsAg) and L protein (for large, also referred to as LHBsAg). S-HBsAg, Μ-HBsAg and L-HBsAg share the same C-terminal extremity (also referred to as S domain, 226 amino acids), which corresponds to the S protein (SHBsAg) and which is crucial for virus assembly and infectivity. S-HBsAg, M-HBsAg and L-HBsAg are synthesized in the endoplasmic réticulum (ER), assembled, and secreted as particles through the Golgi apparatus. The S domain comprises four predicted transmembrane (TM) domains, whereby both the N-terminus as well as the Cterminus of the S domain are exposed to the lumen. The transmembrane domains TM1 and TM2 are both believed necessary for cotranslational protein intégration into the ER membrane and the transmembrane domains TM3 and TM4 are located in the C-terminal third of the S domain. The antigenic loop région of HBsAg is located between the predicted TM3 and TM4 transmembrane domains of the S domain of HBsAg, whereby the antigenic loop région comprises amino acids 101-172 ofthe S domain, which contains 226 amino acids in total (Salisse J. and Sureau C., 2009, Journal of Virology 83: 9321-9328). A déterminant of infectivity résides in the antigenic loop région of HBV envelope proteins. In particular, residues between 119 and 125 of the HBsAg contain a CXXC motif, which îs considered to be important for the mfectivity ot HBV and HDV (Jaoude GA, Sureau C, Journal of Virology, 2005;79:10460-6).
When positions in the amino acid sequence ofthe S domain of HbsAg are referred to herein, such positions are made with reference to the amino acid sequence as set forth in SEQ ID NO; 3 (shown below) or to natural or artificial sequence variants thereof.
MEN1TSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLG QNSQSPTSNHSPTSCPPTCPGYRWMCLRRF11FLFILLLCL1FLLVLLDYQGMLPV CPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFL WEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLP LLPIFFCLWVYI (SEQ IDNO: 3; amino acids 10 J - 172 are shown underlined)
For example, the expression amino acids 101 — 172 ofthe S domain refers to the amino acid residues from positions 101 - 172 of the polypeptide according to SEQ ID NO: 3. However, a person skilled in the art understands that mutations or variations (including, but not limited to, substitution, délétion and/or addition, for exainple, HBsAg of a different génotype or a different HBsAg mutant as described herein) may occur naturally in the amino acid sequence ofthe S domain of HBsAg or be introduced artificiaily into the amino acid sequence ofthe S domain of HBsAg without affecting its biological properties. Therefore, as used herein, the term S domain of HBsAg encompasses ail such polypeptides including, for example, the polypeptide according to SEQ ID NO: 3 and its natural or artificial mutants. In addition, when sequence fragments of the S domain of HBsAg are described herein (e.g. amino acids 101-172 or amino acids 120 -130 of the S domain of HBsAg), they include not only the corresponding sequence fragments of SEQ ID NO: 3, but also the corresponding sequence fragments of its natural or artificial mutants. For example, the phrase amino acid residues from positions 101 - 172 of the S domain of HBsAg encompasses amino acid residues from positions 101 - 172 of SEQ ID NO: 3 and the corresponding fragments of its mutants (natural or artificial mutants). As used herein, the phrases corresponding sequence fragments and corresponding fragments refer to fragments that are located in equal positions of sequences when the sequences are subjected to optimized alignaient, namely, the sequences are aligned to obtain a highest percentage of identity.
The M protein (M-HBsAg) corresponds to the S protein extended by an Nterminal domain of 55 amino acids called pre-S2. The L protein (L-HBsAg) corresponds to the M protein extended by an N-termînal domain of 108 amino acids called pre-Sl (génotype D). The pre-Sl and pre-S2 domains ofthe L protein can be présent either at the inner face of viral particles (on the cytoplasmic side of the ER), and is believed to play a crucial rôle in virus assembly, or on the outer face (on the luminal side ofthe ER), available for the interaction with target cells and important for viral infectivity. Moreover, HBV surface proteins (HBsAgs) are not only incorporated into virion envelopes but also can spontaneously bud from ER-Golgi intermediate compartment membranes to fonn empty subviral particles (SVPs) that are released from the cell by sécrétion.
In some embodiments, a binding protein binds to the antigenic loop région of HBsAg, and is capable of binding to ail of S-HBsAg, Μ-HBsAg and L-HBsAg.
In some embodiments, a binding protein neutralizes infection with hepatitis B virus and hepatitis delta virus. In some embodiments, the binding protein, reduces viral infectivity of hepatitis B virus and hepatitis delta virus.
To study and quantitate virus infectivity (or neutralization) in the laboratory, standard neutralization assays may be utilized. For a neutralization assay, animal viruses are typically propagated in cells and/or cell lines. A neutralization assay wherein cultured cells are incubated with a fixed amount of HBV or HDV in the presence (or absence) of the antibody (or antigen-binding fragment or fusion protein) to be tested may be used. In such an assay, the levels of hepatitis B surface antigen (HBsAg) or hepatitis B e antigen (HBeAg) secreted into the cell culture supernatant may be used and/or HBeAg staining may be assessed to provide a readout. For HDV, for example, delta antigen immunofluorescence staining may be assessed.
In a particular embodiment of an HBV neutralization assay, cultured cells, for example HepaRG cells, such as differentiated HepaRG cells, are incubated with a fixed amount of HBV in the presence or absence of the antibody to be tested. In such and embodiment, incubation may be carried out, for example, for 16 hours at 37°C. That incubation may be performed in a medium (e.g. supplemented with 4% PEG 8000). After incubation, cells may be washed and further cultivated. To measure virus infectivity, the levels of hepatitis B surface antigen (HBsAg) and/or hepatitis B e antîgen (HBeAg) secreted into the culture supernatant, e.g. from day 7 to day 11 postinfection, may be determined by enzyme-linked immunosorbent assay (ELISA). Additionally, HBeAg staining may be assessed in an immunofluorescence assay. In an embodiment of a HDV neutralizatîon assay, essentîally the same assay as for HBV may 10 be used, with the différence that sera from HDV carriers may be used as HDV infection inoculum on differentîated HepaRg cells (instead of HBV). For détection, delta antigen immunofluorescence staining may be used as a readout.
Embodiments of the binding proteins of the disclosure hâve high neutralizing potency. In certain embodiments, the concentration of an antibody as described herein 15 required for 50% neutralizatîon of hepatitis B virus (HBV) and hepatitis delta virus (HDV), is, for example, about 10 gg/ml or less. In other embodiments, the concentration of a binding protein required for 50% neutralizatîon of HBV and HDV is about 5 pg/ml. In other embodiments, the concentration of a binding protein as described herein required for 50% neutralizatîon of HBV and HDV is about l pg/ml. In 20 still other embodiments, the concentration of a binding protein required for 50% neutralizatîon of HBV and HDV is about 750 ng/ml. In yet further embodiments, the concentration of a binding protein as described herein required for 50% neutralizatîon of HBV and HDV is 500 ng/ml or less. In such embodiments, the concentration of a binding protein as described herein required for 50% neutralizatîon of HBV and HDV 25 may be selected from 450 ng/ml or less, 400 ng/ml or less, 350 ng/ml or less, 300 ng/ml or less, 250 ng/ml or less, 200 ng/ml or less, 175 ng/ml or less, 150 ng/ml or less, 125 ng/ml or less, 100 ng/ml or less, 90 ng/ml or less, 80 ng/ml or less, 70 ng/ml or less, 60 ng/ml or less, 50 ng/ml or less, or less than 20 ng/ml, preferably 15 ng/ml or less, more preferably 10 ng/ml or less, such as 7 ng/ml or less.
Binding proteins according to the présent disclosure, which can neutralize both H BV and HD V, are useful in the prévention and treatment of hepatitis B and hepatitis D. Infection with HDV typically occurs simultaneously with or subséquent to infection by HBV (e.g, inoculation with HDV in the absence of HBV does not cause hepatitis D since HDV requires the support of HBV for its own réplication) and hepatitis D is typically observed in chronic HBV carriers.
Embodiments of disclosed binding proteins promote clearance of HBsAg and HBV. In particular embodiments, binding proteins promote clearance of both HBV and subviral particles of hepatitis B virus (SVPs). Clearance of HBsAg or of subviral ] 0 particles may be assessed by measuring the level of HBsAg for example in a blood sample, e.g. from a hepatitis B patient. Similarly, clearance of HBV may be assessed by measuring the level of HBV for example in a blood sample, e.g. from a hepatitis B patient.
[n the sera of patients infected with HBV, in addition to înfectious particles (HBV), there is typically an excess (typically l ,000- to 100,000-fold) of empty subviral particles (SVP) composed solely of HBV envelope proteins (HBsAg) in the form of relative!y smaller spheres and filaments of variable length. Subviral particles hâve been shown to strongly enhance intracellular viral réplication and gene expression of HBV (Bruns M. et al. 1998 J Virol 72(2): 1462-1468). This is also relevant in the context of infectivity of sera containing HBV, since the infectivity dépends not only on the number of viruses but also on the number of SVPs (Bruns M. et al. 1998 J Virol 72(2): 1462-1468). Moreover, an excess of subviral particles can serve as a decoy by absorbîng neutralizing antibodies and therefore delay the clearance of infection.
Achievement of hepatitis B surface antigen (HBsAg) loss is considered in some instances to be an idéal endpoint of treatment and the ciosest outcome to cure chronic hepatitis B (CHB).
Embodiments of binding proteins of the présent disclosure may promote clearance of HbsAg. In certain embodiments, the binding proteins may promote clearance of subviral particles of hepatitis B virus. In some embodiments, the binding 30 proteins may be used to treat chronic hepatitis B.
In any ofthe presently disclosed embodiments, a binding protein of the présent disclosure is capable of binding an HBsAg of a génotype selected from the HBsAg génotypes A, B, C, D, E, F, G, H, I, and J, or any combination thereof.
In certain embodiments, binding proteins of the présent disclosure are capable of 5 binding to any 1,2,3, 4, 5, 6, 7, 8, 9 or 10 of the HBsAg génotypes A, B, C, D, E, F, G, H, I, and J. Examples of different HBsAg génotypes of include the following: GenBank accession number J02203 (HBV-D, ayw3); GenBank accession number FJ899792.1 (HBV-D, adw2); GenBank accession number AM282986 (HBV-A); GenBank accession number D23678 (HBV-BI Japan); GenBank accession number AB117758 (HBV-CI Cambodia); GenBank accession number AB205192 (HBV-E Ghana); GenBank accession number X69798 (HBV-F4 Brazil); GenBank accession number AF160501 (HBV-G USA); GenBank accession number AY090454 (HBV-H Nicaragua); GenBank accession number AF241409 (HBV-I Vietnam); and GenBank accession number AB486012 (HBV-J Bornéo). Exemplary amino acid sequences ofthe antigenic loop région of the S domain of HBsAg of different génotypes are described herein (e.g., SEQ ID NOs.: 5-15).
In some embodiments, a binding protein is capable of binding to one or more, and in some cases at least 6 of the 10 HBsAg génotypes A, B, C, D, E, F, G, FI, I, and J. In certain embodiments, a binding protein is capable of binding to at least 8 of the 10 20 HBsAg génotypes A, B, C, D, E, F, G, H, I, and J. In some embodiments, a binding protein is capable of binding to ail 10 of the 10 HBsAg génotypes A, B, C, D, E. F, G, H, I. and J. HBV is differentiated into several génotypes, according to genome sequence. To date, eight well-known génotypes (A-H) of the HBV genome hâve been defined. Moreover, two other génotypes, I and J, hâve also been identifïed (Sunbul M., 25 2014, World J Gastroenterol 20(18): 5427-5434). The génotype is known to affect the progression of the disease and différences between génotypes in response to antiviral treatment hâve been determined.
In some embodiments, a binding protein according to the présent disclosure is capable of binding to 1,2, 3,4, 5,6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 ofthe
HBsAg mutants having mutations in the antigenic loop région, with such mutant(s) being selected from one or more of HBsAg Y100C/P120T, HBsAg P120T, HBsAg P120T/S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg Ml33H, HBsAg Ml33L, HBsAg Ml33T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R and HBsAg NI46A. These mutants are naturally occurring mutants based on the S domain of HBsAg Génotype D, Genbank accession no. FJ899792 (SEQ ID NO.: 4). The mutated amino acid residue(s) in each of the mutants noted herein are indicated in the name.
SEQ ID NO.: 4:
MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLG QNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPV ÇPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFL WEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSTLSPFLP LLPIFFCLWVYI (the antigenic loop région, i.e. amino acids ΙΟΙ - 172, is shown underlîned).
Amino acid sequences of the antigenic loop région of the S domain of HBsAg of different mutants are shown in SEQ ID NOs.: 16 — 33.
In certain embodiments, a binding protein as disclosed herein is capable of binding to at one or more, and in some cases at least 12 infectious HBsAg mutants selected from HBsAg Y100C/PI20T, HBsAg P120T, HBsAg P120T/S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg Ml33H, HBsAg Ml33L, HBsAg M133T, HBsAg KI4IE, HBsAg P142S, HBsAg Sl43K,HBsAg D144A, HBsAg Gl45R and HBsAg N146A. In some such embodiments, a binding protein is capable of binding to at least 15 infectious HBsAg mutants selected from HBsAg Y100C/P120T, HBsAg P120T, HBsAg P120T/S143L, HBsAg CI2IS, HBsAg R122D, HBsAg RI22I, HBsAg Tl 23N, HBsAg QI29H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg Ml33T, HBsAg KI41E, HBsAg PI42S, HBsAg S143K, HBsAg D144A, HBsAg G145R and HBsAg NH46A. In some embodiments, a binding protein is capable of binding to each ofthe following infections HBsAg mutants: HBsAg Y100C/PI20T; HBsAg PI20T; HBsAg PI20T/S143L; HBsAg C121 S; HBsAg R122D; HBsAg R122I; HBsAg T123N; HBsAg
Q129H; HBsAg Q129L; HBsAg M133H; HBsAgMl33L; HBsAgMl33T, HBsAg K141E; HBsAg P142S; HBsAg S143K; HBsAg D144A; HBsAg GI45R; and HBsAg N146 A.
In certain embodiments, the binding protein (e.g., including an antibody or antigen binding fragment thereoQ is capable of reducing a sérum concentration of HBV DNA in a mammal having an HBV infection. In certain embodiments, the binding protein is capable of reducîng a sérum concentration of HBsAg in a mammal having an HBV infection. In certain embodiments, the binding protein is capable of reducing a sérum concentration of HBeAg in a mammal having an HBV infection. In certain embodiments, the binding protein is capable of reducing a sérum concentration of HBcrAg in a mammal having an HBV infection. In some embodiments, the binding protein is capable of reducing the sérum concentration of HBV DNA, HBsAg, HBeAg, and/or HBcrAg in the mammal for about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days following a single administration of the binding protein.
The term epitope or antigenic epitope includes any molécule, structure, amino acid sequence, or protein déterminant that is recognized and specîfically bound by a cognate binding molécule, such as an immunoglobulin, chimeric antigen receptor, or other binding molécule, domain or protein. Epitopic déterminants generally contain chemically active surface groupings of molécules, such as amino acids or sugar side chains, and can hâve spécifie three dimensional structural characteristics, as well as spécifie charge characteristics.
In some embodiments, a binding protein is capable of binding to an epitope comprising at least one, at least two, at least three, or at least four amino acids of the antigenic loop région of EIbsAg. In certain embodiments, a binding protein is capable of binding at least two amino acids selected from amino acids 115 - 133 of the S domain of HbsAg, amino acids 120- 133 of the S domain of HbsAg, or amino acids 120 - 130 of the S domain of HbsAg. In certain embodiments, a binding protein is capable of binding at least three amino acids selected from amino acids 115 — 133 of the S domain of HbsAg, amino acids 120 - 133 of the S domain of HbsAg, or amino acids 120 - 130 of the S domain of HbsAg. In some embodiments, a binding protein is capable of binding at least four amino acids selected from amino acids 115 - 133 of the S domain of HbsAg, amino acids 120-133 ofthe S domain of HbsAg, or amino acids 120- 130 ofthe S domain of HbsAg. As used herein, the position of the amino acids (e.g. 115- 133, 120- 133, 120 - 130) refers to the S domain of HBsAg as described 5 above, which is présent in ail three HBV envelope proteins S-HBsAg, M-HBsAg, and L-HBsAg, whereby S-HBsAg typically corresponds to the S domain of HBsAg.
The term formed by as used herein in the context of an epitope, means that the epitope to which the binding protein binds to may be linear (continuous) or conformational (discontinuous). A linear or a sequential epitope is an epitope that is ] 0 recognized by an antibody according to its linear sequence of amino acids, or primary structure. A conformational epitope may be recognized according to a threedimensional shape and protein structure. Accordingly, if the epitope is a linear epitope and comprises more than one amino acid located at positions selected from amino acid positions 115 -133 or from amino acid positions 120—133 ofthe S domain of HBsAg, 15 the amino acids comprised by the epitope may be located in adjacent positions of the primary structure (e.g., are consecutive amino acids in the amino acid sequence). In the case of a conformational epitope (3D structure), the amino acid sequence typically forms a 3D structure as epitope and, thus, the amino acids forming the epitope may be or may be not located in adjacent positions of the primary structure (i.e. maybe or may 20 be not consecutive amino acids in the amino acid sequence).
In certain embodiments, an epitope to which a binding protein binds to a conformational epitope. In some embodiments, a binding protein binds to an epitope comprising at least two amino acids of the antigenic loop région of HBsAg, wherein the at least two amino acids are selected from amino acids 120 - 133 or from from amino 25 acids 120- 130, of the S domain of HbsAg, and wherein the at least two amino acids are not located in adjacent positions (of the primary structure). In certain embodiments, a binding protein binds to an epitope comprising at least three amino acids of the antigenic loop région of HBsAg, wherein the at least three amino acids are selected from amino acids 120 - 133 or from from amino acids 120 - 130, ofthe S domain of 30 HbsAg, and wherein at least two of the three amino acids are not located in adjacent positions (ofthe primary structure). In some embodiments, a binding protein binds to an epîtope comprising at least four amino acids of the antigénie loop région of HBsAg, wherein the at least four amino acids are selected from amino acids 120 - 133 or from from amino acids 120 — 130, of the S domain of HbsAg, and wherein at least two of the four amino acids are not located in adjacent positions (of the primary structure).
Amino acids to which a presently disclosed antibody, antigen binding fragment, or fusion protein binds (i.e. the amino acids forming the epitope), which are not located in adjacent positions of the primary structure, are in some cases spaced apart by one or more amino acids, to which the antibody, antigen binding fragment, or fusion protein does not bind. In some embodiments, at least one, at least two, at least three, at least four, or at least five amino acids may be located between two of the amino acids not located in adjacent positions comprised by the epitope.
In certain embodiments, a binding protein binds to an epitope comprising at least amino acids P120, C121, R122 and C ] 24 of the S domain of HBsAg. In other embodiments, a binding protein of the présent disclosure binds to an epitope comprising an amino acid sequence according to SEQ ID NO.: 115:
PCRXC wherein X is any amino acid or no amino acid; X is any amino acid; X is T, Y, R, S, or F; X is T, Y or R; or X is T or R.
In other embodiments, a binding protein ofthe présent disclosure binds to an epitope comprising an amino acid sequence according to SEQ ID NO.: 107:
TGPCRTC or to an amino acid sequence sharing at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO.: 107.
In other embodiments, a binding protein of the présent disclosure binds to an epitope comprising an amino acid sequence according to SEQ ID NO.: 112:
STTSTGPCRTC or to an amino acid sequence sharing at least 80%, at least 90% or a1 least 95% sequence identity with SEQ ID NO.: 112.
In certain embodiments, a binding protein of the présent disclosure binds to an epitope comprising an amino acid sequence comprising at least amino acids 145 - 151 of the S domain of HBsAg:
GNCTCIP (SEQIDNO.: 108).
In still other embodiments, a binding protein of the présent disclosure binds to an epitope comprising an amino acid sequence according to SEQ ID NO: 107 and an amino acid sequence according to SEQ ID NO.; 108.
In other embodiments, a binding protein of the présent disclosure binds to an epitope comprising an amino acid sequence according to SEQ ID NO.: 112 and/or an amino acid sequence according to SEQ ID NO.: 114.
As described above, an epitope to which a binding protein of the présent disclosure binds may be linear (continuous) or conformât!onal (discontinuous). In some embodiments, a binding protein of the disclosure binds to a conformatîonal epitope, and in certain such embodiments, the conformatîonal epitope is présent only under nonreducing conditions.
)n certain embodiments, binding protein ofthe présent disclosure, binds to a linear epitope. In certain such embodiments, the the linear epitope is présent under both, non-reducing conditions and reducing conditions.
In particular embodiments, a binding protein of the présent disclosure binds to an epitope in the antigenic loop of HBsAg formed by an amino acid sequence according to SEQ IDNO.: I:
Xi X2 X3 TC X4 X5 X6A x7g wherein Xi, X2, Xj, X4, X5, Xô and X7 may be any amino acid (SEQ ID NO.: I).
In some embodiments, Xi, X2, X3, X4, X5, Xô and X7 are amino acids, which are conservatively substituted in comparison to amino acids 120 - 130 of SEQ ID NO.: 3. In some embodiments. Xi, X2, X3, X4, Xs, Xô and X7are amino acids, which are conservatively substituted in comparison to amino acids 20 - 30 of any of SEQ ID NOs.: 5-33.
In spécifie embodiments, Xi of SEQ ID NO.: 1 Xi is a small amino acid. A small amino acid, as used herein, refers to any amino acid selected from the group consisting of alanine, aspartic acid, asparagine, cysteine, glycine, proline, serine, threonine and valine. In certain such embodiments, Xi is proline, serine or threonine.
In certain embodiments, X2 of SEQ ID NO.: 1 X2 is a small amino acid. In certain embodiments, X2 may be selected from cystein or threonine.
In some embodiments, X3 of SEQ ID NO.: 1 is a charged amino acid or an aliphatic amino acid. A charged amino acid, as used herein, refers to any amino acid selected from the group consisting of arginine, lysine, aspartic acid, glutamic acid and histidine. A aliphatic amino acid, as used herein, refers to any amino acid selected from the group consisting of alanine, glycine, îsoleucine, leucine, and valine. In certain embodiments, X3 is selected from arginine, lysine, aspartic acid or îsoleucine.
In some embodiments, X4 of SEQ ID NO.: I is a small amino acid and/or a hydrophobie amino acid. A hydrophobie amino acid, as used herein, refers to any amino acid selected from the group consisting of alanine, îsoleucine, leucine, phenylalanine, valine, tryptophan, tyrosine, méthionine, proline and glycine. In certain embodiments, X4 is seiected from méthionine or threonine.
In some embodiments, Xs of SEQ ID NO.; I X5 is a small amino acid and/or a hydrophobie amino acid. In certain embodiments, X5 is selected from threonine, alanine or Îsoleucine.
Jn some embodiments, Xô of SEQ ID NO.: 1 Xé is a small amino acid and/or a hydrophobie amino acid. In certain embodiments, Xô is selected from threonine, proline or leucine.
In some embodiments, X7 of SEQ ID NO.: 1 is a polar amino acid or an aliphatic amino acid. A polar amino acid, as used herein, refers to any amino acid selected from the group consisting of aspartic acid, asparagine, arginine, glutamic acid, histidine, lysine, glutamine, tryptophan, tyrosine, serine, and threonine. In certain such embodiments, X7 is glutamine, histidine or leucine.
In some embodiments, abinding protein according to the présent disclosure binds to an epîtope in the antigenic loop of HBsAg formed by an amino acid sequence according to SEQ ID NO.: 2:
Xi X2 X3 TC X4 Xi XôA X7G wherein
Xi is P, T or S,
X2 is C or S,
X3 is R, K, D or I,
X4 is M or T,
X5 is T, A or I,
Xô is T, P or L, and
X7 is Q, H or L (SEQ ID NO.: 2).
With regard to the epitopes formed by the amino acid sequences according to SEQ ID NO.: 1 or 2, it is noted that the term formed by as used herein is not intended to împly that a disclosed binding protein necessarily binds to each and every amino acid of SEQ IDNO.: 1 or 2. In particular, a binding protein may bind only to some ofthe amino acids of SEQ ID NO.: 1 or 2, whereby other amino acid residues may act as spacers.
In particular embodiments, a binding protein according to the présent disclosure binds to an epîtope in the antigenic loop of HBsAg formed by one or more, two or more, three or more, or four or more amino acids of an amino acid sequence selected from SEQ ID NOs.: 5-33 shown below in Table 4.
In some embodiments, binding protein according to the présent disclosure binds to an antigenic loop région of HBsAg having an amino acid sequence according to any one or more of SEQ ID NOs.: 5-33 shown below in Table 4, or to a sequence variant thereof. In certain embodiments, a binding protein according to the présent disclosure binds to ail of the antigenic loop variants of HBsAg having an amino acid sequence according to any of SEQ ID NOs.: 5-33 shown below in Table 4.
Table 4: Amino acid sequences of the antigenic loop région of the S domain of HBsAg (residues 101-172 of the S domain of HBsAg-except for SEQ ID NO: 16, which refers to residues 100-l 72 ofthe S domain of HBsAg in order to include the relevant mutation) of the different génotypes and mutants as used herein.
| Name | SEQ ID NO. | Amino acid sequence |
| J02203 (D, ayw3) | 5 | QGMEPVCPLIPGSSTTSTGPCRTCMTTAQGTS MYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| FJ899792 (D, adw2) | 6 | QGMLPVCPLIPGSSTTGTGPCRTCTTP AQGTSMYPSCCCTKPSDGNCTCIPIPS SWAFGKFLWEWASARFSW |
| AM282986 (A) | 7 | QGMLPVCPLIPGTTTTSTGPCKTCTTPAQGNS MFPSCCCTKPSDGNCTCIPIPSSWAFAKYLWE WASVRFSW |
| D23678(Bl) | 8 | QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGTS MFPSCCCTKPTDGNCTCiPIPSSWAFAKYLWE WASVRFSW |
| ABU7758(Cl) | 9 | QGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTS MFPSCCCTKPSDGNCTCIPIPSSWAFARFLWE WASVRFSW |
| AB205192 (E) | 10 | QGMLPVCPL1PGSSTTSTGPCRTCTTLAQGTS MFPSCCCSKPSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| X69798 (F4) | il | QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTS MFPSCCCSKPSDGNCTCIPIPSSWALGKYLWE WASARFSW |
| AF160501 (G) | 12 | QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNS MYPSCCCTKPSDGNCTCIPIPSSWAFAKYLWE WASVRFSW |
| Name | SEQ ID NO. | Amino acid sequence |
| AY090454 (H) | 13 | QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTS MFPSCCCTKPSDGNCTCIPIPSSWAFGKYLWE WASARFSW |
| AF24l409(I) | 14 | QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNS MYPSCCCTKPSDGNCTC1PIPSSWAFAKYLWE WASARFSW |
| AB486012 (J) | 15 | QGMLPVCPLLPGSTTTSTGPCRTCTITAQGTS MFPSCCCTKPSDGNCTCIPIPSSWAFAKFLWE WASVRFSW |
| HBsAg Y100C/P120T | 16 | CQGMLPVCPLIPGSSTTGTGTCRTCTTPAQGT SMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLW EWASARFSW |
| HBsAgP120T | 17 | QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTS MYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| HBsAg P120T/S143L | 18 | QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTS MYPSCCCTKPLDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| HBsAg C121S | 19 | QGMLPVCPLIPGSSTTGTGPSRTCTTPAQGTS MYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| HBsAg R122D | 20 | QGMLPVCPLIPGSSTTGTGPCDTCTTPAQGTS MYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| HBsAg RI 221 | 21 | QGMLPVCPL ÏPGS STTGTGPCITCTTPAQGTS M YPSCCCTKPSDGNCTCIP1PSSWAFGKFLWEW ASARFSW |
| HBsAg Tl 23N | 22 | QGMLPVCPLIPGSSTTGTGPCRNCTTPAQGTS MYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| Name | SEQ ID NO. | Amino acid sequence |
| HBsAg Q129H | 23 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAHGTS MYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| HBsAg Q129L | 24 | QGMLPVCPLIPGSSTTGTGPCRTCTTPALGTS MYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| HBsAg Ml33H | 25 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTS HYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| HBsAg M133L | 26 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSL YPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEW ASARFSW |
| HBsAg Ml 33T | 27 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTST YPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEW ASARFSW |
| HBsAg K141E | 28 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTS MYPSCCCTEPSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| HBsAg P142S | 29 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTS MYPSCCCTKSSDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| HBsAg S143K | 30 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTS MYPSCCCTKPKDGNCTCIPIPSSWAFGKFLWE WASARFSW |
| HBsAg Dl 44A | 31 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTS MYPSCCCTKPSAGNCTCIPIFSSWAFGKFLWE WASARFSW |
| HBsAg GI45R | 32 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTS MYPSCCCTKPSDRNCTCIPIPSSWAFGKFLWE WASARFSW |
| Name | SEQ ID NO. | Amino acid sequence |
| HBsAgN146A | 33 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTS MYPSCCCTKPSDGACTCIPÏPSSWAFGKFLWE WASARFSW |
Fc Moiety
In some embodiments, a binding protein (e.g., antibody or an antigen binding fragment thereof) of the présent disclosure comprises an Fc moiety (also referred to as an Fc polypeptide). In certain embodiments, the Fc moiety may be derived trom human origin, e.g., from human IgGl, IgG2, lgG3, and/or IgG4, or from another [g class or isotype. In spécifie embodiments, an antibody or antigen binding fragment can comprise an Fc moiety derived from human IgGl. In particular embodiments, the Fc 10 moiety comprises, or is derived from (e.g., comprises one or more mutations relative to), lgGlml7, l (IgHGl *01) allotype.
As used herein, the term Fc moiety refers to a sequence comprising, consisting of, consisting essentially of, or derived from a portion of an immunoglobulîn heavy chain beginning in the hinge région just upstream of the papain cleavage site (e.g., residue 216 by EU numberîng in native IgG, taking the first residue of heavy chain constant région to be 114) and ending at the C-terminus of the immunoglobulîn heavy chain. Accordingly, an Fc moiety may be a complété Fc moiety or a portion (e.g., a domain) thereof. In certain embodiments, a complété Fc moiety comprises a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216-446). As noted herein, an additional lysine residue (K) is sometimes présent at the extreme Cterminus of the Fc moiety, but is often cleaved from a mature antibody. Amîno acid positions within an Fc moiety hâve been numbered according to the EU numberîng system of Kabat, see e.g., Rabat et al., Sequences of Proteins of Immunologîcal Interest, U.S. Dept. Health and Human Services, 1983 and 1987. Amino acid positions of an Fc moiety can also be numbered according to the IMGT numberîng system (including unique numbering for the C-domain and exon numbering) and the Rabat numbering system.
In some embodiments, an Fc moiety comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge région) domain, a CH2 domain, a CH3 domain, or a variant, portion, or fragment thereof. In some embodiments, an Fc moiety comprises at least a hinge domain, a CH2 domain or a CH3 domain. In further embodiments, the Fc moiety is a complété Fc moiety. The amino acid sequence of an exemplary Fc moiety of human IgGl isotype is provided in SEQ ID NO.:73. The Fc moiety may also comprise one or more amino acid insertions, délétions, or substitutions relative to a naturally occurring Fc moiety. For example, at least one of a hinge domain, CH2 domain, or CHS domain, or a portion thereof, may be deleted. For example, an Fc moiety may comprise or consist of: (i) hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), (ii) a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iii) a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), (iv) a hinge domain (or a portion thereof), (v) a CH2 domain (or a portion thereof), or (vi) a CH3 domain or a portion thereof.
An Fc moiety of the présent disclosure may be modified such that it varies in amino acid sequence from the complété Fc moiety of a naturally occurring immunoglobulin molécule, while retaining or enhancing at least one désirable fonction conferred by the naturally occurring Fc moiety, and/or reducing an undesired fonction of a natorally occurring Fc moiety. Such functions include, for example, Fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC fonction, protein A binding, protein G binding, and complément binding. Portions of naturally occurring Fc moieties which are involved with such functions hâve been described in the art.
For example, to activate the complément cascade, the Clq protein complex can bind to at least two molécules of IgGl or one molécule of IgM when the immunoglobulin molecule(s) is attached to the antigenic target (Ward, E. S., and Ghetie, V., Ther. Immunol. 2 (1995) 77-94). Burton, D. R., described (Mol. Immunol. 22 (1985) 161-206) that the heavy chain région comprising amino acid resîdues 318 to
337 is invol ved in complément fixation. Duncan, A. R.., and Winter, G. (Nature 332 (l 988) 738-740), using site directed mutagenesis, reported that Glu3l8, Lys320 and Lys322 form the binding site to Clq. The rôle of Glu318, Lys320 and Lys 322 residues in the binding ofClq was confirmed by the ability of a short synthetic peptide containing these residues to inhibit complément mediated lysis.
For example, FcR binding can be mediated by the interaction of the Fc moiety (of an antibody) with Fc receptors (FcRs), which are specialized cell surface receptors on cells including hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily, and shown to médiate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of érythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody, via antibody dépendent cell mediated cytotoxicity (ADCC; Van de Winkel, J. G., and Anderson, C. L., J. Leukoc. Biol. 49 (l991 ) 511-524). FcRs are defined by their specificity for immunoglobulin classes; Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FcsR, for IgA as FcaR and so on and néonatal Fc receptors are referred to as FcRn. Fc receptor binding is described for example in Ravetch, J. V., and K inet, J. P., Annu. Rev. Immunol. 9 (I99l) 457-492; Capel, P. J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et aL, JLab. Clin. Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76(1998) 231-248.
Cross-linking of receptors by the Fc domain of native IgG antibodies (FcyR) triggers a wide variety of effector functions including phagocytosis, antibody-dépendent cellular cytotoxicity, and release of inflammatory mediators, as well as immune complex clearance and régulation of antibody production. Fc moieties providing crosslinking of receptors (e.g., FcyR) are contemplated herein. In humans, three classes of FcyR hâve been characterized to-date, which are: (î) FcyRl (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcyRII (CD32), which binds complexed IgG with medium to low affinity, is wîdely expressed, in particular on leukocytes, is believed to be a central player in antibody-mediated immunity, and which can be divided into FcyRl IA, FcyRl IB and FcyRl IC, which perform different functions in the immune
System, but bind with similar low affinity to the IgG-Fc, and the ectodomaîns of these receptors are highly homologuous; and (iii) FcyRIII (CD! 6), which binds IgG with medium to low affinity and has been found în two forms: FcyRIIIA, which has been found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and is belîeved to médiate ADCC; and FcyRIlIB, which is highly expressed on neutrophils.
FcyRIIA is found on many cells involved in kïlling (e.g. macrophages, monocytes, neutrophils) and is believed to actîvate the killing process. FcyRIIB is belîeved to play a rôle in inhibitory processes and is found on B-cells, macrophages and on mast cells and eosinophils. Importantly, it has been shown that 75% of ail FcyRIIB is found în the liver (Ganesan, L. P. et al., 2012: “FcyRIIb on liver sinusoïdal endothélium clears small immune complexes,” Journal of Immunology I89; 498l4988). FcyRIlB is abundantly expressed on Liver Sinusoïdal Endothélium, called LSEC, and in Kupffer cells in the liver and LSEC are the major site of small immune complexes clearance (Ganesan, L. P. et al., 2012: FcyRIIb on liver sinusoïdal endothélium clears small immune complexes. Journal of Immunology 189: 49814988).
In some embodiments, the antibodies disclosed herein and the antigen binding fragments thereof comprise an Fc moiety for binding to FcyRIIb, în particular an Fc région, such as, for example IgG-type antibodies. Moreover, it is possible to engineer the Fc moiety to enhance FcyRIlB binding by introducing the mutations S267E and L328F as described by Chu, S. Y. et al., 2008: Inhibition ofB cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRlIb with Fc-engineered antibodies. Molecular immunology 45, 3926-3933. Thereby, the clearance of immune complexes can be enhanced (Chu, S., et al., 2014; Accelerated
Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, An Fc-Engineered Antibody With Enhanced Affinity For Inhibitory Receptor FcyRIIb. Am J Respir Crït, American Thoracic Society International Conférence Abstracts). In some embodiments, the antibodies of the présent disclosure, or the antigen binding fragments thereof, comprise an engineered Fc moiety with the mutations S267E and L328F, in particular as described by Chu, S. Y. et al., 2008: Inhibition ofB cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fcengineered antibodies. Molecular Immunology 45, 3926-3933.
On B cells, FcyRIIB seems to function to suppress further immunoglobulin production and isotype switching to, for example, the IgE class. On macrophages, FcyRIIB is thought to inhibit phagocytosis as mediated through FcyRIIA. On eosinophils and mast cells, the B form may help to suppress activation of these cells through IgE binding to its separate receptor.
Regarding FcyRI binding, modification in native IgG of at least one of E233G236, P238, D265, N297, A327 and P329 can reduce binding to FcyRI. IgG2 residues ai positions 233-236, substituted into corresponding positions IgGl and IgG4, reduces binding of IgGl and IgG4 to FcyRI by lO^fold and eîiminated the human monocyte response to antibody-sensitîzed red blood cells (Armour, K. L., et al. Ew. J. Immunol. 29(1999) 2613-2624).
Regarding FcyRIl binding, reduced binding for FcyRIIA is found, e.g., for IgG mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414.
Two allelic forms of human FcyRIIA are the H 131 variant, which binds to IgG I Fc with high affinity, and the R131 variant, which binds to IgGl Fc with low affinity. See, e.g., Bruhns et al., Blood 723:3716-3725 (2009).
Regarding FcyRIII binding, reduced binding to FcyRIIIA is found, e.g., for mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V3O3, A327, K338 and D376. Mapping ofthe binding sites on human IgGl for Fc receptors, the above-mentioned mutation sites, and methods for measuring binding to FcyRI and FcyRIIA, are described in Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604.
Two allelic forms of human FcyRIIIA are the F 158 variant, which binds to IgGl Fc with low affinity, and the VI58 variant, which binds to IgGl Fc with high affinity. See, e.g., Bruhns et al., Blood 773:3716-3725 (2009).
Regarding binding to FcyRIl, two régions of native IgG Fc appear to be involved în interactions between FcyRIIs and IgGs, namely (i) the lower hinge site of
IgG Fc, in particular amino acid residues L, L, G, G (234 - 237, EU numbering), and (ii) the adjacent région of the CFI2 domain of IgG Fc, in particular a loop and strands in the upper CH2 domain adjacent to the lower hinge région, e.g. in a région of P33l (Wines, B.D., étal., J. Immunol. 2000; 164: 5313 - 5318). Moreover, FcyRI appears to bind to the same site on IgG Fc, whereas FcRn and Protein A bind to a different site on IgG Fc, which appears to be at the CH2-CH3 interface (Wines, B.D., et al., J. Immunol. 2000; 164:5313 -5318).
Also contemplated are mutations that increase binding affinity of an Fc moiety of the présent disclosure to a (i.e., one or more) Fcy receptor (e.g., as compared to a reference Fc moiety or antibody containing the same that does not comprise the mutation(s)). See, e.g., Delillo and Ravetch, Cell 161(5):1035-1045 (2015) and Ahmed étal., J. Struc. Biol. 194(1):78 (2016), the Fc mutations and techniques of which are incorporated herein by reference.
In any of the herein disclosed embodiments, a binding protein can comprise a (e.g., IgGl or IgGl-derived) Fc moiety comprising a mutation selected from (EU numbering) G236A; S239D; A33OL; and I332E; or a combination comprising any two or more ofthe same; e.g., S239D/I332E; S239D/A330L/I332E; G236A/S239D/I332E; G236A/A330L/I332E (also referred to herein as GAALIE); or G236A/S239D/A330L/I332E. In some embodiments, the Fc moiety does not comprise S239D. In some embodiments, the Fc moiety comprises a native Serine at position 239.
In certain embodiments, the Fc moiety may comprise or consist of at least a portion of an Fc moiety that is involved in binding to FcRn binding. In certain embodiments, the Fc moiety comprises one or more amino acid modifications that împrove binding affinity for (e.g., enhance binding to) FcRn (e.g., at a pH of about 6.0) and, in some embodiments, thereby extend in vivo half-life of a molécule comprising the Fc moiety (e.g., as compared to a reference Fc moiety or antibody that is otherwise the same but does not comprise the modîfication(s)). In certain embodiments, the Fc moiety comprises or is derived from a IgG Fc and a half-life-extending mutation comprises any one or more of: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I Q3111; D376V; T307A; E380A (EU numbering). In certain embodiments, a half-life-extending mutation comprises M428L/N434S (also referred to herein as MLNS). In certain embodiments, a half-life-extending mutation comprises M252Y/S254T/T256E. In certain embodiments, a half-life-extending mutation comprises T250Q/M428L. In certain embodiments, a half-life-extending mutation comprises P257I/Q31II. In certain embodiments, a half-iife-extending mutation comprises P2571/N434H. In certain embodiments, a half-life-extending mutation comprises D376V/N434H. In certain embodiments, a half-life-extending mutation comprises T307A/E380A/N434A.
In some embodiments, a binding protein includes a Fc moiety that comprises the substitution mtuations M428L/N434S. In some embodiments, a binding protein includes a Fc moiety that comprises the substitution mtuations G236A/A330L/I332E. In certain embodiments, a binding protein includes a (e.g., IgG) Fc moiety that comprises a G236A mutation, an A330L mutation, and a I332E mutation (GAALIE), and does not comprise a S239D mutation (e.g., comprises a native S at position 239). In particular embodiments, a binding protein includes an Fc moiety that comprises the substitution mutations: M428L/N434S and G236A/A330L/I332E, and optionally does not comprise S239D (e.g., can comprise a native S at position 329). In certain embodiments, a binding protein includes a Fc moiety that comprises the substitution mutations: M428L/N434S and G236A/S239D/A330L/I332E. In certain further embodiments, a binding protein comprises substitution mutations in a Fc moiety, wherein the substitution mutations consist of, or consist essentially of: M428L/N434S, G236A/S239D/A33OL/I332E, or G236A/S239D/A330L/I332E/M428L/N434S.
In any of the presently disclosed embodiments, a binding protein of the présent disclosure includes a Fc moiety comprising a GAALIE mutation and has enhanced binding to a human FcyRIIa and/or a human FcyRIIIa, as compared to a reference polypeptide (i.e., a polypeptide, which may be a binding protein, that includes a Fc moiety that does not comprise the GAALIE mutation).
In certain embodiments, the reference polypeptide includes a Fc moiety that is a wild-type Fc moiety (e.g., of the same isotype) or is a Fc moiety that comprises one or more substitution mutation (or insertion or délétion), provided that the substitution mutation is not or does not comprise GAALIE. In certain embodiments, the reference polypeptide does not comprise a substitution mutation that is known or believed to affect binding to a human FcyRIIa and/or to a human FcyRIIIa.
Binding between polypeptides, such as binding between a Fc moiety (or a binding protein comprising the same) and a human Fcy Receptor, such as human FcyRIIA, human FcyRIllA, or human Fc FcyRIIB, or a complément protein, such as Clq, can be determined or detected using methods known in the art. For example, a biolayer interferometry (BLI) assay can be performed using an Octet® RED96 (ForteBio, Fremont, California USA) instrument according to manufacturer’s instructions to détermine real-time association and dissociation between a first polypeptide of interest (e.g., a Fc moiety comprising a GAALIE mutation) and a second polypeptide of interest (e.g., a FcyRIIA (Hl31), a FcyRIIA (RI31), a FcyRHIA (Fl58), a FcyRIllA (VI58), or a FcyRIIb) that is captured on a sensor substrate.
In certain embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation and has enhanced binding to a human FcyRIIA (H13 I), a human FcyRIIA (R131 ), a human FcyRIllA (F 158), a human FcyRIllA (VI58), or any combination thereof, as compared to a reference polypeptide that includes a Fc moiety that does not comprise the GAALIE mutation. In certain embodiments, enhanced binding is determined by an increase (e.g·, one or more of: a higher peak signal; a greater rate of association; a slower rate of dissociation; a greater area under the curve) in signal shift versus the reference binding protein in a BLI assay. In certain embodiments, the BLI assay comprises use of Octet(R) RED96 (ForteBio, Fremont, California USA) instrument. In further embodiments, the BLI assay comprises a tagged human FcyR captured onto an anti-penta-tag sensor and exposed to the binding protein. In some embodiments, the binding protein comprises a IgG Fab and the BLI assay further comprises exposîng the captured human FcyR to the binding protein in the presence of an anti-IgG Fab binding fragment to cross-link the binding proteins through the Fab fragment.
In certain embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation and has enhanced binding to a human FcyRIIA (H131), a human
FcyRIlA (R131), a human FcyRIIIA (Fl58), and/or a human FcyRIIIA (VI 58) as compared to a reference polypeptide, wherein the enhanced binding can comprise a signal shift (nanometers) in a BLI assay of 1.5, 2, 2.5, 3, or more times greater than the signal shift observed using the reference binding protein.
In certain embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation and has enhanced binding to a human FcyRIlA (H131 ), a human FcyRIIA (RI31 ), a human FcyRIIIA (Fl 58), and a human FcyRIIIA (VI58), as compared to a reference polypeptide.
In any ofthe presently disclosed embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation and has reduced binding to a human FcyRIIB, as compared to a reference polypeptide. In certain embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation and does not bind to a human FcyRIIB, as determined, for example, by the absence of a statisticaliy significant signal shift versus baseline in a BLI assay.
In any of the presently disclosed embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation and has reduced binding to a human Clq (complément protein), as compared to a reference polypeptide. In certain embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation and does not bind to a human Clq, as determined by the absence of a statisticaliy significant signal shift versus baseline in a BLI assay.
In any of the presently disclosed embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation and activâtes a human FcyRIlA, a human FcyRIIIA, or both, to a greater degree than does a reference polypeptide (i.e., a polypeptide, which may be a HBsAg-specific binding protein, that includes a Fc moiety that does not comprise the GAALIE mutation). In certain embodiments, the reference polypeptide includes a Fc moiety that is a wild-type Fc moiety or that comprises one or more substitution mutation, provided that the substitution mutation is not GAALIE.
Activation of a human FcyR can be determined or detected using methods known in the art. For example, a well-validated, commercially available bioreporter assay involves incubating a HBsAg-specific binding protein with a recombinant HBsAg
(Engerix B, GlaxoSmithKIine) in the presence of Jurkat effector cells (Promega; Cat. no: G9798) stably expressing (i) a FcyR of interest and (ii) firefly luciferase reporter under the control of a NFAT response element. Binding of Fc to cell surface-expressed FcyR drives NFAT-mediated expression of luciferase reporter gene. Luminescence is then measured with a luminometer (e.g., Bio-Tek) using the Bio-GIo-™ Luciferase Assay Reagent (Promega) according to the manufacturer’s instructions. Activation is expressed as the average of relative luminescence units (RLU) over the background by applying the following formula: (RLU at concentration [x] of binding protein (e.g., niAbs) - RLU of background).
In certain embodiments, a binding protein includes a Fc moiety comprising a
GAALIE mutation activâtes a human FcyRIIA (Hl3l), a human FcyRIIA (Rl3l), a human FcyRIIIA (Fl58), and/or a human FcyRIIIA (V158) to a greater degree than does a reference polypeptide. In certain embodiments, a greater degree of activation refers to a higher peak luminescence and/or a greater luminescence area under the curve, as determined using a luminescence bioreporter assay as described herein. In certain embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation and activâtes a human FcyRIIA (H131), a human FcyRIIA (RI 31 ), and a human FcyRIIIA (F158) to a greater degree than does a reference polypeptide, wherein the greater degree of activation comprises to a peak RLU that is L5, 2, 2.5, 3, or more times greater than the peak RLU observed using the reference binding protein.
In any ofthe presently disclosed embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation does not activate a human FcyRHB, as determined by the absence of a statistically significant and/or measurable RLU in a luminescence bioreporter assay as described above.
In any of the presently disclosed embodiments, a binding protein includes a Fc moiety comprising a GAALIE mutation and activâtes a human natural killer (NK) cell in the presence of HBsAg to a greater degree than does a reference polypeptide. In certain embodiments, activation of a NK cell is determined by CDl 07a expression (e.g., by flow cytometry). In certain embodiments, the NK cell comprises a cell that
comprises V158/V158 homozygous, a F158/F158 homozygous, or a V158/F158 heterozygous FcyRIIIa génotype.
It will be appreciated that any binding protein including a Fc moiety comprising a GAALIE mutation according to the présent disclosure can perform or possess any one or more of the features described herein; e.g., enhanced binding to a human FcyRIIA and/or a human FcyRIIIA as compared to a reference polypeptide; reduced binding to a human FcyRIIB as compared to a reference polypeptide (and/or no binding to a human FcyRIIB); reduced binding to a human Clq as compared to a reference polypeptide (and/or no binding to a human Clq); activâtes a FcyRIIA, a human FcyRIUA, or both, 10 to a greater degree than does a reference polypeptide; does not actîvate a human
FcyRIIB; and/or activâtes a human naturel killer (NK) cell in the presence of HBsAg to a greater degree than does a reference polypeptide (e.g., an antibody that is spécifie for HBsAg and includes a Fc moiety that does not comprise a GAALIE mutation).
In certain embodiments, a binding protein of the présent disclosure includes a Fc 15 moiety comprising a GAALIE mutation and: (i) has enhanced binding to a human
FcyRIIA, a human FcyRIUA, or both, as compared to a reference polypeptide that includes a Fc moiety that does not comprise G236A/A330L/I332E, wherein the human FcyRIIA is optionally Hl3l or RI31, and/or the human FcyRIUA is optionally Fl58 or V158; (iî) has reduced binding to a human FcyRIIB, as compared to a reference polypeptide thaï includes a Fc moiety that does not comprise G236A/A330L/I332E; (iii) does not bind to a human FcyRIIB; (iv) has reduced binding to a human Clq, as compared to a reference polypeptide that includes a Fc moiety that does not comprise G236A/A330L/I332E; (v) does not bind to a human Clq; (vi) activâtes a FcyRIIA, a human FcyRIIIA, or both, to a greater degree than does a reference polypeptide that includes a Fc moiety that does not comprise G236A/A330L/I332E, wherein the human FcyRIIA is optionally Hl3l orRl3l, and/or the human FcyRIUA is optionally F158 or
V158; (vii) does not activate a human FcyRIIB; (viii) activâtes a human naturel killer (NK) cell in the presence of HBsAg to a greater degree than does a reference polypeptide thaï includes a Fc moiety that does not comprise G236A/A330L/I332E, wherein the reference polypeptide is optionally an antibody that binds to an HB Ag,
optionally an HBsAg (ix) is capable of binding to an HBsAg variant comprising HBsAg-YI00C/P120T, HBsAg-P120T, HBsAg-Pl20S/Sl43L, HBsAg-CI2lS, HBsAg-RI22D, HBsAg-Rl22I, HBsAg-Tl23N, HBsAg-Q129H, HBsAg-Ql29L, HBsAg-Ml33H, HBsAg-Ml33L, HBsAg-Ml33T, HBsAg-KI4lE, HBsAg-Pl42S,
HBsAg-Sl43K, HBsAg-Dl44A, HBsAg-Gl45R, HBsAg-NJ46A, or any combination thereof; (x) has improved binding to an HBsAg variant comprising HBsAgY100C/P120T, HBsAg-Pl20T, HBsAg-Pl20S/S143L, HBsAg-Cl2lS, HBsAg-Rl22D, HBsAg-Rl22I, HBsAg-Tl23N, HBsAg-Ql29H, HBsAg-Ql29L, HBsAg-Ml33H, HBsAg-Ml33L, HBsAg-Ml33T, HBsAg-K!4lE, HBsAg-PI42S, HBsAg-Sl43K,
HBsAg-Dl44A, HBsAg-Gl45R, HBsAg-Nl46A, or any combination thereof, as compared to a reference antibody or antigen binding fragment that bînds to HBsAg and that includes a Fc moiety that does not comprise G236A/A330L/I332E.
Alternatively or additionally, the Fc moiety of a binding protein ofthe disclosure can comprise at least a portion known in the art to be required for Protein A binding; and/or the Fc moiety of an antibody of the disclosure comprises at least the portion of an Fc molécule known in the art to be required for protein G binding. In some embodiments, a retained function comprises the clearance of HBsAg and HBVg. Accordingly, in certain embodiments, an Fc moiety comprises at least a portion known in the art to be required for FcyR binding. As outlined above, an Fc moiety may thus at least comprise (i) the lower hinge site of native IgG Fc, in particular amino acid residues L, L, G, G (234 - 237, EU numberîng), and (ii) the adjacent région ofthe CH2 domain of native IgG Fc, in particular a loop and strands in the upper CH2 domain adjacent to the lower hinge région, e.g. in a région of P33l, for exampie a région of at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids in the upper CH2 domain of native IgG Fc around P331, e.g. between amino acids 320 and 340 (EU numberîng) of native IgG Fc.
In some embodiments, a binding protein according to the présent disclosure comprises an Fc région. As used herein, the term Fc région refers to the portion of an immunoglobulîn formed by two or more Fc moieties of antibody heavy chains. For example, an Fc région may be monomeric or single-chain Fc région (i.e., a scFc région). Single chain Fc régions are comprised of Fc moieties linked within a single polypeptide chain (e.g., encoded in a single contiguous nucleic acid sequence). Exemplary scFc régions are disclosed in WO 2008/143954 A2, and are incorporated by reference herein. The Fc région can be or comprise a dimeric Fc région; it will be understood that a dimeric Fc région is not the same as an undesired (e.g., antibody:antibody, antibody:antigen-binding fragment, or antigen-binding fragmentantigen-binding fragment) dimer, such as described above and illustrated, in one embodiment, in Figure 7. In certain preferred embodiments, an antibody or antigen-binding fragment comprises a dimeric Fc région, while producing few antibody- or antigen-binding fragment-containing dimers.
A dimeric Fc région or dcFc refers to the dimer formed by the Fc moieties oftwo separate immunoglobulin heavy chains. The dimeric Fc région may be a homodimer of two identical Fc moieties (e.g., an Fc région of a naturally occurring immunoglobulin) or a heterodimer of two non-îdentical Fc moieties (e.g., one Fc monomer of the dimeric Fc région comprises at least one amino acid modification (e.g., substitution, délétion, insertion, or Chemical modification) that is not présent in the other Fc monomer, or one Fc monomer may be truncated as compared to the other).
Presently disclosed Fc moieties may comprise Fc sequences or régions of the same or different class and/or subclass. For example, Fc moieties may be derived from an immunoglobulin (e.g., a human immunoglobulin) of an IgGl, IgG2, IgG3 or lgG4 subclass, or from any combination thereof. In certain embodiments, the Fc moieties of Fc région are of the same class and subclass. However, the Fc région (or one or more Fc moieties of an Fc région) may also be chimeric, whereby a chimeric Fc région may comprise Fc moieties derived from different immunoglobulin classes and/or subclasses. For example, at least two of the Fc moieties of a dimeric or single-chain Fc région may be from different immunoglobulin classes and/or subclasses. In certain embodiments, a dimeric Fc région can comprise sequences from two or more different isotypes or subclasses; e.g., a SEEDbody (strand-exchange engineered domains), see Davis et al.. Protein Eng. Des. Sel. 23(4):195 (2010).
Additionally or altematively, chimeric Fc régions may comprise one or more chimeric Fc moieties. For example, the chimeric Fc région or moiety may comprise one or more portions derived from an immunoglobulin of a first subclass (e.g., an IgGl, IgG2, or IgG3 subclass) while the remainder of the Fc région or moiety is of a different 5 subclass. For example, an Fc région or moiety of an Fc polypeptide may comprise a
CH2 and/or CH3 domain derived from an immunoglobulin of a first subclass (e.g., an IgGl, lgG2 or IgG4 subclass) and a hinge région from an immunoglobulin of a second subclass (e.g., an IgG3 subclass). For example, the Fc région or moiety may comprise a hinge and/or CH2 domain derived from an immunoglobulin of a first subclass (e.g., an 10 IgG4 subclass) and a CH3 domain from an immunoglobulin of a second subclass (e.g., an IgGl, IgG2, or IgG3 subclass). For example, the chimeric Fc région may comprise an Fc moiety (e.g., a complété Fc moiety) from an immunoglobulin for a first subclass (e.g., an IgG4 subclass) and an Fc moiety from an immunoglobulin of a second subclass (e.g., an IgGl, lgG2 or IgG3 subclass). For example, the Fc région or moiety may comprise a CH2 domain from an lgG4 immunoglobulin and a CH3 domain from an IgGl immunoglobulin. For example, the Fc région or moiety may comprise a CHl domain and a CH2 domain from an IgG4 molécule and a CH3 domain from an IgGl molécule. For example, the Fc région or moiety may comprise a portion of a CH2 domain from a particular subclass of antibody, e.g., EU positions 292-340 of a CH2 domain. For example, an Fc région or moiety may comprise amino acids a positions 292-340 of CH2 derived from an IgG4 moiety and the remainder of CH2 derived from an IgGl moiety (altematively, 292-340 of CH2 may be derived from an IgGl moiety and the remainder of CH2 derived from an IgG4 moiety).
It will also be appreciated that any antibody, antigen-binding fragment, or Fc 25 région or moiety of the présent disclosure can be of any allotype and/or haplotype. For example, human Immunoglobulin G allotypes include those disclosed in Jefferis and LeFranc, mAbs 7(4):1-7 (2009), which allotypes (including Glm (l(a); 2(x); 3(f); and 17(z)); G2m (23(n)); G3m (21(gl); 28(g5); 1 l(b0); 5(b2); 13(b3); 14(b4); 10(b5);
15(s); 16(t); 6(c3); 24(c5); 26(u); and 27(v)); A2m (1 and 2); and Km (1; 2; and 3) and 30 haplotypes, and résultant amino acid sequences, and combinations thereof, are incorporated herein by reference. In certain embodiments, an antibody, antigen-binding fragment, or Fc région or moiety of the présent disclosure comprises a IgGl allotype glml7,kl.
Moreover, an Fc région or moiety may (additionally or alternatively) for example comprise a chimeric hinge région. For example, the chimeric hinge may be derived, e.g. in part, from an IgG l, lgG2, or IgG4 molécule (e.g., an upper and lower niîddle hinge sequence) and, in part, from an IgG3 molécule (e.g., an middle hinge sequence). In another example, an Fc région or moiety may comprise a chimeric hinge derived, in part, from an IgGl molécule and, in part, from an IgG4 molécule. In another example, the chimeric hinge may comprise upper and lower hinge domains from an IgG4 molécule and a middle hinge domain from an IgG l molécule. Such a chimeric hinge may be made, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the middle hinge domain of an IgG4 hinge région. In another embodiment, the chimeric hinge can comprise amino acids at EU positions 233-236 are from an IgG2 antibody and/or the Ser228Pro mutation, wherein the remaining amino acids of the hinge are from an IgG4 antibody (e.g., a chimeric hinge of the sequence ESKYGPPCPPCPAPPVAGP (SEQ ID NO.:74)). Further chimeric hinges which may be used in the Fc moiety of the antibody according to the présent disclosure are described in US 2005/0163783 Al.
In some embodiments of the binding proteins disclosed herein, the Fc moiety, or the Fc région, comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., from an Fc région or Fc moiety from a human IgG molécule). However, polypeptides may comprise one or more amino acids from another mammalian species. For example, a primate Fc moiety or a primate binding site may be included in the subject polypeptides. Alternatively, one or more murine amino acids may be présent in the Fc moiety or in the Fc région.
In some embodiments, an antibody is provided that comprises: a heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:75, optionally with the C-termïnal lysine removed, and a light chain (LC), wherein the LC comprises or consists of (i) the VL amino acid sequence set forth in any one of SEQ ID NOs.:58-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.
In some embodiments, an antibody is provided that comprises: a heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:76, optionally with the C-terminal lysine removed, and a light chain (LC), wherein the LC comprises or consists of (i) the VL amino acid sequence set forth in any one of SEQ ID NOs.:58-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.
In some embodiments, an antibody is provided that comprises: a heavy chain (HC) comprising or consisting ofthe amino acid sequence set forth in SEQ ID NO.:77, optionally with the C-terminal lysine removed, and a light chain (LC), wherein the LC comprises or consists of (i) the VL amino acid sequence set forth in any one of SEQ ID NOs.:58-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.
In some embodiments, an antibody is provided that comprises: a heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:78, optionally with the C-terminal lysine removed, and a light chain (LC), wherein the LC comprises or consists of (i) the VL amino acid sequence set forth in any one of SEQ ID NOs.:58-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.
Nucleic acid molecules/Polynucléotides
In another aspect, the disclosure provides a nucleic acid molécule comprising a polynucleotide encoding an antibody, antigen binding fragment, or fusion protein according to the présent disclosure. It will be understood that, for example, a first nucleic acid molécule can encode a heavy chain of an antibody, and a second nucleic acid molécule can encode a light chain of an antibody; these first and second nucleic acid molécules can still be referred-to as a polynucleotide1’ or a nucleic acid molécule that encodes the antibody. In other words, a polynucleotide or nucleic acid molécule includes embodiments, wherein portions (e.g., chains) of an antibody or antigen-binding fragment are encoded by separate nucleic acid molécules and/or by separate portions of nucleci acid molecules.Exemplary polynucleotide sequences are provided in SEQ ID NOs.:80-99. In some embodiments, a polynucleotide encoding an antibody heavy chain comprises or consists of the polynucleotide sequence set forth in
SEQ ID NO.:8l, and a polynucleotide encoding an antibody VL or LC comprises the polynucleotide sequence set forth in any one of SEQ ID NOs.:85-99. In other embodiments, a polynucleotide encoding an antibody heavy chain comprises or consists ofthe polynucleotide sequence set forth in SEQ ID NO.:83, and a polynucleotide encoding an antibody VL or LC comprises the polynucleotide sequence set forth in any one of SEQ ID NOs.:85-99. In still other embodiments, a polynucleotide encoding an antibody heavy chain comprises or consists of the polynucleotide sequence set forth in SEQ ID NO.:84, and a polynucleotide encoding an antibody VL or LC comprises the polynucleotide sequence set forth in any one of SEQ ID NOs.:85-99.
Due to the redundancy of the genetic code, the présent disclosure also comprises sequence variants of these nucleic acid sequences and in particular such sequence variants, which encode the same amino acid sequences.
In certain embodiments, a polynucleotide or nucleic acid molécule comprises a nucléotide sequence sharing at least 50% (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 9l%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or ï 00%) identity to the nucléotide sequence according to any one of SEQ ID NOs.:80-99, wherein the nucléotide sequence is codon optimized for expression by a host cell.
In particular embodiments, a nucleic acid molécule according to the présent disclosure comprises or consists of a nucleic acid sequence according to any one of SEQ ID NOs: SEQ ID NOs.:80-99.
In certain embodiments, a polynucleotide comprises a VH-encoding nucléotide sequence having at least 50% identity to the amino acid sequence set forth in SEQ ID NO.:81 and a VL-encoding nucléotide sequence having at least 50% identity to the amino acid sequence set forth in any one of SEQ ID NOs.:85-97.
In any of the presently disclosed embodiments, a polynucleotide can comprise a VH-CHl -hinge-CH2-CH3-encoding nucléotide sequence according to SEQ ID NO:84. In some embodiments, a polynucleotide comprises a CL-encoding nucléotide sequence having at least 50% identity to the amino acid sequence set forth in SEQ ID NO:98 or 99.
Vectors
Further included within the scope of the disclosure are vectors, for example, expression vectors, that comprise a nucleic acid molécule according to the présent disclosure.
The term vector refers to a construct comprising a nucleic acid molécule. A 5 vector in the context of the présent disclosure is suitable for incorporating or harboring a desired nucleic acid sequence. Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc. A storage vector îs a vector which allows the convenient storage of a nucleic acid molécule. Th us, the vector may comprise a sequence corresponding, e.g., to a desired antibody or antibody fragment thereof according to the présent description.
As used herein, expression vector refers to a DNA construct containing a nucleic acid molécule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molécule in a suitable host. Such control sequences include a promoter (e.g., a heterologous promoter) to effect transcription, an 15 optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termînation of transcription and translation. Any of the éléments of an expression vector that contribute to transcription of a nucleic acid moieeuîe of interest may be heterologous to the vector. The vector may be a plasmid, a phage partie le, a virus, or simply a potential 20 genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, întegrate into the genome itself. In the présent spécification, plasmid, expression plasmid. virus and vector are often used interchangeably.
A cloning vector is typically a vector that contains a cloning site, which may be 25 used to incorporate nucleic acid sequences into the vector. A cloning vector may be, e.g., a plasmid vector or a bactériophage vector.
A transfer vector may be a vector which is suitable for transferring nucleic acid molécules into cells or organisais, for example, viral vectors. A vector in the context of the présent disclosure may be, e.g., an RNA vector or a DNA vector. A vector may be a 30 DNA molécule. For example, a vector in the sense of the présent application comprises
a cloning site, a sélection marker, such as an antibiotic résistance factor, and a sequence suitable for multiplication of the vector, such as an origin of réplication.
In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a lentiviral vector or a γ-retroviral vector). Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, négative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picomavîrus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types l and 2,
Epstein-Barr virus, cytomégalovirus), and poxvirus (e.g-, vaccinîa, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavîrus, flavivîrus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their réplication, In Fundamental Vîrology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, I996).
Retroviruses are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome. Gammaretrovirus refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.
Lentiviral vectors include HIV-based lentiviral vectors for gene delivery, whîch can be intégrative or non-integrative, hâve relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded
linear viral DNA, which is the substrate for viral intégration into the DNA of infected cells.
In certain embodiments, the viral vector can be a gammaretrovirus, e.g., Moloney murine leukemia virus (MLV)-derived vectors. In other embodiments, the viral vector can be a more complex retrovirus-derived vector, e.g., a lentivirus-derîved vector. HIV-1 -derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infections anemîa virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing transgenes are known in the art and hâve been previous described, for example, in: U.S. Patent 8,119,772; Walchlî étal., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et ak, Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression Systems are also commercially available. Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (FISVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).
Other vectors that can be used with the compositions and methods of this disclosure include those derived from baculoviruses and α-viruses. (Jolly, D J. 1999. Emerging Viral Vectors. pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).
When a viral vector genome comprises a plurality of polynucleotides to be expressed in a host cell as separate transcripts, the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include internai ribosome entry sites (1RES), furin cleavage sites, viral 2A peptide, or any combination thereof.
Plasmid vectors, including DNA-based antibody or antigen-binding fragmentencoding plasmid vectors for direct administration to a subject, are described further herein.
Cells
In a further aspect, the présent disclosure also provides a cell (also referred to as a host cell) expressing an antibody, antigen-binding fragment, or fusion protein according to the présent disclosure; or comprising a vector or poiynucleotide according the présent disclosure.
Examples of such cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli. In some embodiments, the cells are mammalian cells. In certain such embodiments, the cells are a mammalian cell line such as CHO cells (e.g., DHFR- CHO cells (Urlaub et al., PNAS 77:4216 (1980), CHO-KSV, ExpiCHO), human embryonîc kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, Sp2/0 cells. NS0 cells, human liver cells, e.g. Hepa RG cells, myeloma cells or hybrîdoma cells. Other examples of mammalian host cell lines include mouse sertoli cells (e.g., TM4 cells); monkey kidney CVI line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); African green monkey kidney cells (VERO-76); monkey kidney cells (CVI); human cervical carcinoma cells (HELA); human lung cells (W138); human liver cells (Hep G2); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Mammalian host cell lines suitable for antibody production also include those described in, for example, Yazaki and Wu. Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ.), pp. 255-268 (2003).
In certain embodiments, a host cell is a prokaryotic cell, such as an E. coli. The expression of peptides in prokaryotic cells such as E. coli is well established (see, e.g., Pluckthun, A. Bio/Technology 9:545-551 (1991). For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria. see, e.g., L.S. Pat. Nos. 5,648,237; 5,789,199; and 5,840,523.
Insect cells useful expressing a binding protein of the present disclosure are known in the art and include, for example, Spodoptera frugipera Sf9 cells, Trichoplusia ni BTI-TN5B1-4 cells, and Spodopterafrugipera SfSWTOl “Mimic™” cells. See, e.g., Palmbergere/oZ., J. Biotechnol. /53(3-4); 160-166 (2011). Numerous baculoviral strains hâve been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugîperda cells.
Eukaryotic microbes such as filamentous fungi or yeast are also suitable hosts for cloning or expressing protein-encoding vectors, and include fungi and yeast strains with “humanized” glycosylation pathways, resulting in the production of an antibody with a partîally or fu 11 y human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215(2006).
Plant cells can also be utilized as hosts for expressing a binding protein ofthe présent disclosure. For example, PLANTIBODIES™ technology (described in, for example, U.S. Pat. Nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; and 6,417,429) 15 employs transgenic plants to produce antibodies.
In some embodiments, a fusion protein is expressed at a cell surface by an immune cell, e.g., a T cell, NK cell, or NK-T cell, or any subtype thereof.
Any protein expression system compatible with the disclosure may be used to produce the disclosed binding proteins. Suitable expression Systems include transgenic animais described in Gene Expression Systems, Academie Press, eds. Fernandez et al., 1999.
In particular embodiments, the cell may be transfected with a vector according to the present description with an expression vector. The term transfection refers to the introduction of nucleic acid molécules, such as DNA or RNA (e.g. mRNA) molécules, into cells, such as into eukaryotic cells. In the context ofthe present description, the term “transfection” encompasses any method known to the skilled person for introducing nucleic acid molécules into cells, such as into eukaryotic cells, including into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g., based on cationic lipids and/or liposomes, calcium phosphate précipitation, nanoparticle based transfection, virus based transfection, or
transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine etc. In certain embodiments, the introduction is non-viral.
Moreover, cells ofthe présent disclosure may be transfected stably or transiently with the vector according to the présent description, e.g. for expressing an antibody, or an antigen binding fragment thereof, according to the présent description. In such embodiments, the cells are stably transfected with the vector as described herein encoding a binding protein. Alternatively, cells may be transiently transfected with a vector according to the présent disclosure encoding a binding protein according to the présent description. In any of the presently disclosed embodiments, a polynucleotide may be heterologous to the host cell.
In a related aspect, the présent disclosure provides methods for producing an antibody, antigen-binding fragment, or fusion protein, wherein the methods comprise culturing a host cell of the présent disclosure under conditions and for a time sufficient to produce the antibody, antigen-binding fragment, or fusion protein.
Accordingly, the présent disclosure also provides recombinant host cells that heterologously express an antibody, antigen-binding fragment, or fusion protein ofthe présent disclosure. For example, the cell may be of a species that is different to the species from which the antibody was fully or partially obtained (e.g., CHO cells expressing a human antibody or an engineered human antibody). In some embodiments, the cell type of the host cell does not express the antibody or antigen binding fragment in nature. Moreover, the host cell may impart a post-transiational modification (PTM; e.g., glysocylation or fucosylatîon) on the binding protein that is not présent in a native State of the binding protein (or in a native State of a parent binding protein from which the subject binding protein was engineered or derived).
Such a PTM may resuit in a functional différence (e.g., reduced immunogenicity). Accordingly, a binding protein of the présent disclosure that is produced by a host cell as disclosed herein may include one or more post-translational modification that is distinct from thebinding protein or parent binding protein in its native state (e.g., a human antibody produced by a CHO cell can comprise a post-translational modification
that is distinct from the antibody when isolated from the human and/or produced by the native human B cell or plasma cell).
Optional Further Features of Antibodies, Antigen-Binding Fragments, and Fusion 5 P ro teins
Antibodies, antigen-binding fragments, and fusion proteins of the disclosure may be coupled, for example, to a drug for delivery to a treatment site or coupled to a détectable label to facilitate imaging of a site comprising cells of interest. Methods for coupling antibodies to drugs and détectable labels are wei! known in the art, as are methods for imaging using détectable labels. Labeled antibodies may be employed in a wide variety of assays, employing a wide variety of labels. Détection ofthe formation of an antîbody-antigen complex between an antibody (or antigen binding fragment or fusion protein) ofthe disclosure and an epitope of interest on HBsAg, in particular on the antigenic loop région of HBsAg, can be facilitated by attaching a détectable substance to the antibody. Suitable détection means include the use of labels such as radionuclides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or co-factors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like. Examples of suitable enzymes include horseradîsh peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avîdin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, lucîferîn, and aequorin; and examples of suitable radioactive material include 125I, 1311, 35S, or 3H. Such labeled reagents may be used in a variety of well-known assays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and the like. Labeled antibodies, antigen binding fragments, and fusion proteins according to the présent disclosure may be thus be used in such assays for example as described in US
3,766,162; US 3,791,932; US 3,817,837; and US 4,233,402.
An antibody, antîgen-binding fragment, or fusion protein according to the présent disclosure may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a radioactive métal ion or radioisotope. Exemples of radioisotopes include, but are not limited to, I-131, 1-123, I-125, Y-90, Re-l88, Re-186, At-211, Cu67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and the like. Such conjugates can be used for modîfying a given biological response; the drug moiety is not to be construed as limited to classîcal Chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin.
Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Arnon et al. (1985) Monoclonal Antibodies for Iinmunotargeting of Drugs în Cancer Therapy, in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et aL (Alan R. Liss, Inc.), pp. 243-256; ed. Hellstrom et al. (1987) Antibodies for Drug Delivery, in Controlled Drug Delîvery, ed. Robinson et al. (2d ed; Marcel Dekker, Inc.), pp. 623-653; Thorpe (1985) Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al. pp. 475-506 (Editrice Kurtis, Milano, Italy, 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy, în Monoclonal Antibodies for Cancer Détection and Therapy, ed. Baldwin et al. (Academie Press, New York, 1985), pp. 303316; and Thorpe et al. (1982) Immunol. Rev. 62:119-158.
Altematively, an antibody, antibody fragment, or fusion protein, can be conjugated to a second antibody, or antibody fragment thereof, (or second fusion protein) to form a heteroconjugate, e.g., as described in US 4,676,980. In addition, linkers may be used between the labels and the antibodies of the description, e.g., as described in US 4,831,175. Antibodies, antîgen-binding fragments, and fusion proteins may be directly labeled with radioactive iodine, indium, yttrium, or other radioactive particle known in the art, e.g., as described in US 5,595,721. Treatment may consist of a combination of treatment with conjugated and non-conjugated antibodies, antigen binding fragments, and/or fusion proteins, adminîstered simultaneously or subsequently e.g., as described în WO00/5203! ; WO00/52473.
Antibodies, antigen-binding fragments, and fusion proteins as described herein may also be attached to a solid support. Additionally, the antibodies of the présent disclosure, functional antibody fragments thereof, or fusion proteins, can be chemically modified by covalent conjugation to a polymer to, for example, increase their circulating half-iife. Examples of polymers, and methods to attach them to peptides, are shown in US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. In some embodiments, the polymers may be selected from polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula: RfO-CHz-CfDnO-R, wherein R can be hydrogen, or a protectîve group such as an alkyl or alkanol group. In certain embodiments, the protectîve group may hâve between 1 and 8 carbons. For example, the protectîve group may be methyl. The symbol n is a positive integer. In one embodiment, n is between 1 and 1,000. In another embodiment n is between 2 and 500. In some embodments, the PEG has an average molecular weight selected from between 1,000 and 40,000, between 2,000 and 20,000, and between 3,000 and 12,000. Furthermore, PEG may hâve at least one hydroxy group, for example the PEG may hâve a terminal hydroxy group. For example, it is the terminal hydroxy group which is activated to react with a free amino group on the inhibitor. However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/antibody of the présent description.
Water-soluble polyoxyethylated polyols may also be utlized in the context ofthe antibodies and antigen binding fragements described herein. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and the like. In one embodiment, POG is used. Without being bound by any theory, because the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animais and humans in mono-, di-, triglycérides, this branching would not necessarily be seen as a foreign agent in the body. POG may hâve a molecular weight in the same range as PEG. Another drug delivery system that
can be used for increasing circulatory half-life is the liposome. Methods of preparing liposome delîvery Systems are known to one of skill in the art. Other drug delivery Systems are known in the art and are described in, for example, referenced in Poznansky et al. (1980) and Poznansky (1984).
Antibodies, antigen-binding fragments, and fusion proteins ofthe disclosure may be provided in purified form. Typically, the antibody, antigen-binding fragment, or fusion protein will be présent in a composition that is substantially free of other polypeptides e.g., where less than 90% (by weight), usually less than 60% and more usually less than 50% of the composition is made up of other polypeptides.
Antibodies, fusion proteins, or antigen-binding fragments of the disclosure may be immunogenic in non-human (or heterologous) hosts e.g., in mice. In particular, the antibodies, antigen-binding fragments, or fusion proteins may hâve an idiotope that is immunogenic in non-human hosts, but not in a human host. In particular, such molécules of the disclosure for human use include those that cannot be easily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and cannot generally be obtained by humanization or from xeno-mice.
Production of antibodies, antigen binding fragments, and fusion proteins
Antibodies, antigen-binding fragments, and fusion proteins according to the 20 disclosure can be made by any method known in the art. For example, the general niethodology for making monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C., 1975; Kozbar et al. 1983). In one embodiment, the EBV immortalization method described in WO2004/076677 is used.
In one embodiment, antibodies are produced using a method described in WO 25 2004/076677. In such methods, B cells producing the antibody are transforined with EBV and a polyclonal B cell activator. Additional stimulants of cellular growth and différentiation may optionally be added during the transformation step to further enhance the efficiency. These stimulants may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, but its use is not essential. The immortalized B cells
produced using these methods can then be cultured using methods known in the art and antibodies isolated therefrom.
Another method for producing antibodies is described in WO 2010/046775. In such a method, plasma cells are cultured in limited numbers, or as single plasma cells in 5 microwell culture plates. Antibodies can be isolated from the plasma cell cultures.
Further, from the plasma cell cultures, RNA can be extracted and PCR can be performed using methods known in the art. The VH and VL régions ofthe antibodies can be amplified by RT-PCR (reverse transcriptase PCR), sequenced and cloned into an expression vector that is then transfected into HEK293T cells or other host cells. The 10 cloning of nucleic acid in expression vectors, the transfection of host cells, the culture of the transfected host cells and the isolation of the produced antibody can be done using any methods known to one of skill in the art.
The antibodies may be further purified, if desired, using filtration, centrifugation and various chromatographie methods such as HPLC or affinity chromatography.
Techniques for purification of antibodies, e.g., monoclonal antibodies, including techniques for producing phannaceutical-grade antibodies, are well known in the art.
Standard techniques of molecular biology may be used to préparé DNA sequences encoding the antibodies, antigen-binding fragments, or fusion proteins of the présent description. Desired DNA sequences may be synthesized completely or in part 20 using oligonucleotide synthesis techniques. Site-directed mutagenesîs and polymerase chain reaction (PCR) techniques may be used as appropriate.
Any suîtable host cell/vector system may be used for expression of the DNA sequences encoding the antibody or fusion protein molécules of the présent disclosure or fragments thereof. Bacterial, for example E. coli, and other microbial Systems may 25 be used. in part, for expression of antibody fragments such as Fab and F(ab’)2 fragments, and especially Fv fragments and single chain antibody fragments, for example, single chain Fvs. Eukaryotic, e.g., mammalian, host cell expression Systems may be used for production of larger antibody molécules, including complété antibody molécules. Suitable mammalian host cells include, but are not limited to, those J.
exemplary host cells and cell lînes disclosed herein.
The présent disclosure also provides a process for the production of an antibody, antigen-binding fragment, or fusion protein molécule according to the présent disclosure comprising culturing a host cell comprising a vector encoding a nucleic acid of the présent disclosure under conditions suitable for expression of protein from DNA 5 encoding the antibody molécule of the présent description, and isolating the antibody molécule.
An antibody molécule or antibody fragment may comprise only a heavy or light chain polypeptide, in which case only a heavy chain or light chain polypeptide coding sequence needs to be used to transfect the host cells. For production of products comprising both heavy and light chains, the cell line may be transfected with two veclors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Altematively, a single vector may be used, the vector including sequences encoding light chaîn and heavy chain polypeptides.
Altematively, antibodies, antigen-binding fragments, and fusion proteins according to the disclosure may be produced by (i) expressing a nucleic acid sequence according to the disclosure in a host cell, e.g. by use of a vector according to the présent description, and (ii) isolating the expressed desired product. Additionally, the method may include (iii) purifying the isolated antibody, antigen-binding fragment, or fusion protein. Transformed B cells and cultured plasma cells may be screened for those producing antibodies, antigen-binding fragments, or fusion proteins of the desired specificity or function.
Screening may be carried out by any immunoassay, e.g., ELISA, by staining of tissues or cells (including transfected cells), by neutralization assay or by one of a number of other methods known in the art for identifying desired specificity or function. The assay may select on the basis of simple récognition of one or more antigens, or may select on the additional basis of a desired function e.g., to select neutralizing antibodies rather than just antigen-binding antibodies, to select antibodies that can change characteristics of targeted cells, such as their signaling cascades, their shape, their growth rate, their capability of influencing other cells, their response to the influence by other cells or by other reagents or by a change in conditions, their différentiation status, or the like.
individual transformed B cell clones may then be produced from the positive transformed B cell culture. The cloning step for separating individual clones from the mixture of positive cells may be carried out using limiting dilution, micromanipulation, single cell déposition by cell sorting or another method known in the art.
Nucleic acid from the cultured plasma cells can be isolated, cloned and expressed in HEK293T cells or other known host cells using methods known in the art.
The immortalized B cell clones or the transfected host-cells of described herein can be used in various ways e.g., as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest, for research, etc.
Inhibitors of HBV Protein Expression and Delivery Systems
The présent disclosure also provides inhibitors of HBV protein expression for use in combination therapy methods and compositions a for treating HBV, wherein the combination therapy comprises a binding protein as provided herein. In certain embodiments, the inhibitor of HBV gene expression is an RNAi agent. As used herein, the term RNA interférence agent or RNAi agent refers to an agent that contains RNA as that term is defined herein, and which médiates the targeted cleavage of an RNA transcrîpt via an RNA-induced silencing complex (RISC) pathway. In some embodiments, an RNAi agent as described herein effects inhibition of expression of an HBV gene.
In one aspect, an RNA interférence agent includes a single-stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound to a particular theory, long double-stranded RNA (dsRNA) introduced into plants and invertebrate cells is broken down into siRNA by a Type III endonuclease known as Dîcer (Sharp, et al., Genes Dev. 15:485 (2001)). Dicer, a rîbonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short iriterfering RNAs (siRNAs) with characteristic two base 3' overhangs (Bernstein, et al., Nature 409:363 (2001)). The siRNAs are then incorporated into an RNA-induced silencîng complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target récognition (Nykanen, et al., Cell 107:309 (2001)). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al, Genes Dev. 15:188 (2001)). Thus, in one aspect the technology described herein relates to a single stranded RNA that promûtes the formation of a RISC complex to effect silencing of the target gene.
The terms silence, inhibit the expression of, down-regulate the expression of, suppress the expression of, and the like, in so far as they refer to an HBV gene, herein refer to the at least partial réduction of the expression of an HBV gene, as manifested by a réduction of the amount of HBV mRNA which can be îsolated from or detected in a first cell or group of cells în which an HBV gene is transcribed and which has or hâve been treated with an inhibitor of HBV gene expression, such that the expression of the HBV gene is înhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or hâve not been so treated (control cells). The degree of inhibition can be measured, by exarnple, as the différence between the degree of mRNA expression in a control cell minus the degree of mRNA expression în a treated cell. Alternatively, the degree of inhibition can be given in terms of a réduction of a parameter that is functionally linked to HBV gene expression, e.g., the amount of protein encoded by an HBV gene, or the number of cells displaying a certain phenotype, e.g., an HBV infection phenotype such as HBV infection, HBV protein expression (such as hepatitis B surface antigen, HBsAg), or changes in cellular gene expression reflecting HBV gene expression (e.g., Smc5/6 expression and localization). The degree of inhibition may also be measured using a cell engîneered to express a reporter gene reflecting HBV RNA expression. In principle, HBV gene silencing can be determined in any cell expressing the HBV gene, e.g., an HBV-infected cell or a cell engîneered to express the HBV gene, and by any appropriate assay.
The level of HBV RNA that is expressed by a cell or group of cells, or the level of circulating HBV RNA, may be determined using any method known in the art for
assessîng mRNA expression, such as the rtPCR method provided in Example 2 of International Application Publication No. WO 2016/077321A1 and U.S. Patent Application No. US2017/0349900A1, which methods are incorporated herein by reference. In some embodiments, the level of expression of an HBV gene (e.g., total
HBV RNA, an HBV transcript, e.g., HBV 3.5 kb transcript) in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., RNA of the HBV gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA préparation kits (Qiagen®), or PAXgene (PreAnalytîx,
Switzerland). Typical assay formats utilizing rîbonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nue. Acids Res. 12:7035), northern blotting, in situ hybridization, and microarray analy sis.
Circulating HBV mRNA may be detected using methods the described in International Application Publication No. WO 2012/177906A1 and U.S. Patent Application No.
US2014/027521 1A1, which methods are incorporated herein by reference.
As used herein, target sequence refers to a contiguous portion of the nucleotîde sequence of an mRNA molécule formed during the transcription of an HBV gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucléotides in iength, e.g., 15-30 nucléotides in length, including ail sub-ranges there between. As non-limiting examples, the target sequence can be from 15-30 nucléotides, 15-26 nucléotides, 15-23 nucléotides, 15-22 nucléotides, 15-21 nucléotides, 15-20 nucléotides, 15-19 nucléotides, 15-18 nucléotides, 15-17 nucléotides, 18-30 nucléotides, 18-26 nucléotides, 18-23 nucléotides, 18-22 nucléotides, 18-21 nucléotides, 18-20 nucléotides, 19-30 nucléotides, 19-26 nucléotides, 19-23 nucléotides, 19-22 nucléotides, 19-21 nucléotides, 19-20 nucléotides, 20-30 nucléotides, 20-26 nucléotides, 20-25 nucléotides, 20- 24 nucléotides,20-23 nucléotides, 20-22 nucléotides, 20-21
nucléotides, 21-30 nucléotides, 21-26 nucléotides, 21-25 nucléotides, 21-24 nucléotides, 21-23 nucléotides, or 21- 22 nucléotides.
As used herein, the term strand comprising a sequence refers to an oligonucleotide comprising a chain of nucléotides that is described by the sequence 5 referred to using the standard nucléotide nomenclature.
As used herein, and unless otherwise îndicated, the term complementary, when used to describe a first nucléotide sequence in relation to a second nucléotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucléotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucléotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. Other conditions, such as physiologîcally relevant conditions as can be encountered inside an organîsm, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultîmate application of the hybridized nucléotides.
Complementary sequences within an RNAi agent, e.g., within an siRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucléotide sequence to an oligonucleotide or polynucleotide comprising a second nucléotide sequence over the entire length of one or both nucléotide sequences. Such sequences can be referred to as fuily complementary with respect to each other herein. However, where a first sequence is referred to as substantially complementary with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultîmate application, e.g., inhibition of gene expression via a RISC palhway.
However, where two oligonucleotides are designed to form, upon hybridization, one or 30 more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the détermination of complementanty. For example, an siRNA comprising one oligonucleotide 21 nucléotides in length, and another oligonucleotide 23 nucléotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucléotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as fully complementary for the purposes described herein.
Complementary sequences, as used herein, can also include, or be formed entirely from non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucléotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.
The ternis complementary, fully complementary, and substantially complementary herein can be used with respect to the base matching between the sense strand and the antisense strand of an siRNA, or between the antisense strand of an RNAi agent and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide that is substantially complementary to at least part of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding an HBV protein). For example, a polynucleotide is complementary to at least a part of an HBV mRNA if the sequence is substantially complementary to a noninterrupted portion of the HBV mRNA.
a, siRNAs
In some embodiments, the RNAi agent comprises an siRNA. The term siRNA, as used herein, refers to an RNAi that includes an RNA molécule or complex of molécules having a hybridized duplex région that comprises two anti-pai allel and substantially complementary nucleic acid strands, which will be referred to as having sense and antisense orientations with respect to a target RNA. The duplex région can be of any length that pennits spécifie dégradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11 ,12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, or 36 and any sub-range there between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18base pairs, 15- 17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-2 i base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, and 21-22 base pairs. siRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length. The term double-stranded RNA or dsRNA, is also used herein synonymously to refer to an siRNA as described above.
One strand of the duplex région of an siRNA comprises a sequence that is substantially complementary to a région of a target RNA. The two strands forming the duplex structure can be from a single RNA molécule having at least one selfcomplementary région, or can be formed from two or more separate RNA molécules. Where the duplex région is formed from two strands of a single molécule, the molécule can hâve a duplex région separated by a single stranded chain of nucléotides (herein referred to as a hairpin loop’1) between the 3'-end of one strand and the 5’-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucléotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucléotides. Where the two substantially complementary strands of an siRNA are comprised by separate RNA molécules, those molécules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a linker.
The term antisense strand or guide strand refers to the strand of an RNAi agent, e.g., an siRNA, which includes a région that is substantially complementary to a
target sequence. As used herein, the term région of complementarity refers to the région on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the région of complementarity is not fuily complementary to the target sequence, the mismatches can be in the internai or 5 terminal régions ofthe molécule.
Generally, the most tolerated mismatches are in the terminal régions, e.g., within 5, 4, 3, or 2 nucléotides of the 5' and/or 3' terminus.
The term sense strand or passenger strand as used herein, refers to the strand of an RNAi that includes a région that is substantially complementary to a région of the 10 antisense strand as that term is defined herein.
In another aspect, the agent is a single-stranded antisense RNA molécule. The antisense RNA molécule can hâve 15-30 nucléotides complementary to the target. For example, the antisense RNA molécule may hâve a sequence of at least 15, 16, 17, 18, 19, 20, 21, or more contiguous nucléotides from one of the antisense sequences 15 disclosed herein.
The skilled artisan will recognize that the term RNA molécule or ribonucleic acid molécule encompasses not only RNA molécules as expressed or found in nature, but also analogs and dérivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or dérivatives as described herein or as known in 20 the art. Strictly speakîng, a ribonucleoside includes a nucleoside base and a ribose sugar, and a ribonucleotide is a ribonucleoside with one, two or three phosphate moieties. However, the terms ribonucleoside and ribonucleotide can be considered to be équivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described in greater detail below.
However, siRNA molécules comprising ribonucleoside analogs or dérivatives retain the ability to form a duplex. As non-limiting examples, an RNA molécule can also include at least one modified ribonucleoside including but not limited to a 2'-O-methyl modified nucleoside, a nucleoside comprising a 5' phosphorothioate group, a terminal nucleoside linked to a cholesteryl dérivative or dodecanoîc acid bisdecylamide group, a 30 locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2’ amino-modified nucleosîde, 2'-alkyl-modîfied nucleoside, morpholino nucleoside, a phosphoramidate, or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molécule can comprise at least two modîfied ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, ai least 10, at least 15, at least 20, or more, up to the entire length ofthe siRNA molécule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molécule. In some embodiments, modîfied RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that hâve the abiiity to form the required duplex structure and that permit or médiate the spécifie dégradation of a target RNA via a RISC pathway.
In some embodiments, a modified ribonucleoside inciudes a deoxyribonucleoside. For example, an RNAÎ agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double-stranded portion of an siRNA. However, the term ”RNAi agent as used herein does not include a fuily DNA molécule.
As used herein, the term nucléotide overhang refers to at least one unpaired nucléotide that protrudes from the duplex structure of an RNAi agent, e.g., an siRNA. For example, when a 3’-end of one strand of an siRNA extends beyond the 5'-end of the other strand, or vice versa, there is a nucléotide overhang. An siRNA can comprise an overhang of at least one nucléotide; alternatively the overhang can comprise at least two nucléotides, at least three nucléotides, at least four nucléotides, at least five nucléotides, or more. A nucléotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be présent on the 5' end, 3' end, or both ends of either an antisense or sense strand of an siRNA.
In some embodiments, the antisense strand of an siRNA has a 1-10 nucléotide overhang at the 3’ end and/or the 5' end. In some embodiments, the sense strand of an siRNA has a 1-10 nucléotide overhang at the 3' end and/or the 5' end. In some other ιοο embodiments, one or more of the nucléotides in the overhang is replaced with a nucleoside thîophosphate.
In some embodiments, at least one end of an siRNA has a sîngle-stranded nucléotide overhang of l to 4, generally l or 2 nucléotides, siRNAs having at least one nucléotide overhang can hâve unexpectedly superior inhibitory properties relative to their blunt-ended counterparts.
The terms blunt or blunt ended as used herein in reference to an siRNA mean that there are no unpaired nucléotides or nucléotide analogs at a given terminal end of an siRNA, Le., no nucléotide overhang. One or both ends of an siRNA can be blunt. Where both ends of an siRNA are blunt, the siRNA is said to be blunt ended. A blunt ended siRNA is an siRNA that is blunt at both ends, i.e., has no nucléotide overhang at either end of the molécule. Most often such a molécule will be doublestranded over its entire length.
In certain embodiments, the combination therapy described herein includes one or more RNAi agents that inhibit the expression of the HBV gene. In some embodiments, the RNAi agent includes short interfering ribonucleic acid (siRNA) molécules for inhibiting the expression of an HBV gene in a rnammal, e.g., in an HBVinfected human, where the siRNA includes an antisense strand having a région of complementarity which is complementary to at least a part of an mRNA formed in the expression of an HBV gene, and where the région of complementarity is 30 nucléotides or less in length, generally 19-24 nucléotides in length, and where the siRNA, upon contact with a cell expressing the HBV gene, inhibits the expression ofthe HBV gene by at least 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of an HBV gene in cell culture or expression of a cellular gene as a surrogate for HBV gene expression (e.g., Smc5/6), such as in COS cells, HeLa cells, primary hépatocytes, HepG2 cells, primary cultured cells or in a biological sample from a subject, can be assayed by measuring HBV mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by immunofluorescence analysis, using, for example, Western Blotting or flow cytométrie techniques.
101
An siRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the siRNA will be used. One strand of an siRNA (the antisense strand) includes a région of complementarity that is substantiel ly complementary, and generally fully complementary, to a target sequence.
The target sequence can be derived from the sequence of an mRNA formed during the expression of an HBV gene. The other strand (the sense strand) includes a région that îs complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suîtable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, 10 yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the région of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucléotides in length, inclusive. In some embodiments, the siRNA is between 15 and 20 nucléotides in length, inclusive, and in other embodiments, the siRNA is between 25 and 30 nucléotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted région of an RNA targeted for cleavage will most often be part of a larger RNA molécule, often an mRNA molécule. Where relevant, a part'* of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for
RNAi-directed cleavage (i.e., cleavage through a RISC pathway). siRNAs having duplexes as short as 9 base pairs can, under some circumstances, médiate RNAidirected RNA cleavage. Most often a target will be at least 15 nucléotides in length. In certain embodiments, the target is 15-30 nucléotides in length.
One of skill in the art will also recognize that the duplex région is a primary functional portion of an siRNA, e.g., a duplex région of 9 to 36, e.g, 15-30 base pairs. Thus, in some embodiments, to the extent that it becomes processed to a functional duplex of e.g., 15- 30 base pairs that targets a desired RNA for cleavage, an RNA molécule or complex of RNA molécules having a duplex région greater than 30 base pairs is an siRNA. Thus, an ordinarily skilled artisan wîll recognize that in some embodiments, then, a miRNA is an siRNA. In some other embodiments, an siRNA is
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not a naturally occurring miRNA. In some embodiments, an RNAi agent useful to target expression of an HBV gene is not generated in the target cell by cleavage of a larger double-stranded RNA.
An siRNA as described herein can be synthesîzed by standard methods known 5 in the art, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
In some embodiments, the RNAi agent comprises an siRNA that targets and inhibits expression of an HBV mRNA. In some embodiments, the RNAi agent comprises an siRNA that targets and inhibits expression of an mRNA encoded by an 10 HBV genome according to NCBl Reference Sequence NC_003977.2 (GenBank Accession No. GI:21326584) (SEQ ID NO:116). Transcription ofthe HBV genome results în polycistronic, overlapping RNAs, and therefore, in some embodiments, an siRNA of the combination therapy targeting a single HBV gene may resuit in significant inhibition of expression of most or ail HBV transcripts. In some embodiments the mRNA target of the siRNA may be an mRNA encoded by: P gene, nucléotides 2309-3182 and 1-1625 of NC 003977.1; S gene (encodîng L, M, and S proteins), nucléotides 2850-3182 and 1-837 ofNC_003977; X protein, nucléotides 1376-1840 ofNC_003977; and/or C gene, nucléotides 1816-2454 ofNC_003977.
In some embodiments, the siRNA targets and inhibits expression of an mRNA 20 encoded by the X gene of HBV. In some embodiments, the RNAi agent or siRNA targets an mRNA encoded by a portion of the HBV genome comprising the sequence GTGTGCACTTCGCTTCAC (SEQ ID NO:117), which corresponds to nucléotides 1579-1597 of NC_003977.2 (GenBank Accession No. GI:21326584) (SEQ ID NO: 116).
In still further embodiments, the siRNA has a sense strand comprising 5'GUGUGCACUUCGCUUCACA -3' (SEQ ID NO: 118) and an antisense strand comprising 5'- UGUGAAGCGAAGUGCACACUU -3' (SEQ ID NO:119).
In certain embodiments, the inhibitor of HBV gene expression comprises an siRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 118, or a sequence that differs by not more than 4,,not more
103 than 3, not more than 2, or not more than l nucléotides from SEQ ID NO: 118; and wherein the antisense strand comprises SEQ ID NO:l 19, or a sequence thaï differs by not more than 4, not more than 3, not more than 2, or not more than l nucléotides from SEQ ID NO:119.
In one aspect, an sîRNA will include at least two nucléotide sequences, a sense and an antisense sequence, whereby: the sense sequence comprises SEQ ID NO: 118, and the corresponding antisense sequence comprises SEQ ID NO: 119. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an HBV gene. As such, in this aspect, an siRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described elsewhere herein and as known in the art, the complementary sequences of an siRNA can also be contained as self-complementary régions of a single nucleic acid molécule, as opposed to being on separate oligonucleotides.
In still further embodiments, the siRNA has a sense strand comprising 5'GGUGGACUUCUCUCAAUUUUA -3’ (SEQ ID NO: 120) and an antisense strand comprising 5 - UAAAAUUGAGAGAAGUCCACCAC -3' (SEQ ID NO: 121).
In certain embodiments, the inhibitor of HBV gene expression comprises an siRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 120, or a sequence that differs by not more than 4, not more than 3, not more than 2, or not more than 1 nucléotides from SEQ ID NO: 120; and wherein the antisense strand comprises SEQ ID NO: 121, or a sequence that differs by not more than 4, not more than 3, not more than 2, or not more than 1 nucléotides from SEQ IDNO:I21.
In one aspect, an siRNA will include at least two nucléotide sequences, a sense and an antisense sequence, whereby: the sense sequence comprises SEQ ID NO: 120, and the corresponding antisense sequence comprises SEQ ID NO:121. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one
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of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an HBV gene. As such, in this aspect, an siRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described elsewhere herein and as known in the art, the complementary sequences of an siRNA can also be contained as self-complementary régions of a single nucleic acid molécule, as opposed to being on separate oligonucleotides.
The skilled person is well aware that siRNAs having a duplex structure of 10 between 20 and 23, but specifically 21, base pairs hâve been hailed as particularly effective in inducing RNA interférence (Elbashir, et al., EMBO 20:6877-88 (2001)). However, others hâve found that shorter or longer RNA duplex structures can be effective as well. In the embodiments described above, siRNAs described herein can include at least one strand of a length of minimally 21 nucléotides. In some ] 5 embodiments, shorter duplexes having one of the sequences of SEQ ID NO: 118, SEQ ID NO;l 19, SEQ ID NO:l20, or SEQ ID NO:l2l minus only a few nucléotides on one or both ends are similarly effective as compared to the siRNAs described above. Hence, siRNAs having a partial sequence ofat least I5, 16, 17, 18, 19, 20, or more contîguous nucléotides from one or both of SEQ ID NO: 118 and SEQ ID NO: 119, and differing in their ability to inhibit the expression of an HBV gene by not more than 5, 10, 15, 20, 25, or 30 % inhibition from an siRNA comprising the full sequence, are contemplated according to the technology described herein. Also within the present disclosure are siRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contîguous nucléotides from one or both of SEQ ID NO: 120 and SEQ ID NO: 121, and differing in their ability to inhibit the expression of an HBV gene by not more than 5, 10, 15, 20, 25, or 30 % inhibition from an siRNA comprising the full sequence, are contemplated according to the technology described herein.
In addition, the siRNAs provided in herein identify a site in an HBV gene transcript that is susceptible to RISC-mediated cleavage. As such, the technology 30 described herein further features RNAi agents that target within one of such sequences.
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As used herein, an RNAi agent îs said to target within a particular site of an RNA transcri pt if the RNAi promûtes cleavage ofthe transcript any where within that particular site. In some embodiments, the RNAi agent includes at least 15 contiguous nucléotides from one or both of the sequences of SEQ ID NO: 118 and SEQ ID NO: 119, 5 coupled to additional nucléotide sequences taken from the région contiguous to the selected sequence in the FIBV gene. In some embodiments, the RNAi agent includes at least 15 contiguous nucléotides from one or both of the sequences of SEQ ID NO: 120 and SEQ IDNO:121, coupled to additional nucléotide sequences taken from the région contiguous to the selected sequence in the HBV gene.
While a target sequence is generally 15-30 nucléotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a window or mask of a 15 given size (as a non-limitîng example, 21 nucléotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence window progressively one nucieotîde upstream or downstream of an initiai target sequence location, the next potential target sequence can be identified, until the complété set of 20 possible sequences is identified for any gîven target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to îdentify those sequences that perform optimally can identify those RNA sequences that, when targeted with an RNAi agent, médiate the best inhibition of target gene expression. It is contemplated that further optimization of inhibition efficiency can be achieved by progressively walking the window one nucléotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
Further, it is contemplated that for any sequence identified, e.g., SEQ ID
NO: H 8, SEQ IDNO:119, SEQ ID NO:120, or SEQ IDNO:121, further optimization could be achieved by systematically either adding or removing nucléotides to generate
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longer or shorter sequences and testing those and sequences generated by w alking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of RNAi agents based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucléotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molécule (e.g., increasing sérum stabîlity or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.
An RNAi agent as described herein can contain one or more mismalches to the target sequence. In some embodiments, an RNAi agent as described herein contains no more than 3 mismatches. In some embodiments, if the antisense strand of the RNAi agent contains mismatches to a target sequence, the area of mismatch is not located in the center of the région of complementarity. In particular embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch is restricted to within the last 5 nucléotides from either the 5' or 3' end of the région of complementarity. For example, for a 23 nucléotide RNAi agent RNA strand which is complementary to a région of an HBV gene, the RNA strand may not contain any mismatch within the central 13 nucléotides. The methods described herein or methods known in the an can be used to déterminé whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of an HBV gene.
Considération of the efficacy of RNAi agents with mismatches in inhibiting expression of an HBV gene is important, especially if the particular région of complementarity în the HBV gene is known to hâve polymorphie sequence variation.
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b. Chemically modifiée! RNAi agents
In some embodiments, the RNA of an RNAi agent, e.g., an siRNA, is chemically modified to enhance stability or other bénéficiai characteristics. The nucleic acids featured in the technology described herein can be synthesized and/or modified by 5 methods well established in the art, such as those described in Current protocols in nucleic acid chemistry, Beaucage, S.L., et al. (Edrs.), John Wiley & Sons, Inc., New York., NY, USA, which methods are incorporated herein by reference.
Modifications include, for example, (a) end modifications, e.g., 5' end modifications (phosphorylation, conjugation, inverted linkages, etc.), 3' end 10 modifications (conjugation, DNA nucléotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilîzîng bases, or bases that base pair with an expanded répertoire of partners, removal of bases (abasic nucléotides), or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, as well as (d) backbone modifications, including modification 15 or replacement of the phosphodiester linkages. Spécifie examples of RNA compounds useful în the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not hâve a phosphorus atom in the backbone. For the purposes of this spécification, and as sometimes referenced in the 20 art, modified RNAs that do not hâve a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will hâve a phosphorus atom in its internucleoside backbone.
(t is not necessary for ail positions in a given compound to be uniformly modified, and in fact more than one of the afo rement ion ed modifications can be incorporated in a single compound or even at a single nucleoside within an RNAi agent. The technology described herein also includes RNAi agent compounds that are chimeric compounds. Chimeric RNAi agent compounds or chimeras, in the context of this disclosure, are RNAi agent compounds, such as siRNAs, which contain two or more chemically distinct régions, each made up of at least one monomer unit, i.e., a nucléotide in the case of an siRNA compound. These RNAi agents typically contain at
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least one région wherein the RNA is modified so as to confer upon the RNAi agent increased résistance to nuclease dégradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An addîtional région of the RNAi agent can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
By way of example, RNase H is a cellular endonucîease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of RNAi agent inhibition of gene expression. Consequently, comparable results can often be obtained with shorter RNAi agents when chimeric siRNAs are used, compared to phosphorothioate deoxy siRNAs
I0 hybridizing to the same target région. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, 15 methyl and other alkyl phosphonates includîng 3'-alkylene phosphonates and chiral phosphonates, pbosphinates, phosphoramidates includîng 3'-amino phosphoramidate and aminoalky [phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thîonoalkylphosphotriesters, and boranophosphates having normal 3'-5' lînkages, 2-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.
Représentative U.S. patents that teach the préparation of the above phosphoruscontaining linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;
5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111 ; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933;
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7,321,029; and US Pat RE39464; each of which is herein incorporated herein by reference.
Modified RNA backbones that do not include a phosphorus atom therein hâve backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclîc internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thiofonnacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having niixed N, O, S, and CH2 component parts.
Représentative U.S. patents that teach the préparation ofthe above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439; each of which is herein incorporated by reference for teachings relevant to such methods of préparation.
In other embodiments, suitable RNA mimetics suitable are contemplated for use in RNAi agents, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucléotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to hâve excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion ofthe backbone. Représentative U.S. patents that teach the préparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262;
HO each of which is încorporated herein by reference for teachings relevant to such methods of préparation. Further teachîng of PNA compounds can be found, for example, in Nielsen, et al. (Science, 254:1497- 1500 (1991)).
Some embodiments featured in the technology described herein include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2-[known as a methylene (methylimino) or MMI backbone], -CH2-O-N(CHa)-CH2-, -CH2-N(CH3)N(CHj)-CH2-, and -N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone is represented as -O-P-O-CH2-] of U.S. Pat. No. 5,489,677, and the amide backbones of U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein hâve morpholino backbone structures of U.S. Pat. No. 5,034,506.
Modified RNAs can also contain one or more substituted sugar moieties. The RNAi agents, e.g., siRNAs, featured herein can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or Oalkyl-O-alkyl; wherein the alkyl, alkenyl, and alkynyl can be substituted or unsubstituted Ci to Cio alkyl or C2 to Cio alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO] mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3. O(CH2)nONH2, and O(CH2)nON[(CH2)nCHî)]2, where n and m are from 1 to about 10. In other embodiments, siRNAs include one of the following at the 2' position: Ci to Cio lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH. SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted siiyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an RNAi agent, or a group for improving the pharmacodynamie properties of an RNAi agent, and other substituents having similar properties. In some embodiments, the modification includes a 2-methoxyethoxy (2’- OCH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or2'-MOE) (Martin, et al., Helv, Chim. Acta 78:486-504 (1995)), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'- dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2 O-dimethylaminoethoxyethyl or 2*-DMAEOE), i.e., 2*-O-CH2-O-CH2-N(CH2)2.
Other exemplary modifications include 2'-methoxy (2-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an RNAi agent, particularly the 3' position of the sugar on the 3' terminal nucléotide or in 2'-5' linked siRNAs and the 5' position ofthe 5' terminal nucléotide. RNAi agents can also hâve sugar mimetîcs such as cyclobutyl moieties in place of the pentofuranosyl sugar.
Représentative U.S. patents that teach the préparation of such modîfied sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920; each of which is incorporated herein by reference for teachings relevant to such methods of préparation.
An RNAi agent can also include nucleobase (often referred to in the art simply as base) modifications or substitutions. As used herein, unmodified or natural nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modîfied nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl dérivatives of adenine and guanine, 2-propyl and other alkyl dérivatives of adenine and guanine, 2-thiouracîl, 2-thiothymîne and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methy ladenine, 8azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenîne, and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modîfied Nucleosides in Biochemistry, Biotechnology and Medicine (Uerdewÿn, P. ed. Wiley-VCH, (2008)); those disclosed in The Concise
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Encyclopédie Of Polymer Science And Engineering (pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons (1990)), those disclosed by Englisch et al. (Angewandte Chemie, International Edition, 30, 613 (1991)), and those disclosed by Sanghvi, Y S. (Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press (1993)). Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the technology described herein. These include 5-substituted pyrimidines, 6-azapyrimîdines and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5propynyluracii and 5-propynylcytosine. 5-methylcytosine substitutions hâve been shown to increase nucleic acid duplex stability by 0.6-E2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, pp. 276-278 (1993)) and are exemplary base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.
Représentative U.S. patents that teachthe préparation of certain ofthe above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, U.S. Pat. No. 3,687,808; U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;
5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088; each of which is incorporated herein by reference for teachings relevant to such methods of préparation.
The RNA of an RNAi agent can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid îs a nucléotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively locks the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in sérum, and to reduce off-target effects (Elmen, J., et al., Nucleic Acids Research 33(1):439-47 (2005); Mook, O.R., et al., Mol Cane Ther
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6(3):833-43 (2007); Grunweller, A., et al, Nucleic Acids Research 31(12):3185-93 (2003)).
Représentative U.S. Patents that teach the préparation of locked nucleic acid nucléotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490;
6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845; each of which is incorporated herein by reference for teachings relevant to such methods of préparation.
In certain embodiments, the combination therapy inciudes an siRNA that is modified to include one or more adenosine-glycol nucleic acid (GNA). A description 10 of adenosine-GNA can be found, for example, in Zhang, et al. (JACS 127(12):4174-75 (2005)).
In some embodiments, the présent disclosure provides methods and related compositions, wherein the RNAi is an siRNA comprising an oligonucleotide sequence having one or more modified nucléotides. Abbreviations for nucléotide monomers in 15 modified nucleic acid sequences as used herein are provided in Table 5.
T able 5. Abbreviations of nucléotide monomers used in modified nucleic acid sequence représentation. It will be understood that, unless otherwise indicated, these monomers, when présent in an oligonucleotide, are mutually linked by 5'-3'20 phosphodiester bonds.
| Abbreviation | Nucleotide(s) |
| A | adenosine-3'-phosphate |
| Af | 2'-fluoroadenosine-3'-phosphate |
| Ais | 2’-fluoroadenosine-3'-phosphorothioate |
| As | adenosine-3'-phosphorothioate |
| C | cytidine-3'-phosphate |
| Cf | 2'-fluorocytidine-3'-phosphate |
| Cfs | 2'-fluorocytidme-3'-phosphorothioate |
| Cs | cytidine-3'-phosphorothioate |
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| Abbreviation | Nucleotide(s) |
| G | guanosine-3'-phosphate |
| Gf | 2'-fluoroguanosine-3'-phosphate |
| Gfs | 2'-fluoroguanosine-3'-phosphorothioate |
| Gs | guanosine-3'-phosphorothîoate |
| T | 5'-m ethyl uridine-3'-phosphate |
| Tf | 2'-tluoro-5-methyluridine-3'-phosphate |
| Tfs | 2’-fluoro-5-methy luridine-3'-phosphorothioate |
| Ts | 5-methyluridine-3'-phosphorothioate |
| U | uridine-3'-phosphate |
| Uf | 2'-fluorouridine-3'-phosphate |
| Ufs | 2'-fluorouridine -3'-phosphorothioate |
| Us | urîdine -3'-phosphorothioate |
| a | 2'-O-methyladenosine-3'-phosphate |
| as | 2'-O-methyladenosine-3'- phosphorothioate |
| c | 2'-O-methylcytidine-3'-phosphate |
| CS | 2'-O-methylcytidine-3'- phosphorothioate |
| g | 2'-0-methylguanosine-3'-phosphate |
| gs | 2'-O-methylguanosîne-3'- phosphorothioate |
| t | 2'-O-methyl-5-methy luridine-3'-phosphate |
| ts | 2'-O-methyl-5-methyluridine-3’-phosphorothioate |
| u | 2'-O-methyluridine-3'-phosphate |
| us | 2'-O-methyluridine-3'-phosphorothioate |
| S | phosphorothioate linkage |
| L96 | N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (also referred to as Hyp-(GalNAc-alkyl)3) |
| (Agn) | adenosine-glycol nucleic acid (GNA) |
| dA | 2'-deoxyadenosîne-3'-phosphate |
| dAs | 2'-deoxyadenosine-3'-phosphorothioate |
| dC | 2'-deoxycytîdine-3'-phosphate |
115
| Abbreviation | Nucleotide(s) |
| dCs | 2'-deoxycytidine-3'-phosphorothioate |
| dG | 2'-deoxyguanosine-3'-phosphate |
| dGs | 2'-deoxyguanosine-3'-phosphorothioate |
| dT | 2*-deoxythyniidine-3'-phosphate |
| dis | 2'-deoxythymidine-3'-phosphorothioate |
| dU | 2'-deoxyurîdine |
| dUs | 2'-deoxyuridîne-3'-phosphorothioate |
In some embodiments, the inhibitor of HBV gene expression comprises an siRNA, whereinthe siRNA has a sense strand comprising 5'gsusguGfcAfCfUfucgcuucacaL96 -3' (SEQ ÏDNO:l22) and an antisense strand comprising 5'- usGfsugaAfgCfGfaaguGfcAfcacsusu -3' (SEQ ID NO:l23).
In still further embodiments, the siRNA has a sense strand comprising 5'gsusguGfcAfCfUfucgcuucacaL96-3’ (SEQ ID NO:l24) and an antisense strand comprising 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO:i25).
In certain embodiments, the inhibitor of HBV gene expression comprises an siRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO:l22 or SEQ ID NO:l24, or a sequence that dîffers by not more than 4, not more than 3, not more than 2, or not more than l nucléotide from SEQ ID NO:I22 or SEQ ID NO;I24, respectîvely.
In certain embodiments, the inhibitor of HBV gene expression comprises an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises SEQ ID NO: 123 or SEQ ID NO:l25, or a sequence that differs by not more than 4, not more than 3, not more than 2, or not more than 1 nucléotide from SEQ ID NO: 123 or SEQ ID NO: 125, respectîvely.
In some embodiments, the inhibitor of HBV gene expression comprises an siRNA, whereinthe siRNA has a sense strand comprising 5'gsgsuggaCfuUfCfUfcucaAfUfuuuaL96-3’ (SEQ ID NO: 126) and an antisense strand comprising 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 127).
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In certain embodiments, the inhibitor of HBV gene expression comprises an siRNA comprising a sense strand and an antîsense strand, wherein the sense strand comprises SEQ ID NO: 126, or a sequence that differs by not more than 4, not more than 3, not more than 2, or not more than l nucléotide from SEQ ID NO:l26.
c. Ligand-conjugated RNAi agents
In some embodiments, the RNAi agent includes modifications involving chemically linking to the RNA one or more ligands, moieties, or conjugales that enhance the activity, cellular distribution, or cellular uptake ofthe RNAi agent. Such moieties include but are not limited to Hpid moieties such as a cholestérol moiety (Letsinger, et al., Proc. Natl. Acid. Scî. USA 86:6553-56 (1989)), cholic acid (Manoharan, et al., Biorg. Med. Chem. Let. 4:1053-60 (1994)), athioether, e.g., berylS-tritylthiol (Manoharan, et al., Ann. N.Y. Acad. Sci. 660:306-9 (1992); Manoharan, et al., Biorg. Med. Chem. Let. 3:2765-70 (1993)), a thiocholesterol (Oberhauser, et al.,
Nucl. Acids Res. 20:533-38 (1992)), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras, et al., EMBO J 10:1111-18 (1991); Kabanov, et al., FEBS Lett. 259:327-30 (1990); Svinarchuk, et al., Biochimie 75:49-54 (1993)), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-dî-O-hexadecylrac-glycero-3-phosphonate (Manoharan, et al., Tetrahedron Lett. 36:3651-54 (1995);
Shea, et al., Nucl. Acids Res. 18:3777-83 (1990)), a polyamine or a polyethylene glycol chain (Manoharan, et al., Nucleosides & Nucléotides 14:969- 73 (1995)), or adamantane acetic acid (Manoharan, et al., Tetrahedron Lett. 36:3651-54 (1995)), a palmityl moiety (Mishra, et al., Biochim. Biophys. Acta 1264:229-37 (1995)), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke, et al., J.
Pharmacol. Exp. Ther. 277:923-37 ( 1996)).
In some embodiments, a ligand alters the distribution, targetîng, or lifetîme of an RNAi agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molécule, cell, or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ, or région of the body, as, e.g., compared to a species absent such a ligand. In such embodiments, the ligands will not take part in duplex pairing in a duplexed nucleic acid.
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Ligands can include a naturally occurring substance, such as a protein (e.g., human sérum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrîn, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molécule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly Lglutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, dîvinyl ether-maleic anhydride copolymer, N-(2hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyuréthane, poly(2-ethylacryllic acid), Nisopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamîne, pseudopeptidepolyamine, peptîdomimetic polyamîne, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary sait of a polyamine, or an alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a liver cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin. bisphosphonate, polyglutamate, polyaspartate, a lipid, cholestérol, a steroid, bile acid, folate, vitamin B12, vîtamin A, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g., acridines), cross- linkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazîne, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilie molécules (e.g., cholestérol, cholic acid, adamantane acetic acid, i-pyrene butyric acid, dihydrotestosterone, 1,3-BisO(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeoi, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid.
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03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), peptide conjugales (e.g., antennapedîa peptide, Tat peptide), alkylatîng agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEGJ2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folie acid), synthetic ribonucleases (e.g., îmîdazole, bisimidazole, histamine, irnidazole clusters, acridine-imidazole conjugales, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, and AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molécules having a spécifie affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectîns, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetylgalactosamine, N-acetyl- gulucosamine multivalent mannose, and multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.
The ligand can be a substance, e.g., a drug, which can increase the uptake of the RNAi agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, Vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g, a liver cell. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folie acid, Bl2, riboilavin, biotin, pyridoxal, or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).
In some embodiments, a ligand attached to an RNAi agent as described herein acts as a pharmacokinetic (PK) modulator. As used herein, a PK modulator refers to a pharmacokinetic modulator. PK modulators include lipophîles, bile acids, steroids, phospholipîd analogues, peptides, protein binding agents, PEG, vitamins, etc,
Exemplary PK modulators include, but are not limited to, cholestérol, fatty acids, cholic
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acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, bîotin, etc. Oligonucleotides that comprise a number of phosphorothioate iinkages are also known to bind to sérum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, comprising multiple of phosphorothioate Iinkages in the backbone are also amenable to the technoiogy described herein as ligands (e.g., as PK modulating ligands). In addition, aptamers that bind sérum components (e.g., sérum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.
(i) Lipid conjugales. In some embodiments, the ligand or conjugate is a lipid or 10 lipid-based molécule. A lipid or lipid-based ligand can (a) increase résistance to dégradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a sérum protein, e.g., HSA. Such a lipid or lipid-based molécule may bind a sérum protein, e.g., human sérum albumin (HSA). An HSA-binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molécules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used.
A lipid based ligand can be used to inhibit, e.g., control the binding ofthe conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA 20 more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
In some embodiments, the lipid based ligand binds HSA. The lipid based ligand may bind to HSA with a sufficient affinity such that the conjugate will be distributed to 25 a non-kidney tissue. In certain particular embodiments, the HSA-lîgand binding is réversible.
In some other embodiments, the lipid based ligand binds HSA weakly or not at ail, such that the conjugate will be distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
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(ii) Cell Perméation Peptide and Agents. In another aspect, the ligand is a cellpermeation agent, such as a helical cell-permeation agent. In some embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, învertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. In some embodiments, the helical agent is an aipha-helical agent. In certain particular embodiments, the helical agent has a lipophilie and a lipophobic phase.
A cell perméation peptide is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A 10 microbial cell-permeating peptide can be, for example, an alpha-helical linear peptide (e.g., LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g., a-defensin, βdefensin, or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin).
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred 15 to herein as an oligopeptidomimetic) is a molécule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to RNAÎ agents can affect phamiacokînetic distribution ofthe RNAi, such as by enhancing cellular récognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 20 25, 30, 35, 40, 45, or 50 amino acids long.
A peptide or peptidomimetic can be, for example, a cell perméation peptide, cationic peptide, amphipathic peptide, or hydrophobie peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constraîned peptide or crosslinked peptide. In another alternative, the peptide moiety 25 can include a hydrophobie membrane translocation sequence (MTS). An exemplary hydrophobie MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:l28). An RFGF analogue (e.g, amino acid sequence AALLPVLLAAP (SEQ ID NO:l29) containing a hydrophobie MTS can also be a targeting moiety. The peptide moiety can be a delivery peptide, which can carry 30 large polar molécules including peptides, oligonucleotides, and proteins across cell
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membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:l30) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWK (SEQ ID N0:131) hâve been found to be capable of functioning as deltverj peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one- compound (OBOC) combinatorial library (Lam, et al., Nature 354:82-84 (1991)).
A cell perméation peptide can also include a nuclear localization signal (NLS). For example, a cell perméation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV- 1 gp41 and the NLS of
SV40 large T antigen (Sîmeoni, et al., Nucl. Acids Res. 31:2717-24 (2003)).
(iii) Carbohydrate Conjugates. In some embodiments, the RNAi agent oligonucleotides described herein further comprise carbohydrate conjugates. The carbohydrate conjugates may be advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use. As used herein, carbohydrate refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up oi'one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Représentative carbohydrates include the sugars (mono-, di-, tri-, and oligosaccharides containing from about 4-9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose, and polysaccharide gums. Spécifie monosaccharides include C5 and above (în some embodiments, C5-C8) sugars; and di- and trisaccharides include sugars having two or three monosaccharide units (in some embodiments, C5-C8).
In some embodiments, the carbohydrate conjugate is selected from the group consisting of:
122
Formula III,
123
Formula V,
Formula VI,
Formula VIII,
124
Formula IX,
ΙΟ
126
Formula XVIII,
Formula XIX,
I0
127
Another représentative carbohydrate conjugale for use in the embodiments described herein includes, but is not limited to,
(Formula XXII), wherein when one of X or Y is an oligonucleotide, the other is a hydrogen.
In some embodiments, the carbohydrate conjugate further comprises another ligand such as, but not limited to, a PK modulator, an endosomolytïc ligand, or a cell I0 perméation peptide.
(iv) Linkers. In some embodiments, the conjugales described herein can be attached to the RNAi agent oligonucleotide with various linkers that can be cleavable or non-cleavable.
The terni linker or linking group means an organic moiety that connects two 15 parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or a chain of aïoms, such as, but not limited to, substituted or unsubstîtuted alkyl, substituted or unsubstîtuted alkenyl, substituted or unsubstîtuted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroaryialkynyl, heterocyclylalkyl, 20 heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, aikynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl,
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alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroary lalkyny l, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroary lalkyny I, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenyl heterocyclylalkyl, alkenylheterocyclylalkenyl, 5 alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroary!, alkenylheteroaryl, and alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclîc; where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In certain embodiments, the linker îs between I-24 atoms, between 4-24 atoms, between 6-l 8 atoms, between 8-18 atoms, or between 8-16 atoms.
A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In certain embodiments, the cleavable linkîng group is cleaved at least 10 times, or at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or sérum).
Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molécules. Generally, cleavage agents are more prévalent or found at higher levels or activities inside cells than in sérum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which hâve no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, présent in cells, that can dégradé a redox cleavable linkîng group by réduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that resuit in a pH of five or lower; enzymes that can hydrolyze or dégradé an acid cleavable linking group by acting as a general acid, peptîdases (which can be substrate spécifie), and phosphatases. A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of
129
human sérum is 7.4, while the average întracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes hâve a more acidic pH, in the range of 5.5-6.Û, and lysosomes hâve an even more acidic pH at around 5.0. Some linkers will hâve a cleavable linking group that is cleaved at a particular pH, thereby releasing the cationic 5 lipid from the ligand inside the cell, or into the desired compartment of the cell.
A linker can include a cleavable linking group that îs cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can dépend on the cell to be targeted. For example, liver-targeting ligands can be linked to the cationic lipids through a linker that inciudes an ester group. Lîver cells are rich in esterases, and 10 therefore the linker will be cleaved more efïîciently in iiver cells than in cell types that are not esterase-rîch. Other cell types rich in esterases include cells of the long, rénal cortex, and testis.
Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synovîocytes.
In general, the suitability of a candidate cleavable linking group can be evaluated by testing the abilîty of a degradative agent (or condition) to cleave the candidate linking group. It can be désirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other nontarget tissue. Thus one can détermine the relative susceptibilîty to cleavage between a 20 first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fiuids, e.g., blood or sérum. The évaluations can be carried out in cell-free Systems, in cells, in cell culture, in organ or tissue culture, or in whole animais. It can be useful to make initial évaluations in cell-free or culture conditions and to confinn by further évaluations in whole animais. In certain embodiments, useful candidate compounds are cleaved at least 2, at least 4, at least 10 or at least i00 times faster in the cell (or under in vitro conditions selected to mimic întracellular conditions) as compared to blood or sérum (or under in vitro conditions selected to mimic extracellular conditions).
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One class of cleavable linking groups are redox cleavable linking groups that are cleaved upon réduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (-S-S-). To détermine if a candidate cleavable linking group is a suîtable reductively cleavable linking group, or for example is suîtable for use with a particular RNAi moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or sérum conditions. In some embodiments, candidate compounds are cleaved by at most 10% in the blood. in certain embodiments, useful candidate compounds are degraded at least 2, at least 4, at least 10, or at least 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extrace llular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
Phosphate-based cleavable linking groups are cleaved by agents that dégradé or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in ceils are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -SP(O)(ORk)-O-, -O- P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)O-, -O-P(O)(Rk)-O-, -O- P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)S-, -O-P(S)( Rk)-S-. In certain embodiments, the phosphate-based linking groups are selected from: -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, O- P(0)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, O- P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, and -O-P(S)(H)-S-. In particular embodiments, the phosphate-linking group is -O-P(O)(OH)-O-, These candidates can be evaluated using methods analogous to those described above.
I3l
Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In some embodiments, acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g,, about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, spécifie low pH organelles, such as endosomes and lysosomes, can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can hâve the general formula -C=N-, C(O)O, or -OC(O). In some embodiments, the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.
Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups hâve the general formula -C(O)O-, or -OC(O)-. These candidates can be evaluated using methods analogous to those described above.
Peptide-based cleavable linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dîpeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a spécial type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptidebased cleavable linking groups hâve the general formula NHCHRAC(O)NHCHRBC(O)- , where RA and RB are the R groups ofthe two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
132
Représentative carbohydrate conjugales with linkers include, but are not limited to.
(Formula XXIII)
(Formula XXIV),
(Formula XXVI),
I33
(Formula XXVII),
(Formula XXXVIII),
(Formula XXIX), and
(Formula XXX),
134
wherein when one of X or Y is an oligonucleotide, the other is a hydrogen.
In certain embodiments of the compositions and methods, a ligand is one or more GalNAc (N-acetylgalactosamine) dérivatives attached through a bivalent or trivalent branched linker. For example, in some embodiments the siRNA is conjugated to a GalNAc ligand as shown in the following structure:
wherein X îs O or S.
In some embodiments, the sense strand of the siRNA is conjugated to a ligand attached at the 3' terminus of the sense strand through a linker as shown in the following structure:
wherein X is O or S.
135
In some embodiments, the combination therapy includes an siRNA that is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI) — (XXXIV):
q2A_r2A γ2Α_|_2Α q2A
-|-2B_|_2B ^p3A_Q3A_R3A
WX, N ^^P^-Q^-R38 γ3Α_|_3Α q3A γ3Β [_3B q3B
Formula XXXI
Formula XXXII
p4A^Q4A_R4A p4B_Q4B_j^4B y4A_|_4A q4A
-----T4B-L4B q4B
Formula XXXIIi, or
Formula XXXIV;
wherein:
q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;
p2A p2B p3A p3B p4A p4B p5A p5B p5C y2A y2B y3A y3B y4A y4B y4A y5B and are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O;
Q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, anc[ Q>C are in(jepenc[ent|y for each occurrence absent, alkylene, or substituted alkylene wherin one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(Rn), C(R')=C(R”), CC or C(O);
136
R.2a, R2b, R3A, R3B, R4a, R , R5a, R , and R5C are each independently for each occurrence absent, NH, O$S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), -C(O)-CH(Ra)S—S.
S—S.
S—S.
or heterocyclyl;
L2A, L2B, L3a, L3B, L4A, L4B, L^, L5B, and L5C represent the ligand; i.e., each independently for each occurrence a monosaccharide (such as GalNAc), disaccharîde. trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra is H or amino acid side chain. Tri valent conjugating GalNAc dérivatives are particuiarly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXIV):
Formula XXXIV wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc dérivative.
Examples of suitable bivalent and tri valent branched linker groups conjugating GalNAc dérivatives include, but are not limited to, the structures recited above as formulas I, VI, X, IX, and XII.
Représentative U.S. patents that teach the préparation of RNA conjugales include U.S. Pat. Nos. 4,828,979; 4,948,882; 5,2 i 8, i 05; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,1 18,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941 ; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,5 12,667;
137
5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371 ; 5,595,726;
5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752;
6,783,931; 6,900,297; and 7,037,646; each of which is incorporated herein by reference for leachings relevant to such methods of préparation.
In certain instances, the RNA of an RNAi agent can be modified by a non- ligand group. A number of non-ligand molécules hâve been conjugated to RNAi agents in order to enhance the activity, cellular distribution or cellular uptake of the RNAi agents, and procedures for performing such conjugations are available în the scientific literature. Such non-ligand moieties hâve included lipid moieties, such as cholestérol (Kubo, T., et al., Biochem. Biophys. Res. Comm. 365(1):54-61 (2007); Letsinger, et al., Proc. Natl. Acad. Sci. USA 86:6553 (1989)), cholic acid (Manoharan, et al., Bioorg. Med. Chem. Lett. 4:1053 (1994)), a thioether, e.g., hexyl-S-trityIthiol (Manoharan, et al., Ann. N.Y. Acad. Sci. 660:306 (1992); Manoharan, et al., Bioorg. Med. Chem. Let. 3:2765 (1993)), a thiocholesterol (Oberhauser, et al., Nucl. Acids Res. 20:533 (1992)), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras, et al., EMBO J. 10:111 (1991); Kabanov, et al., FEBS Lett. 259:327 (1990); Svinarchuk, et al., Biochimie 75:49 (1993)), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan, et al., Tetrahedron Lett. 36:3651 (1995); Shea, et al., Nucl. Acids Res. 18:3777 (1990)), a polyamine or a polyethylene glycol chain (Manoharan, et al., Nucleosides & Nucléotides 14:969 (1995)), or adamantane acetic acid (Manoharan, et al., Tetrahedron Lett. 36:3651 (1 195)), a palmityl moiety (Mishra, et al., Biochim. Biophys. Acta 1264:229 (1995)), or an octadecy lamine or hexylamino-carbonyl-oxycholestérol moiety (Crooke, et al., J. Pharmacol. Exp. Ther. 277:923 (1996)).
Typical conjugation protocols involve the synthesis of an RNAs bearing an aminoiinker at one or more positions of the sequence. The amino group is then reacted with the molécule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification ofthe RNA conjugale by HPLC typically affords the pure conjugale.
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d. RNAi agent delivery
Introducing into a cell, when referring to an RNAi agent, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art.
Absorption or uptake of an RNAi agent can occur through unaided diffusive or active cellular processes, or by auxilîary agents or devices. The meaning of this term is not limited to cells in vitro-, an RNAi agent can also be introduced into a cell, wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, an RNAi agent can be injected into a tissue site or adminîstered systemically. In vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are incorporated herein by reference for teachings relevant to such delivery Systems. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are know n in the art.
The delivery of an RNAi agent to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an RNAi agent, e.g., an siRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the RNAi agent. These alternatives are discussed further below.
In general, any method of delivering a nucleic acid molécule can be adapted for use with an RNAi agent (see, e.g, Akhtar S. and Julian RL., Trends Cell. Biol. 2(5):139-44 (1992) and WO94/02595, which are incorporated herein by reference for teachings relevant to such methods of delivery). Three factors that are particularly important in successfully delivering an RNAi agent in vivo: (a) biological stability of the delivered molécule, (2) preventing nonspecific effects, and (3) accumulation of the delîvered molécule tn the target tissue. The nonspecific effects of an RNAi agent can be minimized by local administration, for example, by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the préparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure ofthe agent to systemic tissues that can otherwise be harmed by the
139 agent or that can dégradé the agent, and permits a lower total dose of the RNAi agent to be administered. Several studies hâve shown successful knockdown of gene products when an RNAi agent is administered locally. For example, intraocular delivery of a VEGF siRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M.J., et al.,
Retina 24:132-38 (2004)) and subretinal injections in mice (Reich, SJ., et al., Mol. Vis. 9:210-16 (2003)) were both shown to prevent neovascularization in an experimental model of age-related macular degeneratîon. In addition, direct intratumoral injection of an sîRNA in mice reduces tumor volume (Pille, J., et al,. Mol. Ther. 11:267-74 (2005)) and can prolong survival of tumor-bearîng mice (Kim, W.J., et al., Mol. Ther. 14:343-
50 (2006); Li, S., étal., Mol. Ther. 15:515-23 (2007)). RNA interférence has also shown success with local delivery to the CNS by direct injection (Dom, G., et al., Nucleic Acids 32:e49 (2004); Tan, P.H., et al., Gene Ther. 12:59-66 (2005); Makimura, H., étal., BMC Neurosci. 3:18 (2002); Shishkina, G.T., et al., Neuroscience 129:521-28 (2004); Thakker, E.R., et al. Proc. Natl. Acad. Sci. U.S.A. 101:17270-75 (2004);
Akaneya,Y., et al., J. Neurophysiol. 93:594- 602 (2005)) and to the iungs by intranasal administration (Howard, K.A., et al.. Mol. Ther. 14:476-84 (2006); Zhang, X., et al., J. Biol. Chem. 279:10677-84(2004); Bitko, V., et al., Nat. Med. 11:50-55 (2005)). For administering an RNAi agent systemicaliy for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid dégradation of the siRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the RNAi agent composition to the target tissue and avoid undesirable off-target effects. RNAi agents can be modified by Chemical conjugation to lipophilie groups such as cholestérol to enhance cellular uptake and prevent dégradation. For example, an RNAi agent directed against ApoB conjugated to a lipophilie cholestérol moiety was injected systemicaliy into mice and resulted in knockdown of apoB mRNA in both the liver and jéjunum (Soutschek, J., et al., Nature 432:173-78 (2004)). In some other embodiments, the RNAi agent can be delivered using drug delivery Systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Posîtively charged cationic delivery Systems typically facilitate binding of an RNAi agent (négatively
140 charged) and enhance interactions at the negatively charged cell membrane to permit efficient uptake of an RNAi agent by the cell. Cationic lipîds, dendrimers, or polymers can either be bound to an RNAi, or induced to form a vesicle or micelle (see, e.g., Kim, S,H., et al., Journal of Controlled Release 129(2):107-16 (2008)) that encases an RNAi agent. The formation of vesicles or micelles further prevents dégradation ofthe RNAi agent when administered systemically. Methods for making and administering cationicRNAi agent complexes are well within the abilîties of one skilled in the art (see, e.g., Sorensen, D.R., étal., J. Mol. Biol 327:761-66 (2003); Verma, U.N., et al., Clin. Cancer Res. 9:1291-1300 (2003); Arnold, A.S. et al., J. Hypertens. 25:197-205 (2007); which methods are incorporated herein by reference). Some non-limiting examples of drug delivery Systems useful for systemic delivery of RNAi agents include DOT AP (Sorensen, D.R., et al. (2003), supra; Verma, U.N., et al., (2003), supra), Oligofectamine, solid nucleic acid iipid particles (Zimmermann, T.S., et al., Nature 441:111-14 (2006)), cardiolipin (Chien, P.Y., et al., Cancer Gene Ther. 12:321-28 (2005); Pal, A., et al., Int J. Oncol. 26: 1087-91 (2005)), polyethylenimine (Bonnet, M.E., et aL, Pharm. Res. 25(12):2972-82; Aigner, A., J. Biomed. BîotechnoL 2006(4):71659 (2006)), Arg-Gly-Asp (RGD) peptides (Liu, S., Mol. Pharm. 3:472-487 (2006)), and polyamidoamines (Tomalia, D.A., et aL, Biochem. Soc. Trans. 35:61-7 (2007); Yoo, H., et aL, Pharm. Res. 16:1799-1804 (1999)).
As used herein, the term SNALP refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipîds coating a reduced aqueous interior comprising a nucleic acid such as an RNAi agent or a plasmid from which an RNAi agent is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. US2006/0240093 and US2007/0135372, and in International Application Publication No. WO 2009/082817. These applications are incorporated herein by reference for teachings relevant to SNALPs.
In some embodiments, an RNAi forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of RNAis and cyclodextrins can be found in U.S. Pat. No. 7, 427, 605, which is incorporated herein by reference for teachings relevant to such compositions and methods. In some
141 embodiments, a gene encoding an RNAi is encoded and expressed from an expression vector. Examples of vectors and their use in deliverying RNAis are described in U.S. Patent Application No. US2017/0349900A1, which examples are incorporated herein by reference.
e. Pharmaceutical Compositions and Formulation of RNAi agents
In some embodiments, provided herein are pharmaceutical compositions containing an RNAi agent, as described herein, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition containing the RNAi agent is useful în a combination therapy to treat HBV infection or reduce HBV viral load in a subject. Such pharmaceutical compositions are formulated based on the mode of delîvery. For example, compositions emay be formulated for systemic administration via parentéral delîvery, e.g., by intravenous (IV) delîvery, or for direct delîvery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.
In some contexts, a pharmaceutically acceptable carrier or excipient is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delîvering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typica! pharmaceutically acceptable carriers or excipients include, but are not limited to, binding agents (e.g., pregelatînized maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate); lubricants (e.g., magnésium stéarate, talc, silica, colloïdal Silicon dioxide, stearic acid, metallic stéarates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulphate).
In some embodiments, pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react
142 with nucleic acids can also be used to formulate the compositions of the présent disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, sait solutions, alcohols, polyethylene glycols, gelatîn, lactose, amylose, magnésium stéarate, talc, silicic acid, viscous paraffm, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.
In certain contexts, formulations for topical administration of nucleic acids can include stérile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents,and other suitable additives. Pharmaceutically acceptable organic or înorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, sait solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnésium stéarate, talc, silicic acid, viscous paraffm, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.
In some embodiments, the pharmaceutical compositions containing an RNAi agent described herein are administered in dosages sufficient to inhibit expression of an HBV gene. In general, a suitable dose of an RNAi agent will be in the range of 0.001 to 200.0 milligrams per kilogram body weight of the récipient per day, and more typicaliy in the range of l to 50 mg per kilogram body weight per day. For example, an siRNA can be administered at 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, l mg/kg, l .5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose. The pharmaceutical composition can be administered once daily, or the RNAi agent can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the RNAi agent contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unît can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the RNAi over a several day period. Sustained release formulations are well known in the art and are particularly
143
useful for delivery of agents at a particular site, such as could be used with the agents of the technology described herein. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The effect of a single dose on the level of expression of an HBV gene can be long-lasting, such that subséquent doses are admînistered at not more than 3, 4, or 5 day intervals, or at not more than i, 2, 3, or 4 week intervals.
The skilled artisan will appreciate that certain factors can influence rhe dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or âge of the subject, and other diseases présent. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a sériés of treatments. Estimâtes of effective dosages and in vivo half-lives for the individual RNAi agents encompassed by the technology described herein can be made using conventional méthodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Mouse models are available for the study of HBV infection, and such models can be used for in vivo testing of RNAi, as well as for determining a dose that is effective at reducing HBV gene expression.
In some embodiments, administration of pharmaceutical compositions and formulations described herein can be topical (e.g., by a transdermal patch), pulmonary (e.g., by inhalation or insufflation of powders or aérosols, including by nebulizer); intratracheal; intranasal; epidermal and transdermal; oral; or parentéral. Parentéral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, and intramuscular injection or infusion; subdermal administration (e.g., via an implanted device); or intracranial administration (e.g., by intraparenchymal, intrathecal, or întraventricular, administration).
In certain embodiments, an RNAi agent used in a combination therapy for treating HBV as disclosed herein is delivered subcutaneously.
In some embodiments, RNAi agents can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hépatocytes of the liver).
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Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like can be necessary or désirable. Coated condoms, gloves, and the like can also be useful. Suitable topical formulations include those in which the RNAis featured in the technology described herein are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dîoleoylphosphatidyl DOPE ethanolamîne, dimyristoylphosphatidyl choline DMPC, dîstearolyphosphatidyl choline), négative (e.g, dimyristoylphosphatîdyl glycerol DM PG), and cationic (e.g., dioîeoyltetramethylaminopropyl DOTAP and dîoleoylphosphatidyl ethanolamîne DOTMA). RNAi agents can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, RNAi agents can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleîc acid, eicosanoic acid, laurîc acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, trîcaprate, monoolein, dilaurin, glyceryl l-monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a Ci.20alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, dîglyceride, or pharmaceutically acceptable sait thereof. Examples of topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference for teachings relevant to such topical formulations.
Vesicles, such as liposomes, may be used in formulations for delivering RNAi agents disclosed herein,; such formulation may hâve désirable properties such as specificity and the duration of action. As used herein, the term liposome means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which hâve a membrane formed from a lipophilie material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes can possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as
145 efficiently with the cell wall, may be taken up by macrophages in vivo. Important considérations in the préparation of liposome formulations are lipid surface charge, vesicle size, and the aqueous volume ofthe liposomes.
In some embodiments, liposomal delivery may hâve the following advantageous properties: being highly déformable and able to pass through fine pores in the skin; biocompatibility and bîodegradabilty; ability to incorporate a wide range of water- and lîpid-soluble drugs; ability to protect encapsulated drugs in their internai compartments from metabolism and dégradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., volume l, p. 245 (1998)); for topical delivery, reduced side-effects related to high systemic absorption of the adminîstered drug, increased accumulation of the adminîstered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobie, into the skin; and ability to deliver agents includîng high-molecular w eight nucleic acids, analgésies, antibodies, and hormones to the skin.
Liposomes fall into two broad classes. Cationîc liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molécules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalîzed în an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang, et al., Biochem. Bîophys. Res. Commun.I47, 980-985 (1987)).
Liposomes that are pH-sensitive or negatively-charged, entrap nucleic acids rather than complex with them. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes hâve been used to deliver nucleic acids to cell monolayers în culture (e.g., Zhou, et al.. Journal of Controlled Release 19, 269-74 (1992)).
In some embodiments, a liposomal composition is formed from phosphatidylcholine (PC), such as, for example, soybean PC and egg PC. In some embodiments, liposomal compositions include phospholipids other than naturallyderived phosphatidylcholine. Neutral liposome compositions, for example, can be
I46 formed from dimyristoyl phosphatidylcholîne (DMPC) or dipalmitoyl phosphatidylcholîne (DPPC). Anionic liposome compositions can be formed from dimyristoyl phosphatidylglyceroi, while anionic fusogenic liposomes can be formed from dioleoyl phosphatidylethanolamine (DOPE). In still other embodiments, a liposomal composition is formed from mixtures of phospholipid and/or phosphatidylcholîne and/or cholestérol.
In some embodiments, liposomal drug formulations are delîvered topically to the skin.
In some embodiments, an RNAi agent used in a combination therapy described herein is fully encapsulated in a lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term SNALP refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term SPLP refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugale). SNALPs and SPLPs may be used for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include pSPLP, which include an encapsulated condensing agent-nucleic acid complex as set forth in International Application Publication No. WO 00/03683. The particles of the technology described herein typically hâve a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, in some embodiments, nucleic acids when présent in the nucleic acid-lipid particles are résistant in aqueous solution to dégradation with a nuclease. Nucleic acid-lipid particles and related methods of préparation are disclosed in, e.g., L.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and International Application Publication No. WO 96/40964.
In some embodiments, the RNAi agent is delivered via a liposome or other lipid formulation, wherein the lipid to drug ratio (mass/mass ratio) (e.g., lipid to siRNA ratio)
147 is in the range oFfrom about l : l to about 50:1, from about l:l to about 25:l, from about 3:1 to about 15:1, from about 4:1 to about J 0:1, from about 5:1 to about 9: l, or about 6:1 to about 9:1.
Pharmaceutical Compositions Comprising Antibodies, Antigen-Binding Fragments, Fusion Proteins, Polynucleotides, Vectors, and/or Host Cells
The present disclosure also provides a pharmaceutical composition comprising an antibody, antigen-binding fragment, or fusion protein, according to the present disclosure, a nucleic acid according to the present disclosure, a vector according to the present disclosure and/or a cell according to the present disclosure. In certain embodiments, a pharmaceutical composition further comprises an inhibitor of HBV protein expression and delivery system (e.g., an RNAi agent).
Pharmaceutical compositions may also contain a pharmaceutically acceptable carrier, diluent and/or excipient. Although the carrier or excipient may facîlitate administration, it should not itself induce the production of antibodies harmful to the individual receiving the composition. Nor should it be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolîc acids, polymeric amino acids, amino acid copolymers and inactive virus partie les. In general, pharmaceutically acceptable carriers in a pharmaceutical composition according to the present disclosure may be active components or inactive components.
Pharmaceutically acceptable salts can be used, for example minerai acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic 25 acids, such as acétates, propionates, malonates and benzoates.
Pharmaceutically acceptable carriers in a pharmaceutical composition may additionally contain liquids such as water, saline, glycerol and éthanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical
148 compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the subject.
Pharmaceutical compositions of the disclosure may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophiîized composition, similar to Synagis™ and Herceptin™, for reconstitution with stérile water containing a preservatîve). The composition may be prepared for topical administration e.g., as an ointment, cream or powder. The composition may be prepared for oral administration e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aurai or ocular administration e.g., as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a subject. For example, a lyophiîized antibody may be provided in kit form with stérile water or a stérile buffer.
In particular embodiments, the active ingrédient in a composition according to the présent disclosure is an antibody molécule, an antibody fragment or variant or dérivative thereof, in particular the active ingrédient in the composition is an antibody, an antibody fragment, a fusion protein, or variants and dérivatives thereof, as described herein. As such. it may be susceptible to dégradation in the gastrointestinal tract. Thus, if the composition is to be admînistered by a route using the gastrointestinal tract, the composition may contain agents which protect the antibody from dégradation but which release the antibody once it has been absorbed from the gastrointestinal tract.
A thorough discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th édition, ISBN: 0683306472.
Pharmaceutical compositions ofthe disclosure may hâve a pH between 5.5 and 8.5, and in some embodiments this may be between 6 and 8. In other embodiments, the pH of a pharmaceutical composition as described herein may be about 7. The pH may
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be maintained by the use of a buffer. The composition may be stérile and/or pyrogen free. The composition may be isotonie with respect to humans. In certain embodiments, pharmaceutical compositions of the disclosure are supplied in hermetically sealed containers.
Within the scope of the disclosure are compositions présent in several forms of administration; the forms include, but are not limited to, those forms suitable for parentéral administration, e.g., by injection or infusion, for example by bol us injection or continuons infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or émulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and/or dispersing agents. Altematively, the antibody molécule may be in dry form, for reconstitution before use with an appropriate stérile liquid. A vehicle is typically understood to be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound, in particular the antibodies according to the présent description. For example, the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, and/or administering a pharmaceutically active compound, în particular the antibodies according to the présent description. Once formulated, the compositions of the présent disclosure can be administered directly to the subject. In one embodiment the compositions are adapted for administration to mainmalian, e.g., human subjects.
The pharmaceutical compositions described herein may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topîcal, subeutaneous, intranasal, enterai, sublingual, intravaginal or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the description. In spécifie embodiments, the pharmaceutical composition may be prepared for oral administration, e.g. as tablets, capsules and the like, for topical administration, or as injectable, e.g. as lîquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be utilîzed, e.g. that the pharmaceutical composition is în lyophilized form.
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For injection, e.g. intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingrédient can be provided be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Preservatives, stabilîzers, buffers, antioxidants and/or other 5 additives may be included, as required.
A composition may be in the form of a solid or liquid. In some embodiments, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aérosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi solid, semî liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be présent; binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnésium stéarate or Sterotex; glidants such as colloïdal Silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
The composition may be in the form of a liquid, for example, an élixir, syrup, solution, émulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to the présent compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition
I5I intended to be admînistered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonie agent may be included.
Liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: stérile diluents such as water for injection, saline solution, preferably physîological saline, Ringer’s solution, isotonie sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acétates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parentéral préparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physîological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably stérile.
A liquid composition intended for either parentéral or oral administration should contain an amount of an antibody or antigen-binding fragment as herein disclosed such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the antibody or antigen-binding fragment in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the antibody or antigen-binding fragment. In certain embodiments, pharmaceutical compositions and préparations according to the présent invention are prepared so that a parentéral dosage unît contains between 0.01 to 10% by weight of antibody or antigen-binding fragment prior to dilution.
The composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, émulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, minerai oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be présent in a composition for
152 topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonîrritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
A composition may include various materials which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingrédients. The materials that form the coating shell are typicaliy inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingrédients may be encased in a gelatin capsule. The composition in solid or liquid form may include an agent that binds to the antibody or antigen-binding fragment of the disclosure and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monocional or polyclonal antibodies, one or more proteins or a liposome. The composition may consist essentially of dosage units that can be administered as an aérosol. The term aérosol is used to dénoté a variety of Systems ranging from those of colloïdal nature to Systems consisting of pressurized packages. Delivery may be by a 1 iquefïed or compressed gas or by a suitable pump system that dispenses the active ingrédients. Aérosols may be delîvered in single phase, bi phasic, or tri phasic Systems in order to deliver the active ingredient(s). Delivery of the aérosol inciudes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue expérimentation, may détermine preferred aérosols.
It will be understood that compositions of the présent disclosure also encompass carrier molécules for polynucleotides, as described herein (e.g., lipid nanoparticles, nanoscale delivery platforms, and the like).
In certain embodiments, a composition comprises a first vector comprising a first plasmid, and a second vector comprising a second plasmîd, wherein the first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH, and a
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second plasmid comprises a poiynucleotide encodîng the cognate light chaîn, VL, or VL+CL of the antibody or antigen-binding fragment thereof. In certain embodiments, a composition comprises a poiynucleotide (e.g., mRNA) coupled to a suitable delivery vehicle or carrier. Exemplary vehîcles or carriers for administration to a human subject 5 include a lipid or lipid-derived delivery vehicle, such as a liposome, solid lipid nanoparticle, oily suspension, submicron lipid émulsion, lipid microbubble, inverse lipid micelle, cochlear liposome, lipid microtubule, lipid microcylinder, or lipid nanoparticle (LNP) or a nanoscale platform (see, e.g., Lî et al. Wilery Interdiscip Rev. Nanomed Nanobiotechnol. 77(2):el53O (2019)). Principles, reagents, and techniques for designing appropriate mRNA and and formulating mRNA-LNP and delivering the same are described in, for example, Pardi et al. (JControlRelease 2/7345-351 (2015)); Thess et al. (Mol Ther 23: 1456-1464 (2015)); Thran et al. (EMBO Mol Med 9(10):1434-1448 (2017); Kose et al. (Sci. Immunol. 4 eaaw6647 (2019); and Sabnis et al. (Mol. Ther. 26:1509-1519 (2018)), which techniques, include capping, codon optimization, nucleoside modification, purification of mRNA, incorporation of the mRNA into stable lipid nanopartîcles (e.g., ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid; ionizable lipid:distearoyl PC:cholesterol:polyethylene glycol lipid), and subcutaneous, întramuscular, intradermal, intravenous, intraperitoneal, and intratracheal administration of the same, 20 are incorporated herein by reference.
The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a composition that comprises an antibody, antigen-binding fragment thereof, or other compositoîn as described herein and optionaliy, one or more of salts, buffers and/or stabilizers, with stérile, disti 1 led water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact wîth the peptide composition so as to facilitate dissolution or homogeneous suspension of the antibody or antigen-binding fragment thereof în the aqueous delivery 30 system.
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Whether it is a polypeptide, peptide, or nucleic acid molécule, cell, or other pharmaceutically useful compound according to the présent disclosure that is to be given to an individual, administration is generally in a prophylactically effective amount or a therapeutically effective amount or an effective amount (as the case may be), this being sufficient to show a benefit to the individual (e.g., improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of lîfe; longer disease-free status; diminishment ofextent of disease, stabilization of disease State; delay of disease progression; rémission; survival; or proionged survival in a statistically significant manner). When referring to an individual active ingrédient, administered alone, a therapeutically effective amount refers to the effects of that ingrédient or cell expressing that ingrédient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of active ingrédients or combined adjunctive active ingrédient with a cell expressing an active ingrédient that results in a therapeutic effect, whether administered serially, sequentially, or simultaneously.
Compositions are administered in an effective amount (e.g., to treat a SARSCoV-2 infection), which will vary depending upon a variety of factors including the activity of the spécifie compound employed; the metabolic stabilîty and ïength of action of the compound; the âge, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excrétion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. In certain embodiments, tollowing administration of thérapies according to the formulations and methods of this disclosure, test subjects will exhibit about a 10% up to about a 99% réduction in one or more symptoms associated with the disease or disorder being treated as compared to placebo-treated or other suitable control subjects.
Generally, a therapeutically effective daily dose of an antibody or antigen binding fragment is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg
155 (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g), Other doses for antibodies or antigenbinding fragments are provided herein.
For polynucleotides, vectors, host cells, and related compositions of the présent disclosure, a therapeutically effective dose may be different than for an antibody or antigen-binding fragment.
The actual amount administered, and rate and time-course of administration, will dépend on the nature and severity of what is being treated. For injection, the pharmaceutical composition according to the présent disclosure may be provided for example in a pre-fïlled syringe.
Pharmaceutical compositions as disclosed herein may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnésium stéarate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried comstarch. When aqueous suspensions are required for oral use, the active ingrédient, i.e. the inventive transporter cargo conjugale molécule as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
The pharmaceutical compositions according to the présent description may also be administered topîcally, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g., including diseases of the skin or of any other accessible épithélial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the pharmaceutical composition may be formulated in a suitable ointment, containing the inventive pharmaceutical composition, particularly its components as defined above, suspended or dissoived in one or more carriers. Carriers for topical administration include, but are not limited to, minerai oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated in a suitable lotion or cream. In the context of the présent description, suitable carriers include, but are not
156 limited to, minera! oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
Doses may be expressed in relation to the bodyweight. Thus, a dose which is expressed as [g, mg, or other unit]/kg (or g, mg etc.) usually refers to [g, mg, or other unit] per kg (or g, mg etc.) bodyweight’’, even if the term bodyweight or body weight is not explicitly mentioned.
In spécifie embodiments, in a single dose, e.g. a daily, weekly or monthly dose, the amount of the antibody, or the antigen binding fragment thereof, in the pharmaceutical compositiondoes not exceed l g. In certain such embodiments, the single dose does not exceed a dose selected from 500 mg, 250 mg, 100 mg, and 50 mg. Further embodiments of doses are provided herein.
In certain embodiments, a method comprises administering the antibody, antîgen-binding fragment, polynucleotide, vector, host cell, or composition to the subject at 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more.
In certain embodiments, a method comprises administering the antibody, antîgen-binding fragment, or composition to the subject a plurality of times, wherein a second or successive administration is performed at about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 24, about 48, about 74, about 96 hours, or more, following a first or prior administration, respectively.
In certain embodiments, a method comprises administering the antibody. antîgen-binding fragment, polynucleotide, vector, host cell, or composition at least one time prior to the subject
Compositions comprising an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition of the présent disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the invention and one or more additional active agents, as well as administration of compositions comprising an antibody or antigen-binding fragment of the disclosure and each active agent in its own separate dosage formulation. For example, an antibody or antigen
157 binding fragment thereof as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations Similarly, an antibody or antigen-binding fragment as described herein and the other active agent can be administered to the subject together in a single parentéral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parentéral dosage formulations. Where separate dosage formulations are used, the compositions comprising an antibody or antigen-binding fragment and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include ail these regimens.
In some embodiments, a composition or kit as described herein further comprises (i) a polymerase inhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovîr, Entecavir, Telbivudine, Tenofovir, or any combination thereof; (ii) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha; (iii) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an anti-PD-1 antibody or antigen binding fragment thereof, an anti-PD-LI antibody or antigen binding fragment thereof, and/or an anti-CTLA4 antibody or antigen binding fragment thereof; (iv) an agonist of a stimulatory immune checkpoint molécule; or (v) any combination of (i)-(iv). In some embodiments, a kit comprises a composition or combination as described herein, and further comprises instructions for using the component to prevent, treat, attenuate, and/or diagnose a hepatitis B infection and/or a hepatitis D infection.
In certain embodiments, a composition of the présent disclosure (e.g., antibody, antigen-binding fragment, host cell, nucleic acid, vector, or pharmaceutical omposition) is used in combination with a PD-l inhibitor, for example a PD-1-spécifie antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly AMP-514), AMP-224, BMS-936558 or any combination thereof. In certain embodiments, a composition of the présent disclosure is used in combination with a PD-Ll spécifie antibody or binding fragment thereof, such as BMS-936559,
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durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof. In certain embodiments, a composition of the présent disclosure is used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof. In certain embodiments, a composition of the présent disclosure is used in combination with an inhibitor of CTLA4. In particular embodiments, an a composition of the présent disclosure is used in combination with a CTLA4 spécifie antibody or binding fragment thereof, such as ipilimumab, tremelimumab, CTLA4-lg fusion proteins (e.g., abatacept, belatacept), or any combination thereof. In certain embodiments, a composition ofthe présent disclosure îs used in combination with a B7-H3 spécifie antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. An anti-B7-H3 antibody binding fragment may be a scFv or fusion protein comprising the same, as described in, for example, Dangaj et al., Cancer Res. 73:4820, 2013, as well as those described in U.S. Patent No. 9,574,000 and PCT Patent Publication Nos.
WO/201640724A1 and WO 2013/025779A1. In certain embodiments, a composition ofthe présent disclosure is used in combination with an inhibitor of CD244. In certain embodiments, a composition ofthe présent disclosure îs used in combination with an inhibitor of BLTA, HVEM, CD 160, or any combination thereof. Anti CD-160 antibodies are described in, for example, PCT Publication No. WO 2010/084158. In certain embodiments, a composition of the présent disclosure îs used in combination with an inhibitor of TIM3. In certain embodiments, a composition of the présent disclosure is used in combination with an inhibitor of Gal9. In certain embodiments, a composition of the présent disclosure is used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor. In certain embodiments, a composition of the présent disclosure is used in combination with an inhibitor of A2aR. In certain embodiments, a composition of the présent disclosure is used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015). In certain embodiments, a composition of the présent disclosure is used in combination with an inhibitor of an inhibitory cytokine (typically, a cytokine other than TGFp) or Treg development or activity. In certain embodiments, a composition of the présent disclosure îs used in
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combination with an IDO inhibitor, such as levo-l-methyl tryptophan. epacadostat (1NCB024360; Lin et al., Blood 775:3520-30,2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino étal., American Association for Cancer Research I04th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (I -MT)-tira-pazamine, or any combination thereof. In certain embodiments, a composition ofthe present disclosure is used in combination with an arginase inhibitor, such as N(omega)-Nitro-L-argînine methyl ester (L-NAME), N-omega-hydroxy-nor-larginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABU), S-(2boronoethyl)-L-cysteine (BEC), or any combination thereof. In certain embodiments, a 10 composition of the present disclosure îs used in combination with an inhibitor of
V1STA, such as CA-170 (Curis, Lexington, Mass.). In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of T1G1T such as, for example, COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD] 55, such as, for example, COM701 (Compugen), or both. In certain embodiments, 15 a composition of the present disclosure is used in combination with an inhibitor of
PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described in, for example, PCT Publication No. WO 2016/134333. Anti-PVRL2 antibodies are described in, for example, PCT Publication No. WO 2017/021526. In certain embodiments, a composition ofthe present disclosure is used in combination with a LAIR1 inhibitor. In 20 certain embodiments a composition of the present disclosure is used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.
In certain embodiments, a composition of the present disclosure is used in combination with an agent that increases the activity (i.e., is an agonist) of a stimulatory 25 immune checkpoint molécule. For example, a composition of the present disclosure can be used in combination with a CD 137 (4-1BB) agonist (such as, for example, urelumab), a CD 134 (OX-40) agonist (such as, for example, MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (such as, for example, CDX-1127), a CD28 agonist (such as, for example, TGN14I2, CD80, or CD86), a
CD40 agonist (such as, for example, CP-870,893, rhuCD40L, or SGN-40), a CD 122
160 agonist (such as, for example, IL-2) an agonist of GITR (such as, for example, humanized monoclonal antibodîes described in PCT Patent Publication No. WO 2016/054638), an agonist of ICOS (CD278) (such as, for example, GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos 314-8, or any combination thereof).
In any of the embodiments disclosed herein, a method may comprise administering a composition of the présent disclosure with one or more agonist of a stimulatory immune checkpoint molécule, including any of the foregoing, singly or in any combination.
An antibody, antigen binding fragment, or fusion protein according to the présent disclosure can be présent either in the same pharmaceutical composition as the additional active component or, the antibody, antigen binding fragment, or fusion protein according to the présent disclosure may be included in a first pharmaceutical composition and the additional active component may be included in a second pharmaceutical composition different from the first pharmaceutical composition.
Uses
In a further aspect, the présent disclosure provides methods for the use of an antibody, an antigen binding fragment, a fusion protein, a nucleic acid, a vector, a cell, a pharmaceutical composition, a combination (e.g, of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of HBV protein expression and delivery system (e.g, an RNAi agent), or a kit according to the présent disclosure in the (i) prophylaxis, treatment or atténuation of hepatitis B and/or hepatitis D; or in (ii) diagnosis of hepatitis B and/or hepatitis D (e.g., in a human subject).
Methods of diagnosis (e.g, in vitro, ex vivo) may include contacting an antibody, antibody fragment (e.g., antigen binding fragment), or fusion protein with a sample. Such samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancréas, kidney, ear, eye, placenta, alimentary tract, heart, ovarîes, pituitary, adrenals, thyroid, brain, skin or blood. The methods of diagnosis may also include the détection of an antigen/antibody or antigen/fusion protein complex, in particular
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following the contacting of an antibody, antibody fragment, or fusion protein with a sample. Such a détection step is typically performed at the bench, i.e. without any contact to the human or animal body. Examples of détection methods are well-known to the person skilled in the art and include, e.g., ELISA (enzyme-linked immunosorbent 5 assay).
The disclosure also provides the use of (i) an antibody, an antibody fragment, fusion protein, or variants and dérivatives thereof according to the disclosure, (ii) host cell (which can be an immortalized B cell) according to the disclosure, (iii) a nucleic acid or a vector according to the présent disclosure (iv) a pharmaceutical composition 10 ofthe présent disclosure or (v) a combination (e.g., of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of HBV protein expression and delivery system (e.g., an RNAi agent)) in (a) the manufacture of a médicament for the prévention, treatment or atténuation of hepatitis B and/or hepatitis D or for (b) diagnosis of hepatitis B and/or hepatitis D.
The term disease as used herein is intended to be generally synonymous, and is used interchangeably with, the terms disorder and condition (as in medical condition), in that ail reflect an abnormal condition of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the affected human or animal to hâve a 20 reduced duration or quality of life.
As used herein, reference to treatment of a subject or patient is intended to include prévention, prophy Iaxis, atténuation, amelioration and therapy, and refers to medical management of a disease, disorder, or condition of a subject. Benefits of treatment can include improved clînical outcome; lessening or allevîation of symptoms 25 associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease; stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof. The terms subject or patient are used interchangeably herein to mean ail mammals, including humans. Examples of subjects include humans, cows, 30 dogs, cats, horses, goats, sheep, pigs, and rabbits. In certain embodiments, the patient is
162 a human. The subjects can be male or femaie and can be any suitable âge, including infant, juvénile, adolescent, adult, and gériatrie subjects.
The disclosure also provides an antibody, antigen binding fragment, or fusion protein according to the présent disclosure, a nucleic acid according to the présent disclosure, a vector according to the présent disclosure, a cell according to the présent disclosure a pharmaceutical composition according, and/or a combination (e.g., of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of HBV protein expression and delîvery System (e.g., an RNAi agent) ofthe présent disclosure for use as a médicament for the prévention or treatment of hepatitis B and/or hepatitis D. It also provides the use of an antibody, antigen binding fragment, or fusion protein of the disclosure în the manufacture of a médicament for treatment of a subject and/or diagnosis in a subject. It also provides a method for treating a subject (e.g., a human subject), comprising administering to the subject an effective amount of a composition or combination as described herein. In some embodiments, the subject may be a human. One way of checking efficacy of therapeutic treatment in volves monitoring disease symptoms after administration of the composition. Treatment can be a single dose schedule or a multiple dose schedule.
In one embodiment, an antibody, antigen-binding fragment, fusion protein, host cell (e.g., immortalized B cell clone, or T cell, NK-T cell, or NK cell that expresses a fusion protein), pharmaceutical composition, or combination according to the disclosure is administered to a subject in need of such treatment. Such a subject includes, but is not limited to, one who is particularly at risk of or susceptible to hepatitis B and/or hepatitis D.
Antibodies, antigen binding fragments, fusion proteins, polynucleotides, vectors, host cells, pharmaceutical compositions, and combinations of the same, according to the présent disclosure may also be used în a kit for the prévention, treatment, atténuation, and/or diagnosis of hepatitis B and/or hepatitis D. In some embodiments, a kit further comprises instructions for using the component to prevent, treat, attenuate, and/or diagnose a hepatitis B infection and/or a hepatitis D infection. Further, the epitope in the antigenic loop région of HBsAg, which is capable of binding an antibody, antigen
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binding fragment, or fusion protein of the disclosure as described herein may be used in a kil for monitoring the efficacy of application procedures by detecting the presence or determining the titer of protective anti-HBV antibodies.
In certain embodiments, a composition or a kit of this disclosure further 5 comprises: a polymerase înhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir, or any combination thereof; (ii) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha; (iii) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an antî-PD-1 antibody or antigen binding fragment thereof, an anti-PD-LI antibody or antigen binding fragment thereof, and/or an anti-CTLA4 antibody or antigen binding fragment thereof; (iv) an agonist of a stimulatory immune checkpoint molécule; or (v) any combination of (vîîi)-(xii).
In some embodiments, an antibody, an antigen binding fragment, or fusion protein according to the présent disclosure, a nucleic acid according to the présent 15 disclosure, the vector according to the présent disclosure, a cell according to the présent disclosure, a pharmaceutical composition according to the présent disclosure, and/or a combination (e.g., of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of FIBV protein expression and delivery system (e.g., an R.NAÎ agent) of the présent disclosure îs used in treatment or atténuation of chronic hepatitis B infection.
In particular embodiments, an antibody, antigen binding fragment, or fusion protein according to the présent disclosure (i) neutralizes HBV infection, (ii) binds to LHBsAg (the large HBV envelope protein, which is présent in infections HBV parti clés), thereby preventing spreading of HBV, (iii) binds to S-HBsAg, thereby pronioting clearance of subviral particles (SVP) and/or (iv) can induce séroconversion, i.e. an active immune response to the virus.
In particular embodiments, an antibody, antigen binding fragment, or fusion protein according to the présent disclosure, a nucleic acid according to the présent disclosure, a vector according to the présent disclosure, a cell according to the présent 30 disclosure, or a pharmaceutical composition according to the présent disclosure, may be
164 used in prévention of hepatitis B (re-)infection after liver transplantation in particular for hepatitis B induced liver failure.
In further embodiments an antibody, antigen binding fragment thereof, or fusion protein according to the présent disclosure, a nucleic acid according to the présent disclosure, a vector according to the description provided herein, a cell according to the présent disclosure, a pharmaceutical composition, and/or a combination (e.g., of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of HBV protein expression and delivery system (e.g., an RNAi agent) according to the présent disclosure, may be used in prevention/prophy Iaxis of hepatitis B in non-immunized subjects. This is for example in case of (an assumed) accidenta! exposure to HBV (post-exposure prophyIaxis). The term non-immunized subjects includes subjects, who never received a vaccination and are, thus, not immunized, and subjects, who did not show an immune response (e.g., no measurable anti-hepatitis B antibodies) after vaccination.
In some embodiments, an antibody, antigen binding fragment, or fusion protein according to the présent disclosure, the nucleic acid according to the présent disclosure, a vector according to the présent disclosure, a cell according to the présent disclosure, a pharmaceutical composition according to the présent disclosure, or a combination (e.g., of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of HBV protein expression and delivery system (e.g., an RNAi agent) ofthe présent disclosure, is used in prophylaxis of hepatitis B in haemodialysed patients.
In some embodiments, an antibody, an antigen binding fragment, or fusion protein according to the présent disclosure, a nucleic acid according to the présent disclosure, a vector according to the présent disclosure, a cell according to the présent disclosure, a pharmaceutical composition according to the présent disclosure, or a combination (e.g., of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of HBV protein expression and delivery system (e.g., an RNAi agent) ofthe présent disclosure, is used in prévention of hepatitis B in a newborn. In such embodiments, an antibody, or an antigen binding fragment thereof, according to the présent disclosure, a nucleic acid according to the présent disclosure, a vector
165 according to the présent disclosure, a cell according to the présent disclosure, a pharmaceutical composition according to the présent disclosure, or a combination (e.g., of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of HBV protein expression and delivery System (e.g., an RNAi agent) ofthe présent disclosure, may be administered at birth or as soon as possible after birth. The administration may be repeated until séroconversion following vaccination.
Moreover, the présent disclosure also provides the use of an antibody, antigen binding fragment, or fusion protein according to the présent disclosure, a nucleic acid according to the présent disclosure, a vector according to the présent disclosure, a cell according to the présent disclosure or a pharmaceutical composition according to the présent disclosure in the diagnosis (e.g. in vitro, ex vivo, or in vivo) of hepatitis B and/or hepatitis D.
In addition, the use of an antibody, antigen binding fragment, or fusion protein according to the présent disclosure, a nucleic acid according to the présent disclosure, a vector according to the présent disclosure, a cell according to the présent disclosure or a pharmaceutical composition according to the présent disclosure in determining whether an isolated blood sample is înfected with hepatitis B virus and/or hepatitis delta virus is provided.
As described above, methods of diagnosis may include contacting an antibody, antibody fragment, or fusion protein with a sample. Such samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavîties, salivary glands, lung, liver, pancréas, kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary, adrenals, thyroid, brain, skîn or blood. The methods of diagnosis may also include the détection of an antigen/antibody complex, in particular following the contacting of an antibody or an antibody fragment with a sample. Such a détection step is typicaliy performed at the bench, i.e. without any contact to the human or animal body. Examples of détection methods are wellknown to the person skilled in the art and include, e.g., ELISA (enzyme-linked immunosorbent assay).
I66
The présent disclosure also provides a method of treating, preventing and/or attenuating hepatitis B and/or hepatitis D in a subject, wherein the method comprises administering to the subject an antibody, antigen binding fragment, or fusion protein according to the présent disclosure, a nucleic acid according to the présent disclosure, a vector according to the présent disclosure, a cell according to the présent disclosure, a pharmaceutical composition according to the présent disclosure, and/or a combination (e.g, of a presently disclosed antibody or antigen-binding fragment with a presently disclosed inhibitor of HBV protein expression and delivery System (e.g., an RNAi agent) of the présent disclosure. In certain embodiments, a method further comprises administering to the subject one or more of: (vii) a polymerase inhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir, or any combination thereof; (viîî) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha; (ix) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an anti-PD-l antibody or antigen binding fragment thereof, an anti-PD-Ll antibody or antigen binding fragment thereof, and/or an antî-CTLA4 antibody or antigen binding fragment thereof; (x) an agonist of a slimulatory immune checkpoint molécule; or (xi) any combination of (vii)-(x).
In some embodiments, the hepatitis B infection is a chronic hepatitis B infection. In some embodiments, the subject has received a lîver transplant. In some embodiments, the subject is non-immunized against hepatitis B. In certain embodiments, the subject îs a newbom. In some embodiments, the subject is undergoing or has undergone hemodialysis.
The présent disclosure also provides a method of treating a subject who has received a liver transplant comprising administering to the subject who has received the liver transplant an effective amount of an antibody, an antigen binding fragment, or fusion protein according to the présent disclosure, a nucleic acîd according to the présent disclosure, a vector according to the présent disclosure, a cell according to the présent disclosure, a pharmaceutical composition according to the présent disclosure, or a combination (e.g., of a presently disclosed antibody or antigen-binding fragment with
167 a presently disclosed inhibitor of HBV protein expression and delivery System (e.g., an RNAi agent) of the présent disclosure.
Also provided herein are methods for detecting the presence or absence of an epitope in a correct conformation in an anti-hepatitis-B and/or an anti-hepatitis-D vaccine, wherein the methods comprise: (i) contacting the vaccine with an antibody, antigen-binding fragment, or fusion protein of any one ofthe présent disclosure; and (ii) determining whether a complex comprising an antigen and the antibody, or comprising an antigen and the antigen binding fragment, or comprising an antigen and the fusion protein, has been formed.
The term vaccine as used herein is typically understood to be a prophylactic or therapeutic material providing at least one antigen, such as an immunogen. The antigen or immunogen may be derived from any material that is suitable for vaccination. For example, the antigen or immunogen may be derived from a pathogen, such as from bacteria particles, virus particles, a tumor (including a solid or liquid tumor], or other cancerous tissue. The antigen or immunogen stimulâtes the body's adaptive immune system to provide an adaptive immune response. In certain embodiments, an antigen or an immunogen refers to a substance which may be recognized by the immune system, e.g. by the adaptive immune System, and which is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigenspecific T cells as part of an adaptive immune response. In some embodiments, an antigen may be or may comprise a peptide or protein which may be presented by an MHC complex (e.g, MHC class I; MHC class II) to T cells. In certain embodiments, the antigen comprises a HBV and/or HBD antigen; e.g., an HBsAg antigen.
Some embodiments of the présent disclosure provide methods of treating chronic HBV infection or an I-IBV-associated disease in a subject in need thereof, comprising: (i) administering to the subject an agent that reduces HBV antigenic load; and (ii) administering to the subject an anti-HBV antibody or antigen-binding fragment thereof. in certain embodiments, the agent that reduces HBV antigenic load is admînistered before the anti-HBV antibody or antigen-binding fragment thereof. In certain embodiments, administering the agent that reduces HBV antigenic load before
168 the anti-HBV antibody or antigen-binding fragment thereof causes the viral load to be reduced when the anti-HBV antibody or antigen-binding fragment thereof is adminîstered. In certain embodiments, the therapeutîcally effective amount ofthe antiHBV antibody or antigen-binding fragment thereof of the combination therapy is less than a therapeutîcally effective amount of the anti-HBV antibody or antigen-binding fragment thereof delivered when the agent that reduces HBV antigenic load has not been adminîstered to the subject (e.g., when the anti-HBV antibody or antigen-binding fragment thereof is adminîstered alone as a monotherapy). In some embodiments, the agent that reduces HBV antigenic load is an RNAi agent (e.g., an siRNA) that inhibits expression of an HBV transcript.
In certain embodiments, the présent disclosure provides a method of treating a chronic HBV infection or HBV-associated disease in a subject in need thereof, comprising: administering to the subject an agent that reduces HBV antigenic load; and administering to the subject an anti-HBV antibody or antigen-binding fragment thereof; and further comprising measuring the amount of HBsAg présent in a blood sample from the subject before and after administering the the agent that reduces HBV antigenic load, wherein a decrease in HBsAg indicates reduced expression of the at least one HBV gene.
In certain embodiments, the présent disclosure provides an agent that reduces HBV antigenic load for use in the treatment of a chronic HBV infection or an HBVassociated disease in a subject, wherein the subject îs subsequently adminîstered an anti-HBV antibody or antigen-binding fragment thereof. In certain other embodiments, the présent disclosure provides an anti-HBV antibody or antigen-binding fragment thereof for use in the treatment of a chronic HBV infection or an HBV-associated disease in a subject, and the subject has been previously adminîstered an agent that reduces HBV antigenic load. In further embodiments, expression ofat least one HBV gene is reduced after administration of the agent that reduces HBV antigenic load, and the anti-HBV antibody or antigen-binding fragment thereof is adminîstered to the subject when expression of the at least one HBV gene is reduced.
169
In certain embodiments, the présent disclosure provides the use of an agent that reduces HBV antigenic load and/or an anti-HBV antibody or antigen-bînding fragment thereof in the manufacture of a médicament for the treatment of a chronic HBV infection or an HBV-associated disease.
Some embodiments of the présent disclosure provide methods of treating chronic HBV infection or an HBV-associated disease in a subject in need thereof, comprising: (i) administering to the subject an inhibitor of HBV gene expression; and (ii) administering to the subject an anti-HBV antibody or antigen-binding fragment thereof. In certain embodiments, the inhibitor of HBV gene expression is administered before the anti-HBV antibody. In certain embodiments, administering the inhibitor of HBV gene expression before the anti-HBV antibody or antigen-bînding fragment thereof causes the viral load to be reduced when the anti-HBV antibody is administered. In certain embodiments, the therapeutically effective amount of the anti-HBV antibody of the combination therapy is less than a therapeutically effective amount of the antiHBV antibody or antigen-binding fragment thereof delivered when the inhibitor of HBV gene expression has not been administered to the subject (e.g., when the antiHBV antibody or antigen-binding fragment thereof îs administered alone as a monotherapy).
In certain embodiments, expression of at least one HBV gene is reduced after administering the inhibitor of HBV gene expression, and the anti-HBV antibody or antigen-binding fragment thereof is administered to the subject when expression of the at least one HBV gene is reduced. In particular embodiments, the at least one HBV gene is HBV X gene and/or HBsAg.
In certain embodiments, the présent disclosure provides a method of treating a chronic HBV infection or HBV-associated disease in a subject in need thereof, comprising: administering to the subject an inhibitor of HBV gene expression; and administering to the subject an anti-HBV antibody or antigen-binding fragment thereof; and further comprising measuring the amount of HBsAg présent in a blood sample from the subject before and after administering the inhibitor of HBV expression, wherein a decrease in HBsAg indicates reduced expression of the at least one HBV gene.
170
In certain embodiments, the présent disclosure provides an inhibitor of HBV gene expression for use in the treatment of a chronic HBV infection or an HBVassociated disease in a subject, wherein the subject is subsequently administered an anti-HBV antibody or antigen-binding fragment thereof. In certain other embodiments, 5 the présent disclosure provides an anti-HBV antibody or antigen-binding fragment thereof for use in the treatment of a chronic HBV infection or an HBV-associated disease in a subject, and the subject has been prevîously administered an inhibitor of gene expression. In further embodiments, expression of at least one HBV gene is reduced after administration of the inhibitor of HBV gene expression, and the anti-HBV 10 antibody or antigen-binding fragment thereof is administered to the subject when expression of the at least one HBV gene is reduced.
in certain embodiments, the présent disclosure provides the use of an inhibitor of HBV gene expression and/or an anti-HBV antibody or antigen-binding fragment thereof in the manufacture of a médicament for the treatment of a chronic HBV infection or an HBV-associated disease.
In any of the above methods, compositions for use, or uses in manufacture, the methods and compositions may be used for treating a chronic HBV infection.
In certain embodiments, the inhibitor of HBV gene expression is administered in a single dose, two doses, three doses, four doses, or five doses. In certain particular 20 embodiments, at least the first dose of the inhibitor of HBV gene expression is administered prior to administering the anti-HBV antibody or antigen-binding fragment thereof.
In certain embodiments, the inhibitor of HBV gene expression is administered in a single dose, two doses, three doses, four doses, or five doses, six doses, seven doses, 25 or eight doses. The dose or doses may be administered, for example, twice daily, once daily, every two days, every three days, twice per week, once per week, every other week, every four weeks, or once per month.
In certain embodiments, administering the anti-HBV antibody or antigenbinding fragment thereof comprises administering the anti-HBV or antigen-binding
I7l fragment thereof antibody twice per week, once per week, every other week, every two weeks, or once a month.
In certain embodiments, administering the anti-HBV antibody or antigenbinding fragment thereof comprises administering at least two doses of a therapeutically effective amount of the anti-HBV antibody or antigen-binding fragment thereof. In certain further embodiments, the at least two doses are administered twice per week, once per week, every other week, every two weeks, or once a month.
In certain embodiments, administering the anti-HBV antibody or antigenbinding fragment thereof begins at least l week after administering the inhibitor of HBV gene expression. In certain embodiments, administering the anti-HBV antibody begins 2 weeks after administering the inhibitor of HBV gene expression. In certain embodiments, administering the anti-HBV antibody or antigen-binding fragment thereof begins 8 weeks after administering the inhibitor of HBV gene expression.
In certain embodiments, the anti-HBV antibody or antigen-binding fragment thereof and the inhibitor of HBV gene expression are each administered subcutaneously.
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the anti-HBV antibody or antigen-binding fragment thereof may recognize HBV génotypes A, B, C, D, E, F, G, H, I, and J.
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the anti-HBV antibody or antigen-binding fragment thereof may be a human antibody or antigen-binding fragment thereof; a monoclonal antibody or antigen-binding fragment thereof; or a bispecific antibody or antigen-binding fragment thereof, with a first specificity for HBsAg and a second specîficîty that stimulâtes an immune effector (e.g., a second specificity that stimulâtes cytotoxicity or a vaccinal effect). In certain other embodiments of the above methods, compositions for use, or uses in manufacture disclosed herein, the anti-HBV antibody is a monoclonal antibody.
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the anti-HBV antibody or antigen-binding fragment thereof comprises a non-natural variant of HBC34 as disclosed herein. For example, in certain
172 embodiments, the anti-HBV antibody (i) a heavy chain variable région (VH) comprising a CDRHl amino acid sequence according to SEQ ID NO.;34, a CDRH2 amino acid sequence according to SEQ ID NO.:35 or 36, and a CDRH3 amino acid sequence according to SEQ ID NO.:37; and (ii) a light chain variable région (VL) comprising a CDRLl amino acid sequence set forth in any one of SEQ ID NOs.:40-43, a CDRL2 amino acid sequence according to any one of SEQ ID NOs:45-53, and a CDRL3 amino acid sequence according to SEQ IDNO.:55 or 56, wherein the CDRs are determined according to the CCG numbering System, and wherein the antibody or antigen-binding fragment thereof is capable of binding to the antigenic loop région of HBsAg and neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, 1, or J, or any combination thereof.
In certain embodiments, the CDRH!, CDRH2, CDRH3, CDRLl, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.: (i) 34, 35, 37, 41, 45, and 55, respectively; (ii) 34, 35, 37, 41, 46, and 55, respectively; (iii) 34, 35, 37, 41,47, and 55, respectively; (iv) 34, 35, 37, 41,48, and 55, respectively; (v) 34, 35, 37, 41, 49, and 55, respectively; (vi) 34, 35, 37, 41, 50, and 55, respectively; (vîi) 34, 35, 37, 41,51, and 55, respectively; (viii) 34, 35, 37, 41,52, and 55, respectively; or (ix) 34, 35, 37, 41,53, and 55, respectively.
In certain further embodiments, antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein: (i) the VH comprises or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.; 38 or 39; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 58-66, 69, 71, or 72.
in some embodiments, the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO.; 38 or 39; and/or the VL comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs.: 58-66, 69, 71, or 72.
173
In particular embodiments, the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 58, respectively; (ii) 38 and 59, respectîvely; (iii) 38 and 60, respectively; (iv) 38 and 61, respectively; (v) 38 and 62, respectively; (vi) 38 and 63, respectively; (vii) 38 and 64, respectively; (vîîi) 38 and 65, 5 respectively; (ix) 38 and 66, respectively; (x) 38 and 71, respectively; or (xi) 38 and 72, respectively.
In another aspect, the présent disclosure provides an antibody or antigen-binding fragment thereof, comprising: a heavy chain variable domain (VH) and a lîght chain variable domain (VL), wherein the VH and the VL comprise or consist of the amino 10 acid sequences set forth in SEQ ID NOs.; (i) 38 and 67, respectively; or (ii) 38 and 68, respectively, wherein the antibody or antigen-binding fragment thereof is capable of binding to the antigenic loop région of HBsAg and neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof.
Also provided is an antibody or antigen-binding fragment comprising a VH 15 according to SEQ ID NO.:38 or 39 and a VL variant of any one of SEQ ID NOs.:57-72 that comprises one or more of the following mutations in framework région 3 relative to SEQ ID NO.:57-72, respectively, as determined by CCG numbering: R60A, R60N, R60K, S64A, I74A. In some embodiments, no further mutation relative to SEQ ID NO. :57-72, respectively, is comprised in the variant.
Also provided is an antibody or antigen-binding fragment comprising a VH according to SEQ ID NO.:38 or 39 and a VL variant of any one of SEQ ID NOs.:57-72 that comprises a substitution mutation (such as, for example, a conservative amino acid substitution, or a mutation to a germline-encoded amino acid) at Q78, D81, or both. In some embodiments, no further mutation relative to SEQ ID NO.:57-72, respectively, is 25 comprised in the variant.
In any of the presently disclosed embodiments, in a sample comprising a plurality of the antibody or antigen-binding fragment, less than 12%, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less of the plurality is comprised in an antibody dirrier when the sample 30 has been incubated for about 120 to about 168 hours at about 40°C, wherein, optionaliy,
174
the presence of antibody dimer is determined by absolute size-exclusion chromatography. As used herein, an antibody dimer or multimer is a complex comprising two or more of an antibody or antigen-binding fragment ofthe présent disclosure (e.g., an antibody:antibody dimer, a Fab:Fab dimer, or an antibody:Fab 5 dimer).
In certain embodiments, a therapeutically effective amount of the antî-HBV antibody or antigen-binding fragment is less than a therapeutically effective amount of the anti-FIBV antibody or antigen-binding fragment delivered when the inhibitor of HBV gene expression has not been administered to the subject. For example, the combination therapy may lower the effective dose of the antî-HBV antibody or antigenbinding fragment, as compared to administration ofthe antî-HBV antibody or antigenbinding fragment alone.
In certain embodiments, the antî-HBV antibody or antigen-binding fragment is administered in at least two separate doses. In particular embodiments, the at least two 15 doses are administered twice per week, once per week, every other week, every two w eeks, or once a month.
In certain embodiments, the subject is a human and a therapeutically effective amount of the antî-HBV antibody is administered; wherein the therapeutically effective amount is from about 3 mg/kg to about 30 mg/kg.
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the the inhibitor is an RNAi agent that inhibits expression of an HBV transcript. In some embodiments, inhibition of expression of an HBV transcript is measured by rtPCR. In some embodiments, inhibition of expression of an HBV transcript is measured by a réduction in protein levels as measured by EL ISA.
In certain embodiments, the RNAi agent comprises a sense strand and an antisense strand forming a double-stranded région, wherein the sense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from nucléotides 1579-1597 of SEQ ID NO:116. In certain embodiments, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises nucléotides 1579-1597 of SEQ ID NO: 116.
175
In particular embodiments of the above methods, compositions for use, or uses in manufacture, at least one strand of the RNAi agent may comprise a 3' overhang of at least l nucléotide or at least 2 nucléotides.
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the double-stranded région of the RNAi agent may be 15-30 nucléotide pairs in length; 17-23 nucléotide pairs in length; 17-25 nucléotide pairs in length; 23-27 nucléotide pairs in length; 19-21 nucléotide pairs in length; or 21-23 nucléotide pairs in length.
In particular embodiments of the above methods, compositions for use, or uses in manufacture, each strand of the RNAi agent may be 15-30 nucléotides or 19-30 nucléotides.
In particular embodiments ofthe above methods, compositions for use, or uses in manufacture, the RNAi agent is an sîRNA. In particular embodiments, the siRNA inhibits expression of an HBV transcript that encodes an HBsAg protein, an HBcAg protein, and HBx protein, or an HBV DNA polymerase protein. In certain embodiments, the siRNA binds to at least 15 contiguous nucléotides of a target encoded by: P gene, nucléotides 2309-3182 and 1 -1625 of NC_003977.2; S gene (encoding L, M, and S proteins), nucléotides 2850-3182 and 1-837 of NC_003977.2; HBx, nucléotides 1376-1840 of NC_003977.2; or C gene, nucléotides 1816-2454 of
NC_003977.2.
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the RNAi agent îs an siRNA, and the antisense strand of the siRNA comprises at least 15 contiguous nucléotides or 19 contiguous nucléotides of the nucléotide sequence of 5'- UGUGAAGCGAAGUGCACACUU -3’ (SEQ ID NO:119).
In some embodiments, the antisense strand of the siRNA comprises the nucléotide sequence of 5'- UGUGAAGCGAAGUGCACACUU -3' (SEQ ID NO:119).In some embodiments, the antisense strand consists of the nucléotide sequence of 5'UGUGAAGCGAAGUGCACACUU -3' (SEQ ID NO:119). In some embodiments, the sense strand ofthe sîRNA comprises the nucléotide sequence of 5'30 GUGUGCACUUCGCUUCACA -3'(SEQ IDNO:118). In some embodiment, the sense
176 strand of the siRNA consists of the nucléotide sequence of 5'GUGUGCACUUCGCUUCACA -3' (SEQ ID NO:118).
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the RNAi agent is an siRNA, and the antisense strand of the siRNA 5 comprises at least 15 contiguous nucléotides or 19 contiguous nucléotides of the nucléotide sequence of 5'- UAAAAUUGAGAGAAGUCCACCAC -3' (SEQ ID NO:l2l). In some embodiments, the antisense strand ofthe siRNA comprises the nucléotide sequence of 5'- UAAAAUUGAGAGAAGUCCACCAC -3' (SEQ ID NO:l2I). In some embodiments, the antisense strand consists of the nucléotide sequence of 5’- UAAAAUUGAGAGAAGUCCACCAC -3’ (SEQ ID NO:l2l). In some embodiments, the sense strand of the siRNA comprises the nucléotide sequence of 5'GGUGGACUUCUCUCAAUUUUA -3' (SEQ ID NO:l20). In some embodiment, the sense strand of the siRNA consists of the nucléotide sequence of 5'GGUGGACUUCUCUCAAUUUUA -3' (SEQ ID NO: 120).
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the RNAi agent is an siRNA, wherein substantîally ail ofthe nucléotides of said sense strand and substantîally ail ofthe nucléotides of said antisense strand are modified nucléotides, and wherein said sense strand is conjugated to a ligand attached at the 3'-terminus. In particular embodiments, the ligand is one or more
GalNAc dérivatives attached through a monovalent linker, bivalent branched linker, or trivalent branched linker. In certain embodiments, the GalNAc dérivative attached through a linker is is or comprises:
\ΊΊ
In particular embodiments, the siRNA is conjugated to the ligand as shown in the following schematic (i.e., the GalNAc dérivative attached through a linker is):
wherein X is O or S.
In particular embodiments of the above methods, compositions for use, or uses în manufacture, the RNAi agent is an siRNA, wherein at least one nucléotide of the siRNA is a modified nucléotide comprising a deoxy-nucleotide, a 3'-terminal deoxy10 thymine (dT) nucléotide, a 2'-O-methyl modified nucléotide, a 2'-fluoro modified nucléotide, a 2'-deoxy-modified nucléotide, a locked nucléotide, an unlocked nucléotide, a conformat!onally restricted nucléotide, a constrained ethyl nucléotide, an abasic nucléotide, a 2'-amino-modified nucléotide, a 2'-O-allyl-modified nucléotide, 2'C-alkyl-modified nucléotide, 2'-hydroxyl-modified nucléotide, a 2'-methoxyethyl modified nucléotide, a 2'-O-alkyl-modified nucléotide, a morpholino nucléotide, a phosphoramidate, a non-natural base comprising nucléotide, a tetrahydropyran modified nucleotîde, a 1,5-anhydrohexitol modified nucléotide, a cyclohexenyl modified nucléotide, a nucléotide comprising a phosphorothioate group, a nucléotide comprising a mcthylphosphonate group, a nucléotide comprising a 5-phosphate, an adenosine20 glycol nucleic acid, or a nucleotîde comprising a 5'-phosphate mimic. In certain embodiments, the siRNA comprises a phosphate backbone modification, a 2' ribose
178 modification, 5' triphosphate modification, or a GalNAc conjugation modification. In certain embodiments, the phosphate backbone modification comprises a phosphorothioate bond. In certain embodiments, the 2' ribose modification comprises a fluoro or -O-methyl substitution.
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the RNAi agent is an siRNA having a sense strand comprising 5'gsusguGfcAfCfUfucgcuucacaL96 -3' (SEQ ID NO:l22) and an antisense strand comprising 5’- usGfsugaAfgCtGfaaguGfcAfcacsusu -3' (SEQ ID NO:I23), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-Omethylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-Omethyluridine-3'-phosphate, respectîvely;
Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'phosphate, 2’-fluoroguanosine-3'-phosphate, and 2'-fluorouridîne-3'-phosphate, respectîvely;
s is a phosphorothioate linkage; and
L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.
In particular embodiments of the above methods, compositions for use, or uses în manufacture, the RNAi agent is an siRNA having a sense strand comprising 5'gsusguGfcAfCfUfucgcuucacaL96 -3' (SEQ ID NO:l24) and an antisense strand comprising 5'- usGfsuga(Agn)gCfGfaaguGfcAfcacsusu -3' (SEQ ID NO:l25) wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-Omethylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-OmethyluιΊdine-3,-phosphate, respectîvely;
Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectîvely;
(Agn) is adenosine-glycol nucleic acid (GNA);
s is a phosphorothioate linkage; and
L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.
179
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the RNAi agent is an siRNA having a sense strand comprising 5'gsgsuggaCfuUfCfUfcucaAfUfuuuaL96 -3' (SEQ IDNOJ26) and an antisense strand comprising 5'- usAfsaaaUfuGfAfgagaAfgUfccaccsasc -3' (SEQ ID NO;l27), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-Omethylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-Omethy l urid i ne-3 ' -phosphate, respecti vely ;
Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytîdine-3'phosphate, 2'-flLioroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively;
s is a phosphorothioate linkage; and
L96 is N-[tris(GalNAc-aIkyl)-amidodecanoyl)]-4-hydroxyprolinol.
In particular embodiments of the above methods, compositions for use, or uses in manufacture, the subject is a human and a therapeutically effective amount of RNAi or siRNA is administered to the subject; and wherein the effective amount of the RNAi or siRNA is from about l mg/kg to about 8 mg/kg.
In some embodiments of the methods, compositions for use, or uses disclosed herein, the siRNA is administered to the subject twice daily, once daily, every two days, every three days, twice per week, once per week, every other week, every four weeks, or once per month. In some embodiments, wherein the siRNA is administered to the subject every four weeks.
In certain embodiments, the methods include administering two inhibitors of HBV gene expression with an anti-HBV antibody. The two inhibitors of HBV gene expression may be two siRNAs, such as two siRNAs that target different HBV genes. The two different HBV genes may, for example, be HBsAg, and HBV X. The two inhibitors of HBV gene expression may be administered simultaneously. In certain embodiments, two siRNAs each directed to an HBV gene are administered, and the first siRNA has an antisense strand comprising SEQ IDNO:l 19, SEQ ID NO:l23, or SEQ
180
ID NO:I25; and the second siRNA comprises an siRNA having a sense strand that comprises at least 15 contiguous nucléotides of nucléotides 2850-3182 of SEQ ID NO:116. In certain embodiments, two siRNAs each directed to an HBV gene are administered, and the first siRNA has an antisense strand comprising SEQ ID NO: 121 or SEQ ID NO: 127; and the second siRNA comprises an siRNA having a sense strand that comprises at least 15 contiguous nucléotides of nucléotides 2850-3182 of SEQ ID NO: 116. In certain embodiments, two siRNAs each directed to an HBV gene are administered, and the first siRNA has an antisense strand comprising SEQ ID NO: 119, SEQ ID NO: 123 or SEQ ID NO: 125; and the second siRNA has an antisense strand comprising SEQ ID NO: 121 or SEQ IDNO:127. In certain embodiments, the first siRNA has a sense strand comprising SEQ ID NO:118, SEQ ID NO: 122, or SEQ ID NO: 124; and the second siRNA has a sense strand comprising SEQ ID NO: 120 or SEQ ID NO: 126.
In certain embodiments, the anti-HBV antibody and the inhibitor of HBV gene 15 expression exhibit a synergistic therapeutic effect. The term synergy is used to describe a combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent. Thus, where the combined effect of two or more agents resuits in synergistic inhibition of an activity or process, it is intended that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent. The term synergistic therapeutic effect refers to a therapeutic effect observed with a combination of two or more thérapies wherein the therapeutic effect (as measured by any of a number of parameters) is greater than the sum of the individual therapeutic effects observed with the respective individual thérapies.
In some embodiments, an RNAi agent targeting an HBV mRNA is administered to a subject having an HBV infection, and/or an HBV-assocîated disease, such that the expression of one or more HBV genes, HBV ccc DNA levels, HBV antigen levels, HBV viral load levels, ALT, and/or AST, e.g., in a cell, tissue, blood,, or fluid ofthe subject are reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%,
I8l
33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,
48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,
62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.
In some embodiments, an RNAi agent targeting an HBV mRNA is administered to a subject having an HBV infection, and/or an HBV-associated disease, and inhibits HBV gene expression by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or about 100%, i.e., to below the level of détection of the assay.
In some embodiments, the combination therapy according to the présent disclosure comprises administering a nucleot(s)ide analog as a third component. As used herein, the term nucelot(s)ide analog (or polymerase inhibitor or reverse transcriptase inhibitor) is an inhibitor of DNA réplication that is structurally similar to a nucléotide or nucleoside and specifically inhibits réplication ofthe HBV cccDNA and does not significantly inhibit the réplication of the host (e.g., human) DNA. Such inhibitors include tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine (FTC), clevudine, ritonavir, dipivoxil, lobucavir, famvir, N-Acetyl-Cysteine (NAC), PC 1323, theradigm-HBV, thymosîn-alpha, ganciclovir, besifovir (ANA-380/LB80380), and tenofvir-exaliades (TLX/CMX157). ïn certain embodiments, the nucelot(s)ide analog is entecavir (ETV). Nucleot(s)ide analogs are commercially available from a number of sources and are used in the methods provided herein according to their label indication (e.g., typically orally administered at a spécifie dose) or as determined by a skilled practitioner Ên the treatment of HBV.
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The anti-HBV antibody or the inhibitor of HBV gene expression can be présent either în the same pharmaceutical composition as the third active component or, the anti-HBV antibody, the inhibitor of HBV gene expression, and the third active component are présent in three different pharmaceutical compositions. Such different 5 pharmaceutical compositions may be administered either combined/simultaneously or at separate times or at separate locations (e.g., separate parts of the body).
The présent disclosure also provides the following Embodiments:
Embodiment l. An antibody, or an antigen-binding fragment thereof, comprising: (i) a heavy chain variable région (VH) that comprises therein the amino 10 acid sequence of SEQ ID NO.:34, the amino acid sequence of SEQ ID NO.:35 or SEQ ID NO.:36, and the amino acid sequence of SEQ ID NO.:37; and (ii) a light chain variable région (VL) that comprises therein the amino acid sequence any one of SEQ ID NOs.:41,40, 42, and 43, the amino acid sequence according to any one of SEQ ID NOs:49,44-48, and 50-53, and the amino sequence according to SEQ ID NO.:55 or 56, 15 wherein, optionally, the VL comprises a R60N substitution mutation, a R60A substitution mutation, a R60K substitution mutation, a S64A substitution mutation, a I74A substitution mutation, or any combination thereof, relative to SEQ ID NO.:58 and wherein the amino acid numbering of the substitution mutation(s) îs according to SEQ ID NO.:58, and still further optionally wherein the VL does not comprise any further mutation(s) relative to SEQ ID NO.:58, and wherein the antibody or antigen-binding fragment thereof is capable of binding to the antigenic loop région of HBsAg and, optionally, neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, I, θΓ Λ or any combination thereof.
Embodiment 2. The antibody or antigen-binding fragment of Embodiment 25 1, comprising: (i) in the VH, the amino acid sequences according to SEQ ID NOs.:34,
35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:41,49, and 55, respectively; (ii) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, and in the VL, the amino acid sequences according to SEQ ID NOs.: 41, 46, and 55, respectively; (iii) in the VH, the amino acid sequences 30 according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid
183 sequences according to SEQ ID NOs.: 41, 47, and 55, respectively; (iv) m the VH, the amino acid sequences according to SEQ ID NOs,:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ lDNOs.:41,48, and 55, respectively; (v) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:41, 45, and 55, respectively; (vi) in the VH, the amino acid sequences according to SEQ ID NOs,:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:41, 50, and 55, respectively; (viî) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ IDNOs,:41,51, and 55, respectively; (viii) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:41, 52, and 55, respectively; or (ix) in the VH, the amino acid sequences according to SEQ ID NOs.:34, 35, and 37, respectively, and in the VL, the amino acid sequences according to SEQ ID NOs.:41, 53, and 55, respectively.
Embodiment 3. An antibody, or an antigen-binding fragment thereof, comprising: (i) a heavy chain variable région (VH) comprising a CDRHl amino acid sequence according to SEQ ID NO.:34, a CDRH2 amino acid sequence according to SEQ ID NO.:35 or 36, and a CDRH3 amino acid sequence according to SEQ ID NO.:37; and (ii) a light chain variable région (VL) comprising a CDRJ J amino acid sequence according to any one of SEQ ID NOs.:40-43, a CDRL2 amino acid sequence according to any one of SEQ ID NOs:49, 44-48, and 50-53, and a CDRL3 amino acid sequence according to SEQ ID NO.:55 or 56, wherein CDRs are defined according to the CCG nnmbering system, and wherein the antibody or antigen-binding fragment thereof is capable of binding to the antigenic loop région of HBsAg and, optionally, neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, 1, or J, or any combination thereof, provided that the antibody or antigen-binding fragment does not comprise CDRHl, CDRH2, CDRH3, CDRLI, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs.:34, 35, 37, 41, 44, and 45, respectively.
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Embodiment 4. The antibody or antigen-binding fragment of Embodiment 3, wherein the CDRHl, CDRH2, CDRH3, CDRLI, CDRL2, and CDRL3 amino acid sequences are according to SEQ ID NOs.: (i) 34, 35, 37, 41,49, and 55, respectively; (ii) 34, 35, 37, 41,46, and 55, respectiveiy; (iiî) 34, 35, 37, 41,47, and 55, respectively; 5 (iv) 34, 35, 37, 41, 48, and 55, respectively; (v) 34, 35, 37, 41,45, and 55, respectively; (vi) 34, 35, 37, 41, 50, and 55, respectively; (vîî) 34, 35, 37, 41, 5 1, and 55, respectively; (viii) 34, 35, 37, 41,52, and 55, respectively; (ix) 34, 35, 37, 41, 53, and 55, respectively; or (x) 34, 35, 37, 41,44, and 55, respectively.
Embodiment 5. An antibody, or an antigen-binding fragment thereof, 10 comprising a heavy chain variable région (VH) and a light chain variable région (VL), wherein the VH and the VL comprise CDRHl, CDRH2, CDRH3 and CDRLI, CDRL2, CDRL3, respectively, according to: HBC34-v40; HBC34-v36; HBC34-v37; HBC34v38; HBC34-v39; HBC34-v41 ; HBC34-v42; HBC34-v43; HBC34-v44; HBC34-v45; HBC34-v46; HBC34-v47; HBC34-v48; HBC34-v49; or HBC34-v50, wherein the CDRs are defined according to IMGT numbering, optionally wherein the VL further comprises a R60N substitution mutation, a R60A substitution mutation, a R60K substitution mutation, a S64A substitution mutation, a I74A substitution mutation, or any combination thereof, relative to SEQ ID NO.:58 and wherein the amino acid numbering of the substitution mutation(s) îs according to SEQ
ID NO.:58, and further optionally wherein the VL does not comprise any further mutation(s) relative to SEQ ID NO.:58.
Embodiment 6. An antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable région (VH) and a light chain variable région (VL), wherein the VH and the VL comprise CDRHl, CDRH2, CDRH3 and CDRLI, CDRL2, 25 CDRL3, respectively, according to: HBC34-v40; HBC34-v36; HBC34-v37; HBC34v38; HBC34-v39; HBC34-v41; HBC34-v42; HBC34-v43; HBC34-v44; HBC34-v45; HBC34-v46; HBC34-v47; HBC34-v48; HBC34-v49; or HBC34-v50, wherein the CDRs are defined according to CCG numbering, optionally wherein the VL further comprises a R60N substitution mutation, a R60A substitution mutation, a R60K substitution mutation, a S64A substitution mutation, a I74A substitution
185 mutation, or any combination thereof, relative to SEQ ID NO.:58 and wherein the amino acid numbering of the substitution mutation(s) is according to SEQ ID NO.:58, and further optionally wherein the VL does not comprise any other mutation(s) relative to SEQ IDNO.:58.
Embodiment 7. The antibody or antigen-binding fragment of any one of Embodiments l -6, wherein: (i) the VH comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 90% identity to lhe amino acid sequence set forth in any one of SEQ ID NOs.: 62, 5861,63-66, 69, 7 Land 72.
Embodiment 8. The antibody or antigen-binding fragment of any one of Embodiments 1-7, wherein: (i) the VH comprises or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9830, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and/or (îi) the VL comprises or consists of an amino acid sequence having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value therebetween) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 62, 58-61, 63-66, 69, 71, and 72.
Embodiment 9. The antibody or antigen-binding fragment of any one of Embodiments 1-8, wherein the VH and the VL comprise or consist of amino acid sequences having at least 90% (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any non-integer value therebetween) identity to the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 62, respectively; (ii) 38 and 59, respectively; (iii) 38 and 60, respectively; (iv) 38 and 61, respectively; (v) 38 and 58, respectively; (vi) 38 and 63, respectively; (vii) 38 and 64, respectively; (viii) 38 and 65, respectively; (ix) 38 and 66, respectively; (x) 38 and 71, respectively; or (xi) 38 and 72, respectively.
Embodiment 10. An antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable région (VH) that comprises or consists of the amino acid sequence of SEQ ID NO.:38 or 39, and a lîght chaîn variable région (VL) that comprises a variant of any one of SEQ ID NOs.:62, 57-61, and 63-72, wherein the
186
variant comprises any one or more of the following mutations: R60A; R60N; R60K; S64A; and I74A, and wherein, optionaliy, the VL variant does not comprise any further mutations as compared to SEQ ID NO.:62, 57-61, and 63-72, respectively.
Embodiment 11. An antibody, or an antigen-binding fragment thereof, 5 comprising a heavy chain variable région (VH) that comprises or consists of the amino acid sequence of SEQ ID NO.:38 or 39, and a light chain variable région (VL) that comprises a variant of any one of SEQ ID NOs.:62, 57-61, and 63-72, wherein the variant comprises a substitution mutation (such as, for example, a conservative amino acid substitution, or a mutation to a germline-encoded amino acid) at Q78, D8l, or both, and wherein, optionaliy, the VL variant does not comprise any further mutations as compared to SEQ ID NO.:62, 57-61, and 63-72, respectively.
Embodiment 12. The antibody or antigen-binding fragment of any one of Embodiments 1 -9, wherein: the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 38 or 39; and/or the VL comprises or consists of the amino 15 acid sequence set forth in any one of SEQ IDNOs.: 62, 58-61, 63-66, 69, 71, or 72.
Embodiment 13. Lhe antibody or antigen-binding fragment of any one of Embodiments 1-9 and 12, wherein the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 62, respectively; (ii) 38 and 59, respectively; (iii) 38 and 60, respectively; (iv) 38 and 61, respectively; (v) 38 and 58, 20 respectively; (vi) 38 and 63, respectively; (vii) 38 and 64, respectively; (viii) 38 and 65, respectively; (ix) 38 and 66, respectively; (x) 38 and 71, respectively; or (xi) 38 and 72, respectively.
Embodiment 14. An antibody or antigen-binding fragment, comprising a heavy chain variable région (VH) and a light chain variable région (VL), wherein the 25 VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 62, respectively; (ii) 38 and 66, respectively; (iii) 38 and 67, respectively; (iv) 38 and 68, respectively; or (v) 38 and 72, respectively, wherein the antibody or antigen-binding fragment thereof is capable of binding to the antigenic loop région of HBsAg and neutralizing infection by a hepatitis B virus 30 (HBV) of génotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof.
187
Embodiment 15. The antibody or antigen-binding fragment ofany one of Embodiments l-I4, which is capable of neutralizing infection by a hepatitis D virus (HDV).
Embodiment 16. The antibody or antigen-binding fragment of any one of Embodiments l-l5, wherein, in a sample comprising a pluralîty of the antibody or antigen-binding fragment, less than 12%, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less of the pluralîty is comprised in a dimer when the sample has been incubated for about 120 to about 168 hours at about 40°C, wherein, optionally, the presence of dimer is determined by absolute size-exciusion chromatography.
Embodiment 17. The antibody or antigen-binding fragment of any one of
Embodiments l -16, wherein incubation of a pluralîty of the antibody or antigen-binding fragment results in reduced fonnation of a dîmer as compared to incubation of a pluralîty of a reference antibody or antigen-binding fragment, wherein the reference antibody or antigen-binding fragment comprises CDRHl, CDRH2, CDRH3, CDRLl, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 41,44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57, and wherein, optionally, the presence of antibody dimer is determined by absolute size-exclusion chromatography.
Embodiment 18. The antibody or antigen-binding fragment of any one of Embodiments l -l 7, which forms a lower amount of dimer, and/or forms diiners at a reduced frequency and/or as a lower percentage of total antibody or antigen-binding fragment molécules in a sample or composition as compared to a reference antibody:
(i) in a 5-day, a 15-day, and/or a 32-day incubation at 4°C;
(ii) in a 5-day, a 15-day, and/or a 32-day incubation at 25°C; and/or (îii) in a 5-day, a 15-day, and/or a 32-day incubation at 40°C, wherein the reference antibody or antigen-binding fragment comprises CDRHl, CDRH2, CDRH3, CDRLl, CDRL2, and CDRL3 amino acid sequences according to
188 the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.;38 and the VL amino acid sequence set forth in SEQ ID NO.:57.
Embodiment 19. The antibody or antigen-binding fragment of any one of Embodiments 1-18, wherein apercentage of antibody orantigen-binding fragment molécules in a composition that are comprised in a dimer is less than 4/5, less than 3/4, less than 1/2, less than 1/3, less than 1/4, less than 1/5, less than 1/6, less than 1/7, less than 1/8, less than 1/9, or less than 1/10 the percentage of reference antibody molécules in a composition that are présent in a dimer, respectively.
Embodiment 20. The antibody or antigen-binding fragment of any one of Embodiments 1-19, wherein a host cell transfected with a polynucleotide encoding the antibody or antigen-binding fragment provides L5x or more, 2x or more, 3x or more, or 4x or more the amount of antibody or antigen-binding fragment, respectively, than a reference host cell transfected with a polynucleotide encoding a reference antibody or antigen-binding fragment, wherein the reference antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57.
Embodiment 21. The antibody or antigen-binding fragment of any one of Embodiments 1-20, wherein the antibody or antigen-binding fragment thereof is produced in transfected cells at a higher titer as compared to a reference antibody or antigen-binding fragment is produced in reference transfected cells, wherein the reference antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 41,44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57.
Embodiment 22. The antibody or antigen-binding fragment of any one of Embodiments 1-21, wherein the antibody or antigen-binding fragment thereof is
189 produced in transfected cells at titers of at least l .5-fold, at least 2-fold, at least 3-fold, or at least 4-fold, higher than the titer at which a reference antibody or antigen-binding fragment is produced, wherein the reference antibody or antigen-binding fragment comprises CDRHl, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs/34, 35, 37, 4I, 44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57.
Embodiment 23. The antibody or antigen-binding fragment of any one of Embodiments l-22, wherein the antibody or antigen-binding fragment is capable of binding to a HBsAg (adw) with an EC50 (ng/ml) of about 3.2 or less, less than 3.0, less than 2.5, less than 2.0, less than L5, or less than L0.
Embodiment 24. The antibody or antigen-binding fragment of any one of Embodiments l-23, wherein the antibody or antigen-binding fragment is capable of binding to a HBsAg (e.g., of subtype adw) with an EC50 (ng/ml) of less than 3.5, less than 3.4, less than 3.3, less than 3.2, less than 3.1, less than 3.0, less than 2.9, less than 2.8, less than 2.7, less than 2.6, less than 2.5, less than 2.4, less than 2.3, less than 2.1, less than 2.0, less than l .9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than l .2, less than 1.1, or less than 1.0.
Embodiment 25. The antibody or antigen-binding fragment of any one of Embodiments 1-24, wherein the antibody or antigen-binding fragment is capable of binding to a HBsAg (e.g, of subtype adw) with an EC50 (ng/ml) of between 0.9 and 2.0, or of between 0.9 and 1.9, or of between 0.9 and 1.8, or of between 0.9 and 1.7, or of between 0.9 and 1.6, or of between 0.9 and 1.5, or of betw'een 0.9 and l .4, or of between 0.9 and 1.3, or of between 0.9 and 1.2, or of between 0.9 and Ll,orof between 0.9 and 1.0, or of between 1.0 and 2.0.
Embodiment 26. The antibody or antigen-binding fragment of any one of Embodiments 1 -25, wLerein the antibody or antigen-binding fragment is capable of binding to a HBsAg (adw) with an EC50 (ng/ml) of 2.0 or less.
190
Embodiment 27. The antibody or antigen-binding fragment of any one of Embodiments [-26, which has a hepatitis B virus neutralization of infection EC50 of less than 20 ng/ml, preferably 15 ng/ml or less, more preferably 10 ng/mL or less.
Embodiment 28. The antibody or antigen-binding fragment of any one of
Embodiments I -27, wherein the antibody or antigen-binding fragment thereof is capable of neutralizing hepatitis B virus infection with a neutralization of infection EC50ofl8, 17, 16, 15, 14, 13, 12, H, 10, 9, 8, or 7 ng/mL.
Embodiment 29. The antibody or antigen-binding fragment of any one of Embodiments l-28, wherein the antibody or antigen-binding fragment thereof is capable of neutralizing hepatitis B virus infection with a neutralization of infection EC50 thaï is lower than the neutralization of infection EC50 of a reference antibody or antigen-binding fragment that comprises CDRHl, CDRH2, CDRH3, CDRLl, CDRL2, and CDRL3 amino acid sequences according to the amino acid sequences set forth in SEQ ID NOs.:34, 35, 37, 41,44, and 55, respectively, and optionally comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ IDNO.:57.
Embodiment 30. The antibody or antigen-binding fragment of any one of Embodiments l-29, wherein the antibody, or the antigen-binding fragment thereof, comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, a Fab, a Fab’, a F(ab’)2, a Fv, or a scFv.
Embodiment 31. The antibody or antigen-binding fragment of any one of Embodiments l-30, wherein the antibody or antigen-binding fragment is a multi-specific antibody or antigen-binding fragment.
Embodiment 32. The antibody or antigen-binding fragment of any one of
Embodiments l -31, wherein the antibody or antigen-binding fragment is a bispecific antibody or antigen-binding fragment.
Embodiment 33. The antibody of any one of Embodiments l-32, or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment comprises a Fc moiety.
I9l
Embodiment 34. The antibody or antigen-binding fragment of Embodiment 33, wherein the Fc moiety comprises a mutation that enhances binding to FcRn as compared to a reference Fc moiety that does not comprise the mutation.
Embodiment 35. The antibody or antigen-binding fragment of Embodiment 33 or 34, wherein the Fc moiety comprises a mutation that enhances binding to a FcyR, preferably a FcyRIIA and/or a FcyRIIIA, as compared to a reference Fc moiety that does not comprise the mutation.
Embodiment 36. The antibody or antigen-binding fragment of any one of Embodiments 33-35, wherein the Fc moiety is an IgG isotype, such as IgGl, or is derîved from an IgG isotype, such as IgGl.
Embodiment 37. The antibody or antigen-binding fragment of any one of Embodiments 33-36, which comprises or is derîved from Ig Glml7, l (IgHGI*0l).
Embodiment 38. The antibody or antigen-binding fragment of any one of Embodiments 34-37, wherein the mutation that enhances binding to FcRn comprises: (î) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q31II; (v) P257I/N434H; (vi) D376V/N434H; (vii) T307A/E380A/N434A; or (viîi) any combination of (i)-(vii), wherein amino acid numberîng of the Fc moiety is according to the EU numbering system.
Embodiment 39. The antibody or antigen-binding fragment of Embodiment 38, wherein the mutation that enhances binding to FcRn comprises M428L/N434S.
Embodiment 40. The antibody or antigen-binding fragment of any one of Embodiments 35-39, wherein the mutation that enhances binding to a FcyR comprises S239D; I332E; A330L; G236A; or any combination thereof, wherein amino acid numberîng of the Fc moiety is according to the EU numbering system.
Embodiment 4]. The antibody or antigen-binding fragment of Embodiment40, wherein the mutation that enhances binding to a FcyR comprises: (i) S239D/I332E; (ii) S239D/A330L/I332E; (iii) G236A/S239D/1332E; or (iv) G236A/A330L/I332E.
Embodiment 42. The antibody or antigen-binding fragment of Embodiment 40 or 41, wherein the mutation that enhances binding to a FcyR comprises or consists of
192
G236A/A330L/I332E, and optionally wherein the antibody or antigen-binding fragment does not comprise S239D, and wherein the antibody or antigen-binding fragment further optionally comprises a native S at position 239.
Embodiment 43. The antibody or antigen-binding fragment of any one of Embodiments 33-42, wherein the Fc moiety comprises the amino acid substitution mutations: M428L; N434S; G236A; A330L; and I332E, and optionally does not comprise S239D.
Embodiment 44. The antibody or antigen-binding fragment of any one of Embodiments l-43, comprising a light chain constant région (CL) that comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.:79.
Embodiment 45. The antibody or antigen-binding fragment of any one of Embodiments 1 -44, comprising a CH1-CH2-CH3 that comprises or consists of an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.:73, or a variant thereof that comprises one or more of the following amino acid substitutions (EU numbering): G236A; A330L; I332E; M428L; N434S.
Embodiment 46. The antibody or antigen-binding fragment of Embodiment
45, wherein the CH1-CH2-CH3 has a C-terminal lysine removed.
Embodiment 47. An antibody comprising: a heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:75, optionally with the C-terminal lysine removed; and a light chain (LC), wherein the LC comprises or consists of (i) the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 5861, and 63-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.
Embodiment 48. The antibody of Embodiment 47, wherein the LC comprises the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 66, 67, and 72.
Embodiment 49. An antibody comprising: a heavy chain (HC) comprising or consislîng of the amino acid sequence set forth in SEQ ID NO,:76, optionally with the C-terminal lysine removed, and a light chain (LC), wherein the LC comprises or
193 consists of (i) the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 5861, and 63-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.
Embodiment 50. The antibody of Embodiment 49, wherein the LC comprises the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 66, 67, and 72.
Embodiment 51. An antibody comprising: a heavy chain (HC) comprising or consîsting of the amino acid sequence set forth in SEQ ID NO.:77, optionally wirh the C-terminal lysine removed, and a light chain (LC), wherein the LC comprises or consists of (i) the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 5861, and 63-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.
Embodiment 52. The antibody of Embodiment 51, wherein the LC comprises the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 66, 67, and 72.
Embodiment 53. An antibody comprising: a heavy chain (HC) comprising or consîsting of the amino acid sequence set forth in SEQ ID NO.:78, optionally with the C-tenninal lysine removed, and a light chain (LC), wherein the LC comprises or consists of (i) the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 5861, and 63-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.
Embodiment 54. The antibody of Embodiment 53, wherein the LC comprises the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 66, 67, and 72.
Embodiment 55. The antibody or antigen-binding fragment of any one of Embodiments 1-54, wherein the antibody or the antigen-binding fragment is capable of binding an HBsAg of a génotype selected from the HBsAg génotypes A, B, C, D, E, F, G, H, I, and J, or any combination thereof.
Embodiment 56. The antibody or antigen-binding fragment of any one of Embodiments 1-55, wherein the antibody or antigen-binding fragment is capable of reducing a sérum concentration of HBV DNA in a mammal having an HBV infection.
194
Embodiment 57, The antibody or antigen-binding fragment of any one of Embodiments 1 -56, wherein the antibody or antigen-binding fragment is capable of reducing a sérum concentration of HBsAg in a mammal having an E1BV infection.
Embodiment 58. The antibody or antigen binding fragment of any one of Embodiments 1-57, wherein the antibody or antigen-binding fragment is capable of reducing a sérum concentration of HBeAg in a mammal having an HBV infection.
Embodiment 59. The antibody or antigen binding fragment of any one of Embodiments 1 -58, wherein the antibody or antigen binding fragment is capable of reducing a sérum concentration of HBcrAg in a mammal having an HBV infection.
Embodiment 60. A polynucleotide comprising a nucleotîde sequence that encodes the antibody, or the antigen-binding fragment, of any one of Embodiments 159.
Embodiment 61. A polynucleotide encoding a light chain variable région (VL) and, optionally, a light chain constant région (CL) of the antibody, or the antigenbinding fragment, of any one of Embodiments 1-59.
Embodiment 62. The polynucleotide of Embodiment 61, wherein the nucleotîde sequence that encodes the antibody or the antigen-binding fragment is codon optimized for expression in a host cell.
Embodiment 63. The polynucleotide of Embodiment 62, comprising a nucleotîde sequence having at least 50% identity to the nucleotîde sequence according to any one of SEQ ID NOs:89, 85-88, and 90-99.
Embodiment 64. The polynucleotide of any one of Embodiments 60-63, comprising (i) the polynucleotide sequence set forth in SEQ ID NO.:8I or SEQ ID NO.:82, and (ii) the polynucleotide sequence set forth in any one or more of SEQ ID NOs.:89, 85-88, and 90-99.
Embodiment 65. The polynucleotide of any one of Embodiments 60-63, comprising (i) the polynucleotide sequence set forth in SEQ ID NO.:83, and (ii) the polynucleotide sequence set forth in any one or more of SEQ ID NOs.:89, 85-88, and 90-99.
195
Embodiment 66. The poiynucleotide of any one of Embodiments 60-63, comprising (i) the poiynucleotide sequence set forth in SEQ ID NO.:84, and (ii) the poiynucleotide sequence set forth in any one or more of of SEQ ID NOs.:89, 85-88, and 90-99.
Embodiment 67. A vector comprising the poiynucleotide of any one of
Embodiments 60-66.
Embodiment 68. The vector of Embodiment 67, wherein the vector comprises a lentiviral vector or a retroviral vector.
Embodiment 69. A host cell comprising the poiynucleotide of any one of
Embodiments 60-66 and/or the vector of Embodiment 67 or 68.
Embodiment 70. A pharmaceutical composition comprising: (i) the antibody or antigen binding fragment of any one of Embodiments I -59; (ii) the poiynucleotide according to any one of Embodiments 60-66; (iii) the vector according to Embodiment 67 or 68; (iv) the host cell of Embodiment 69; or (v) any combination 15 of (i)-(iv), and a pharmaceutically acceptable excipient, diluent or carrier.
Embodiment 71. A kit comprising:
(a) a component selected from: (i) the antibody or antigen-binding fragment of any one of Embodiments l-59; (ii) the poiynucleotide according to any one of Embodiments 60-66; (iii) the vector according to Embodiment 67 or 68;
(iv) the host cell of Embodiment 69; (v) the pharmaceutical composition of
Embodiment 70; or (vi) any combination of (i)-(vi); and (b) (l) instructions for using the component to prevent, treat, attenuate, and/or diagnose a hepatitis B infection and/or a hepatitis D infection and/or (2) a means for administerîng the component to the subject, such as a syringe.
Embodiment 72. The composition of Embodiment 70 or the kit of
Embodiment 71, further comprising: (i) a polymerase inhibitor, wherein the polymerase inhibitor optionaliy comprises Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir, or any combination thereof; (ii) an interferon, wherein the interferon optionaliy comprises IFNbeta and/or IFNalpha; (iîî) a checkpoint inhibitor, wherein the checkpoint inhibitor optionaliy comprises an anti-PD-l antibody or antigen binding
196 fragment thereof, an anti-PD-Ll antibody or antigen binding fragment thereof, and/or an antî-CELA4 antibody or antigen binding fragment thereof; (iv) an agonist of a stimulatory immune checkpoint molécule; or (v) any combination of (i)-(iv).
Embodiment 73. The composition or kit of Embodiment 72, wherein the polymerase inhibitor comprises lamivudine.
Embodiment 74. A method of producing the antibody or antigen binding fragment of any one of Embodiments l-59, comprising culturing the host cell of Embodiment 69 under conditions and for a time sufficient to produce the antibody or antigen-binding fragment.
Embodiment 75. Use of: (i) the antibody or antigen-binding fragment of any one of Embodiments l-59; (ii) the polynucleotide of any one of Embodiments 6066; (iii) the vector of Embodiment 67 or 68; (iv) the host cell of Embodiment 69; and/or (v) the pharmaceutical composition of Embodiment 70, 72, or 73, in the manufacture of a médicament to prevent, treat, attenuate, and/or diagnose a hepatitis B infection and/or a hepatitis D infection in a subject.
Embodiment 76. A method of treating, preventing, and/or attenuating a hepatitis B and/or hepatitis D infection in a subject, comprising administering to the subject an effective amount of: (i) the antibody or antigen-binding fragment of any one of Embodiments l -59; (ii) the polynucleotide of any one of Embodiments 60-66; (iii) the vector of Embodiment 67 or 68; (iv) the host cell of Embodiment 69; and/or (v) the pharmaceutical composition of Embodiment 70, 72, or 73.
Embodiment 77. The method of Embodiment 76, further comprising administering to the subject one or more of: (vi) a polymerase inhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovir, Entecavir,
Telbivudine, Tenofovir, or any combination thereof; (vii) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha; (viii) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an anti-PD-l antibody or antigen binding fragment thereof, an anti-PD-Ll antibody or antigen binding fragment thereof, and/or an anti-CTLA4 antibody or antigen binding fragment thereof; (ix) an agonist of a stimulatory immune checkpoint molécule; or (x) any combination of (vi)-(ix).
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Embodiment 78. The method of Embodiment 76 or 77, wherein the hepatitis B infection is a chronic hepatitis B infection.
Embodiment 79. The method of any one of Embodiments 76-78, wherein the subject has received a liver transplant.
Embodiment 80. The method of any one of Embodiments 76-79, wherein the subject is non-immunized against hepatitis B.
Embodiment 81. The method of any one of Embodiments 76-80, wherein the subject is a newborn.
Embodiment 82. The method of any one of Embodiments 76-81, wherein 10 the subject is undergoing or has undergone hemodialysis.
Embodiment 83. The method of any one of Embodiments 76-82, wherein the method comprises administering to the subject a single dose of a pharmaceutical composition comprising the antibody or antigen-binding fragment.
Embodiment 84. The method of Embodiment 83, wherein the single dose 15 ofthe pharmaceutical composition comprises the antibody in a range from 2 to 18 mg/kg (subject body weight).
Embodiment 85. The method of Embodiment 83 or 84, wherein the single dose of the pharmaceutical composition comprises up to 6 mg, up to 10 mg, up to 15 mg, up to 18 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up 20 to 50 mg, up to 55 mg, up to 60 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of the antibody, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 3000 mg, or in a range from 5 mg to 3000 mg, or in a range from 10 mg to 3000 mg, or in a range from 25 mg to 3000 25 mg, or in a range from 30 mg to 3000 mg, or in a range from 50 mg to 3000 mg, or in a range from 60 mg to 3000 mg, or in a range from 75 mg to 3000 mg, or in a range from 90 mg to 3000 mg, or in a range from 100 mg to 3000 mg, or in a range from 150 mg to 3000 mg. or in a range from 200 mg to 3000 mg, or in a range from 300 mg to 3000 mg, or in a range from 500 mg to 3000 mg, or in a range from 750 mg to 3000 mg, or in
198 •
a range from 900 mg to 3000 mg, or in a range from 1500 mg to 3000 mg, or in a range from 2000 mg to 3000 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from l mg to 900 mg, or in a range from 5 mg to 900 mg, or in a range from 10 mg to 900 mg, or in a range from 25 mg to 900 mg, or in a range from 30 mg to 900 mg, or in a range from 50 mg to 900 mg, or in a range from 60 mg to 900 mg, or in a range from 75 mg to 900 mg, or in a range from 90 mg to 900 mg, or in a range from 100 mg to 900 mg, or in a range from 150 mg to 900 mg, or in a range from 200 mg to 900 mg, or in a range from 300 mg to 900 mg, or in a range from 500 mg to 900 mg, or in a range from 750 mg to 900 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from l mg to 500 mg, or in a range from 5 mg to 500 mg, or in a range from 10 mg to 500 mg, or in a range from 25 mg to 500 mg, or in a range from 30 mg to 500 mg, or in a range from 50 mg to 500 mg, or in a range from 60 mg to 500 mg, or in a range from 75 mg to 500 mg, or in a range from 90 mg to 500 mg, or in a range from 100 mg to 500 mg, or in a range from 150 mg to 500 mg, or in a range from 200 mg to 500 mg, or in a range from 300 mg to 500 mg, or in a range from 400 mg to 500 mg, or wherein the single dose of the pharmaceutical composition comprises lhe antibody in an amount that is in a range from 1 mg to 300 mg, or in a range from 5 mg to 300 mg, or in a range from 10 mg to 300 mg, or in a range from 25 mg to 300 mg, or in a range from 30 mg to 300 mg, or in a range from 50 mg to 300 mg, or in a range from 60 mg to 300 mg, or in a range from 75mgto 300 mg, or in a range from 90 mg to 300 mg, or in a range from 100 mg to 300 mg, or in a range from 150 mg to 300 mg, or in a range from 200 mg to 300 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 200 mg, or in a range from 5 mg to 200 mg, or in a range from 10 mg to 200 mg, or in a range from 25 mg to 200 mg, or 30 in a range from 30 mg to 200 mg, or in a range from 50 mg to 200 mg, or in a range
199 from 60 mg to 200 mg, or in a range from 75 mg to 200 mg, or in a range from 90 mg to 200 mg, or in a range from 100 mg to 200 mg, or în a range from 150 mg to 200 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is în a range from l mg to 100 mg, or în a range from 5 mg to 100 mg, or in a range from 10 mg to 100 mg, or in a range from 25 mg to 100 mg, or in a range from 30 mg to 100 mg, or in a range from 50 mg to 100 mg, or în a range from 60 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 90 mg to 100 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from l mg to 25 mg, or in a range from 5 mg to 25 mg, or in a range from 10 mg to 25 mg, or în a range from 15 mg to 25 mg, or in a range from 20 mg to 25 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 50 mg, or in a range from 1 mg to 25 mg, or in a range from 5 mg to 50 mg, or in a range from 5 mg to 25 mg, or în a range from 10 to 50 mg, or in a range from 10 to 25 mg, or in a range from 1 to 1 5 mg, or in a range from 5 mg to 15 mg, or in a range from 10 mg to 15 mg,or wherein the single dose of the pharmaceutical composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25,30,35,40,45, 50,55,60,65, 70, 75,80, 85,90, 95, 100, 105, 110, i 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265,
270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350,
355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435,
440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520,
525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605,
610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690,
695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775,
780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860,
865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945,
950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 mg, or more, ofthe antibody,
200 •
or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is less than 3000 mg, less than 2500 mg, less than 2000 mg, less than 1500 mg, less than 1000 mg, less than 900 mg, less than 500 mg, less than 300 mg, Less than 200 mg, less than 100 mg, less than 90 mg, less than 75 mg, less than 50 5 mg, less than 25 mg, or less than 10 mg, but is more than 1 mg, more than 2 mg, more than 3 mg, more than 4 mg, or more than 5 mg.
Embodiment 86. The method of any one of Embodiments 83-85, wherein the single dose of the pharmaceutical composition comprises the antibody at a concentration in a range from 100 mg/mL to 200 mg/mL, such as 100 mg/mL, 110 10 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, or 200 mg/mL, preferably 150 mg/mL.
Embodiment 87. The method of any one of Embodiments 83-86, wherein the single dose of the pharmaceutical composition comprises about 75 mg of the antibody.
Embodiment 88. The method of any one of Embodiments 83-87, wlierein the single dose ofthe pharmaceutical composition comprises about 90 mg of the antibody.
Embodiment 89. The method of any one of Embodiments 83-88, wherein the single dose ofthe pharmaceutical composition comprises up to 300 mg of the 20 antibody.
Embodiment 90. The method of any one of Embodiments 83-89, wherein the single dose of the pharmaceutical composition comprises up to 900 mg of the antibody.
Embodiment 91. The method of any one of Embodiments 83-90, wherein 25 the single dose of the pharmaceutical composition comprises up to 3,000 mg of the antibody.
Embodiment 92. The method of any one of Embodiments 83-91, wherein the method comprises administering the single dose by subcutaneous injection, optionally wherein the single dose comprises or consists of 6 mg of the antibody or 18 30 mg of the antibody.
201
Embodiment 93. The method of any one of Embodiments 83-92, wherein the method comprises administering the single dose by intravenous injection.
Embodiment 94. The method of any one of Embodiments 83-93, wherein the pharmaceutical composition further comprises water, optionally U SP water.
Embodiment 95. The method of any one of Embodiments 83-94, wherein the pharmaceutical composition further comprises histidine, optionally at a concentration in a range from 10 mM to 40 mM, such as 20 mM, in the pharmaceutical composition.
Embodiment 96. The method of any one of Embodiments 83-95, wherein the pharmaceutical composition further comprises a disaccharide, such as sucrose, optionally at 5%, 6%, 7%, 8%, or 9%, preferably about 7% (w/v).
Embodiment 97. The method of any one of Embodiments 83-96, wherein the pharmaceutical composition further comprises a surfactant or a triblock copolymer, optionally a polysorbate or poloxamer-l 88, preferably polysorbate 80 (PS80), wherein, optionally, the polysorbate or poloxamer-l88 is présent in a range from 0.01% to 0.05% (w/v), preferably 0.02% (w/v).
Embodiment 98. The method of any one of Embodiments 83-97, wherein the pharmaceutical composition has a pH in a range from 5.8 to 6.2, in a range from 5.9 to 6. L, or of 5.8, of 5.9, of 6.0, of 6.1, or of 6.2.
Embodiment 99. The method of Embodiment 98, wherein the pharmaceutical composition comprises:
(î ) the antibody at 150 mg/mL;
(ii) USP water;
(iii) 20 mM histidine;
(iv) 7% sucrose; and (v) 0.02% PS80, wherein the pharmaceutical composition comprises a pH of 6.
Embodiment 100. The method of any one of Embodiments 83-99, wherein the subject is an adult.
202
Φ
Embodiment ΙΟΙ. The method of Embodiment 100, wherein the subject is in a l'ange from 18 years of âge to 65 years of âge.
Embodiment 102. The method of any one of Embodiments 83-101, wherein the subject weighs from 40 kg to 125 kg and/or the subject has a body mass 5 index (BMI) from 18 to 35 kg/m2.
Embodiment 103. The method of any one of Embodiments 83-102, wherein the subject has a chronic HBV infection; e.g., defined by positive sérum HBsAg, HBV DNA, and/or HBeAg on 2 occasions, wherein the 2 occasions are at least 6 months apart.
Embodiment 104. The method of any one of Embodiments 83-103, wherein the subject does not hâve cirrhosis.
Embodiment 105. The method of Embodiment 104, wherein absence of cirrhosis is determined by:
Fibroscan évaluation (e.g., within 6 months prior to administering the single 15 dose of the pharmaceutical composition); or liver biopsy (e.g., within 12 months prior to administering the single dose of the pharmaceutical composition), wherein, preferably the absence of cirrhosis is determined by the absence of Metavir F3 fîbrosis or the absence of F4 cirrhosis.
Embodiment 106. The method of any one of Embodiments 83-105, wherein the subject has received a nucleos(t)ide reverse transcriptase inhibitor (NRTI), optionally within 120 days, further optionally within 60 days, prior to the single dose being administered.
Embodiment 107. The method of Embodiment 106, wherein the NRTI 25 comprises one or more of: tenofovir; tenofovir disoproxil (e.g., tenofovir disproxil fumarate); tenofovir alafenamide; Entecavir; Lamivudine; Adefovir; and adefovir dipîvoxîl
Embodiment 108. The method of any one of Embodiments 83-107, wherein the subject has a sérum HBV DNA concentration of less than 100 lU/mL no more than 28 days prior to the single dose being administered.
203
Embodiment 109. The method of any one of Embodiments 83-l08, wherein the subject has a sérum HBsAg concentration of less than 3,000 lU/mL prior to the single dose being administered, and optionally less than 1,000 IU/mL prior to the single dose being administered.
Embodiment 110. The method of any one of Embodiments 83-109, wherein the subject has a sérum HBsAg concentration of greater than or equal to 3,000 IU/mL no more than 28 days prior to the single dose being administered, and optionally greater than or equal to 1,000 IU/mL no more than 28 days prior to the single dose being administered.
Embodiment 111. The method of any one of Embodiments 83-110, wherein the subject was HB e-antigen (HBeAg)-negative no more than 28 days prior to the single dose being administered.
Embodiment 112. The method of any one of Embodiments 83-111, wherein the subject was négative for anti-HB antibodies no more than 28 days prior to the single dose being administered.
Embodiment 113. The method of any one of Embodiments 83-1 12, wherein the subject, prior to administration of the single dose:
(i) does not hâve fibrosis and/or does not hâve cirrhosis; and/or (ii) has alanine aminotransferase (ALT) <2 x Upper Limit of Normal (ULN). Embodiment 114. The method of any one of Embodiments 83-113, wherein at 56 days following administration of the single dose, the subject has a > 2fold réduction in sérum HBsAg (e.g., concentration of HBsAg in sérum, e.g., as determined using an Abbott ARCHITECT assay) as compared to the subject’s sérum HBsAg at from 0 days to 28 days prior to administration of the single dose.
Embodiment 115. The method of any one of Embodiments 83-114, wherein following administration of the single dose (e.g., at 56 days following administration of the single dose), the subject has:
(i ) has reduced or less severe intrahepatic spread of HBV as compared lo a reference subject; and/or (ii) comprises an adaptive immune response against HBV.
204
Embodiment 116. the subject is male.
Embodiment 117. the subject is female.
Embodiment 118.
The method of any one of Embodiments 83-115, wherein
The method of any one of Embodiments 83-l 15, wherein
A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of Embodiments l-59 at a concentration ranging from 100 mg/mL to 200 mg/mL, such as 100 mg/mL, 110 mg/mL, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL, or 200 mg/mL, preferably 150 mg/mL, and a pharmaceutically acceptable carrier, excipient, or diluent.
Embodiment I 19. The pharmaceutical composition of Embodiment l 18, wherein the pharmaceutical composition comprises up to 6 mg, up to 18 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of the antibody.
Embodiment 120. The pharmaceutical composition of Embodiment 118 or 119, wherein the pharmaceutical composition comprises about 75 mg of the antibody.
Embodiment I2l. The pharmaceutical composition of Embodiment l I8 or 119, wherein the pharmaceutical composition comprises about 90 mg of the antibody.
Embodiment 122. The pharmaceutical composition of Embodiment l 18 or 119, wherein the pharmaceutical composition comprises about 300 mg of the antibody.
Embodiment 123. The pharmaceutical composition of Embodiment 118 or l ] 9, wherein the pharmaceutical composition comprises about 900 mg of the antibody.
Embodiment 124. The pharmaceutical composition of Embodiment 118 or ] 19, wherein the pharmaceutical composition comprises about 3,000 mg of the antibody.
Embodiment 125. The pharmaceutical composition of any one of Embodiments 118-l24, wherein the pharmaceutical composition comprises water, optionallv USP water.
205
Embodiment 126. The pharmaceutical composition of any one of Embodiments 118-125, wherein the pharmaceutical composition comprises histîdine, optionally at a concentration from 10 mM to 40 mM, such as 20 mM, in the pharmaceutical composition.
Embodiment 127. The pharmaceutical composition of any one of Embodiments 118-126, wherein the pharmaceutical composition comprises a disaccharide, such as sucrose, optionally at 5%, 6%, 7%, 8%, or 9%, preferably about 7% (w/v).
Embodiment 128. The pharmaceutical composition of any one of Embodiments 118-127, wherein the pharmaceutical composition comprises a surfactant, optionally a polysorbate, preferably polysorbate 80 (PS80), wherein, optionally, the polysorbate is présent in a range from 0.01% to 0.05% (w/v), preferably 0.02% (w/v).
Embodiment 129. The pharmaceutical composition of any one of Embodiments 118-128, wherein the pharmaceutical composition has a pH ranging from 5.8 to 6.2, ranging from 5.9 to 6.1, or of 5.8, of 5.9, of 6.0, of 6.1, or of 6.2.
Embodiment 130. The pharmaceutical composition of any one of Embodiments 118-129, wherein the pharmaceutical composition comprises:
(i) the antibody at 150 mg/mL;
(ii) USP water;
(iii) 20 mM histidine;
(iv) 7% sucrose; and (v) 0.02% PS80, wherein the pharmaceutical composition comprises a pH of 6.
Embodiment 131. The method of any one of Embodiments 83-117, wherein following administration of the single dose, sérum HBsAg of the subject îs reduced as compared to baseline by 1.0 log10 lU/mL, 1.5 log10 lU/mL, or more, wherein, optionally, the réduction persists for 1, 2, 3, 4, 5, 6, 7, 8, or more days following administration ofthe single dose.
206
Embodiment 132. The method of any one of Embodiments 83-11Ί and 131, wherein following administration of the single dose, sérum HBsAg of the subject is reduced as compared to baseline for at least 8, at least ] 5, at least 22, or at least 29 days.
Embodiment 133. A method for in vitro diagnosis of a hepatitis B and/or a hepatitis D infection, the method comprising:
(i) contacting a sample from a subject with an antibody or antigen-binding fragment of any one of Embodiments l-59; and (ii) detecting a complex comprising an antigen and the antibody, or comprising an antigen and the antigen-binding fragment.
Embodiment 134. The method of Embodiment 133, wherein the sample comprises blood isolated from the subject.
Embodiment 135. A method for detecting the presence or absence of an epitope in a correct conformation in an anti-hepatitis-B and/or an anti-hepatitis-D vaccine, the method comprising:
(i) contacting the vaccine with an antibody or antigen-binding fragment of any one of Embodiments 1 -59; and (ii) determinîng whether a complex comprising an antigen and the antibody, or comprising an antigen and the antigen binding fragment, has been formed.
Embodiment 136. The antibody or antigen-binding fragment of any one of 20 Embodiments 1 -59, wherein the antibody or antigen-binding fragment:
(i) has enhanced binding to a human FcyRIIA, a human FcyRIllA, or both, as compared to a reference polypeptide that includes a Fc moiety that does not comprise G236A/A330L/I332E, wherein the human FcyRlIA is optionally H13I orRI3l, and/or the human FcyRIllA is optionally Fl58 or VI58;
(ii) has reduced binding to a human FcyRIlB, as compared to a reference polypeptide that includes a Fc moiety that does not comprise G236A/A330L/I332E;
(îii) does not bind to a human FcyRIlB;
(iv) has reduced binding to a human Clq, as compared to a reference polypeptide that includes a Fc moiety that does not comprise G236A/A330L/I332E;
207 (v) does not bind to a human C l q; (vi) activâtes a FcyRIIA, a human FcyRIIIA, or both, to a greater degree than does a reference polypeptide that includes a Fc moiety that does not comprise G236A/A330L/I332E, wherein the human FcyRIlA is optionally Hl31 or Rl31, and/or the human FcyRIllA is optionally Fl58 or VI58;
(vii) does not activate a human FcyRlIB;
(viii) activâtes a human natural killer (NK) cell în the presence of HBsAg to a greater degree than does a reference polypeptide that includes a Fc moiety that does not comprise G236A/A330L/I332E, wherein the reference polypeptide is optionally an antibody that binds to an HB Ag, optionally an HBsAg;
’(ix) is capable of binding to an HBsAg variant comprising HBsAgYI00C/P120T, HBsAg-PI20T, HBsAg-Pl20S/Sl43L, HBsAg-Cl2lS, HBsAg-Rl22D, HBsAg-R 122L HBsAg-TJ 23N, HBsAg-Q 129H, HBsAg-Q 129L, HBsAg-M 133H, HBsAg-MI33L, HBsAg-Ml33T, HBsAg-K14lE, HBsAg-Pl42S, HBsAg-Sl43K, HBsAg-Dl44A, HBsAg-Gl45R, HBsAg-Nl46A, or any combination thereof; and/or (x) has improved binding to an HBsAg variant comprising HBsAg-Yl00C/Pl20T, HBsAg-PI20T, HBsAg-Pl20S/Sl43L, HBsAg-Cl2lS, HBsAg-Rl22D, HBsAg-RI22l. HBsAg-Tl23N, HBsAg-QI29H, HBsAg-Ql29L, HBsAg-Ml33H, HBsAg-Ml33L, HBsAg-Ml33T, HBsAg-Kl4lE, HBsAg-Pl42S, HBsAg-S143K, HBsAg-D144A, HBsAg-GI45R, HBsAg-N146A, or any combination thereof, as compared to a reference antibody or antigen binding fragment that bînds to HBsAg and that includes a Fc moiety that does not comprise G236A/A330L/1332E.
Embodiment 137. A method of treating chronic HBV infection in a subject in need thereof, comprising:
administering to the subject an agent that reduces HBV antigenic load; and administering to the subject an anti-HBV antibody from any one of Embodiments 1-59.
Embodiment 138. A method of treating chronic HBV infection in a subject in need thereof, comprising:
administering to the subject an inhibitor of HBV gene expression; and
208 administering to the subject an anti-HBV antibody from any one of Embodiments l-59.
Embodiment 139. The method according to Embodiments 137 or 138, wherein the RNAi agent comprises a sense strand and an antisense strand forming a double-stranded région, wherein the sense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from nucléotides 1579-1597 of SEQ ID NO:Il6.
Embodiment 140. The method according to any one of Embodiments 137139, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand comprises nucléotides 1579-1597 of SEQ IDNO:116.
Embodiment 141. The method according to any one of Embodiments 137140, wherein at least one strand of the RNAi agent comprises a 3' overhang of at least 1 nucléotide.
Embodiment 142. The method according to any one of Embodiments 137140, wherein at least one strand of the RNAi agent comprises a 3' overhang of at least 2 nucléotides.
Embodiment 143. The method according to any one of Embodiments 137142, wherein the double-stranded région of the RNAi agent is 15-30 nucléotide pairs in lengîh.
Embodiment 144. The method according to any one of Embodiments 137142, wherein the double-stranded région of the RNAi agent is 17-23 nucléotide pairs in length.
Embodiment 145. The method according to any one of Embodiments 137142, wherein the double-stranded région ofthe RNAi agent îs 17-25 nucléotide pairs in length.
Embodiment 146. The method according to any one of Embodiments 137142, wherein the double-stranded région of the RNAi agent is 23-27 nucléotide pairs in length.
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Embodiment I47. The method according to any one of Embodiments I37142, wherein the double-stranded région of the RNAi agent is 19-21 nucléotide pairs in length.
Embodiment 148. The method according to any one of Embodiments 1375 142, wherein the double-stranded région of the RNAi agent is 21 -23 nucléotide pairs in length.
Embodiment 149. The method according to any one of Embodiments 137142, wherein each strand of the RNAi agent has 15-30 nucléotides.
Embodiment 150. The method according to any one of Embodiments 13710 142, wherein each strand of the RNAi agent has 19-30 nucléotides.
Embodiment 151. The method according to any one of the Embodiments 137-150, wherein the RNAi agent is an siRNA.
Embodiment ! 52. The method according to Embodiment 151, wherein the siRNA inhibits expression of an HBV transcrîpt that encodes an HBsAg protein, an 15 HBcAg protein, and HBx protein, or an HBV DNA polymerase protein.
Embodiment 153. The method according to Embodiment 151 or Embodiment i 52, wherein the siRNA binds to at least 15 contiguous nucléotides of a target encoded by: P gene, nucléotides 2309-3182 and 1-1625 of NC_003977.2; S gene (encodîng L, M, and S proteins), nucléotides 2850-3182 and 1-837 of NC_003977.2;
HBx, nucléotides 1376-1840 of NC_003977.2; or C gene, nucléotides 1816-2454 of NC 003977.2.
Embodiment 154. The method according to Embodiment 151 or Embodiment 152, wherein the antisense strand of the siRNA comprises at least 15 contiguous nucléotides of the nucléotide sequence of 5'25 UGUGAAGCGAAGUGCACACUU -3’ (SEQ ID NO: 119).
Embodiment 155. The method according to Embodiment 151 or 152, wherein the antisense strand of the siRNA comprises at least 19 contiguous nucléotides ofthe nucléotide sequence of 5'- UGUGAAGCGAAGUGCACACUU -3' (SEQ ID NO:II9).
210
Embodiment 156. The method according to Embodiment L 51 or 152, wherein the antisense strand of the siRNA comprises the nucléotide sequence of 5’UGUGAAGCGAAGUGCACACUU -3' (SEQ ID NO:l 19).
Embodiment 157. The method according to Embodiment L5l or 152, wherein the antisense strand of the siRNA consists of the nucieotide sequence of 5'UGUGAAGCGAAGUGCACACUU -3' (SEQ ID NO:l 19).
Embodiment 158. The method according to any one of Embodiments 154157, wherein the sense strand of the siRNA comprises the nucieotide sequence of 5'GUGUGCACUUCGCUUCACA -3’ (SEQ ID NO:l 18).
Embodiment 159. The method according to any one ofEmbodiments 154157, wherein the sense strand of the siRNA consists of the nucieotide sequence of 5’GUGUGCACUUCGCUUCACA -3' (SEQ ID NO: 118).
Embodiment J60. The method according to Embodiment I5l or 152, wherein the antisense strand of the siRNA comprises at least 15 contiguous nucléotides ofthe nucieotide sequence of 5'- UAAAAUUGAGAGAAGUCCACCAC -3' (SEQ ID NO:l2I).
Embodiment I6l. The method according to Embodiment I5l or 152, wherein the antisense strand of the siRNA comprises at least 19 contiguous nucléotides ofthe nucieotide sequence of 5'- UAAAAUUGAGAGAAGUCCACCAC -3' (SEQ ID NO:12I).
Embodiment 162. The method according to Embodiment I5I or 152, wherein the antisense strand ofthe siRNA comprises the nucieotide sequence of 5'UAAAAUUGAGAGAAGUCCACCAC -3' (SEQ ID NO:l2l).
Embodiment 163. The method according to Embodiment I5l or 152, wherein the antisense strand of the siRNA consists of the nucieotide sequence of 5'UAAAAUUGAGAGAAGUCCACCAC -3’ (SEQ ID NO:l2l).
Embodiment I64. The method according to any one ofEmbodiments 154157, wherein the sense strand of the siRNA comprises the nucieotide sequence of 5'GGUGGACUUCUCUCAAUUUUA -3’ (SEQ ID NO: 120).
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Embodiment 165. The method, composition for use, or use according to any one of Embodiments 154-157, wherein the sense strand of the siRNA consists of the nucléotide sequence of 5'- GGUGGACUUCUCUCAAUUUUA -3' (SEQ ID NO: J 20).
Embodiment 166. The method according to any one of Embodiments 15l5 165, wherein substantially all of the nucléotides of said sense strand and substantially all of the nucléotides of said antisense strand are modified nucléotides, and wherein said sense strand is conjugated to a ligand attached at the 3'-terminus.
Embodiment 167. The method according to Embodiment 166, wherein the ligand is one or more GalNAc dérivatives attached through a monovalent linker, 10 bivalent branched linker, or trivalent branched linker.
Embodiment 168. The method according to Embodiment I66 or 167,
Embodiment 169. The method according to Embodiment 168, wherein the siRNA is conjugated to the ligand as shown in the following structure:
212 wherein X is O or S.
Embodiment 170. The method according to any one of Embodiments I5l169, wherein at least one nucléotide of the siRNA is a modîfied nucléotide comprising a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucléotide, a 2'-O-methyl modîfied 5 nucléotide, a 2'-fluoro modîfied nucieotîde, a 2'-deoxy-modifîed nucléotide, a locked nucléotide, an unlocked nucieotîde, a conformationally restricted nucieotîde, a constrained ethyl nucieotîde, an abasic nucieotîde, a 2'-amino-modified nucieotîde, a 2'O-allyl-modified nucieotîde, 2'-C-alkyl-modified nucieotîde, 2'-hydroxyl-modîfied nucieotîde, a 2'-methoxyethyl modîfied nucieotîde, a 2’-O-alkyl-modified nucieotîde, a 10 morpholino nucieotîde, a phosphoramidate, a non-natural base comprising nucieotîde, a tetrahydropyran modîfied nucieotîde, a l,5-anhydrohexitol modîfied nucieotîde, a cyclohexenyl modîfied nucieotîde, a nucieotîde comprising a phosphorothioate group, a nucieotîde comprising a methylphosphonate group, a nucieotîde comprising a 5’phosphate, an adenosine-glycol nucleic acid, or a nucieotîde comprising a 5'~phosphate 15 mimic.
Embodiment 171. The method according to any one of Embodiments 151 I69, wherein the siRNA comprises a phosphate backbone modification, a 2' ribose modification, 5' triphosphate modification, or a GalNAc conjugation modification.
Embodiment 172. The method according to Embodiment 171, wherein the 20 phosphate backbone modification comprises a phosphorothioate bond.
Embodiment 173. The method according to Embodiment I7l or Embodiment 172, wherein the 2' rîbose modification comprises a fluoro or -O-methyl substitution.
Embodiment 174. The method according to any one of Embodiments 151 25 159 and 166-173, wherein the siRNA has a sense strand comprising 5'gsusguGfcAfCfUfucgcuucacaL96 -3' (SEQ ID NO:l22) and an antisense strand comprising 5 - usGfsugaAfgCfGfaaguGfcAfcacsusu -3' (SEQ ID NO:i23), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-Omethylcytîdine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O30 methyluridine-3'-phosphate, respectively;
2I3
Af, Cf, Gf, and Ufare 2'-fluoroadenosÎne-3'-phosphate, 2'-fluorocytidine-3'phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorourîdine-3'-phosphate, respectively;
s is a phosphorothioate iinkage; and
L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.
Embodiment 175. The method according to any one of Embodiments 151 159 and 166-173, wherein the siRNA has a sense strand comprising 5'gsusguGfcAfCfUfucgcuucacaL96 -3' (SEQ ID NO:l24) and an antisense strand comprising 5'- usGfsuga(Agn)gCfGfaaguGfcAfcacsusu -3' (SEQ ID NO:125) wherein a, c, g, and u are 2'-0-methyladenosine-3'-phosphate, 2-0methyIcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-Omethyluridine-3'-phosphate, respectively;
Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytÎdine-3'phosphate, 2'-fluoroguanosîne-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively;
(Agn) is adenosine-glycol nucleic acid (GNA);
s is a phosphorothioate Iinkage; and
L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.
Embodiment 176. The method, compositions for use, or use according to any one of Embodiments 151-153 and 160-173, wherein the siRNA has a sense strand comprising 5'- gsgsuggaCfuUfCfUfcucaAfUfuuuaL96 -3' (SEQ ID NO:]26) and an antisense strand comprising 5'- usAfsaaaUfuGfAfgagaAfgUfccaccsasc -3' (SEQ ID NO: 127), wherein a, c, g, and u are 2'-O-methy!adenosine-3'-phosphate, 2'-Omethylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-Omethyluridine-3'-phosphate, respectively;
Af, Cf, Gf, and Uf are 2'-fluoroadenosîne-3'-phosphate, 2'-fluorocytidine-3'phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively;
s îs a phosphorothioate Iinkage; and
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Φ
L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.
Embodiment 177. The method according to any one of Embodiments I37176, wherein the subject is a human and a therapeutically effective amount of RNAi agent or siRNA is administered to the subject; and wherein the effective amount of the 5 RNAi agent or siRNA is from about i mg/kg to about 8 mg/kg.
Embodiment 178. The method according to any one of Embodiments 137177, wherein the RNAi agent or siRNA is administered to the subject twice daîly, once daily, every two days, every three days, twice per week, once per week, every other week, every four weeks, or once per month.
Embodiment 179. The method according to any one of Embodiments 137-
177, wherein the RNAi agent or siRNA is administered to the subject every four weeks.
Embodiment 180. The method according to any one of Embodiments 151179, wherein two siRNAs each directed to an HBV gene are administered, and the first siRNA has an antisense strand comprising SEQ ID NO:l 19, SEQ ID NO:l20, or SEQ 15 ID NO:l26; and the second siRNA comprises an siRNA having a sense strand that comprises at least 15 contîguous nucléotides of nucléotides 2850-3182 of SEQ ID NO:116.
Embodiment 181. The method according to any one of Embodiments 151179, wherein two siRNAs directed to an HBV gene are administered, wherein the two 20 siRNAs comprise: an siRNA directed to an HBV X gene and an siRNA directed to an HBV S gene.
Embodiment 182. The method according to any one of Embodiments 151179, wherein two siRNAs each directed to an HBV gene are administered, and the first siRNA has an antisense strand comprising SEQ ID NO:119, SEQ ID NO: 123, or SEQ 25 ID NO: 125; and the second siRNA has an antisense strand that comprises SEQ ID NO:121 or SEQ IDNO:127.
Embodiment 183. The method according to Embodiment 181, wherein the first siRNA has a sense strand comprising SEQ ID NO:l 18, SEQ ID NO: 122, or SEQ ID NO: 124; and the second siRNA has a sense strand comprising SEQ ID NO: 120 or 30 SEQIDNO:126.
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Embodiment 184. The method according to any one of Embodiments 179183, wherein the two sîRNAs are admînistered simultaneously.
Embodiment 185. The method according to any one of the Embodiments 137-184, further comprising administering a nucleot(s)ide analog to the subject, or 5 wherein the subject is also admînistered a nucleot(s)ide analog.
Embodiment 186. The method, composition for use, or use according to Embodiment 185, wherein the nucleot(s)ide analog is tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), lamivudine, adefovir dipivoxil, entecavîr (ETV), telbivudine, AGX-1009, emtrîcitabine (FTC), clevudine, ritonavir, dipivoxil, lobucavir, 10 famvir, N-Acetyl-Cysteine (NAC), PC 1323, theradigm-FIBV, thymosin-alpha, and ganciclovir, besifovir (ANA-380/LB-80380), or tenofvir-exaliades (TLX/CMX157).
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In some instances, éléments of the antibodies, antigen-binding fragments, fusion proteins, nucleic acids, cells, compositions, combinations, uses, and methods provided herein are described or listed with reference to embodiments or examples. However, it should be understood that the examples and embodiments described herein may be combined in various ways to create addîtional embodiments.
EX AMPLE S
In the following, particular examples illustrating various embodiments and aspects ofthe disclosure are presented. However, the présent disclosure shall not to be limited in scope by the spécifie embodiments described herein.
Example 1 : Dimer Formation b y an anti-HBV Antibody
Anti-HBV antibodies are disclosed in PCT Publication No. WO 2017/060504. Engineering anti-HBV antibody HBC34-v7 produced, inter alia, antibody HBC34v35 (PCT Publication No. WO 2020/132091), having VH and VL amino acid sequences according to SEQ ID NOs.:38 and 57, respectively. HBC34-v35 binds to HBsAg with picomolar affinity and potently neutralizes ten (10) HBV génotypes and Hepatitis D virus, binding to a conserved conformational epitope. Représentative binding and neutralizatîon data for HBC34-v35 (expressed as IgGl and includîng the Fc mutations G236A, A330L, I332E, M428L, and N434S (EU numbering; collectively referred-to as ”GAALIE-MLNS or GAALIE+MLNS or MLNS-GAALIE or MLNS -- GAALIE)) are shown in Figure I.
HBC34-v35 was expressed as recombinant IgG (allotype GlmI7, 1) in a host cell line, purified from supernatant, and formulated for administration. Size-exclusion chromatography analysis of the formulation following a 1-week incubation revealed a peak corresponding to antibody monomer (i.e., single antibody molécules comprising two heavy chains and two light chaîns) and a high molecular w eight species
217 •
corresponding to an antibody dimer (i.e., aggregate formed by two single antibody molécules) (Figure 2).
It was hypothesized that the dimer formation was mediated via Fab-Fab interactions, and that recombinant Fab shouid also dimerize. Size exclusion chromatography was used to purify enriched IgG dimer and Fab dimer. Figure 3 shows that Fab dimer fraction slowly increases over time; dimer formation kinetics also increased with température (data not shown).
Different modes of Fab dimerization hâve been described (see, e.g., Plath et al. MAbs 5(5):928-940 (2016)). Depending on the mode of Fab dimerization, the Fab will 10 either retain or lose the abi!ity to bind antigen. For example, an IgG dimer could be expected to lose a maximum 50% of binding capacity, with two of the four Fabs being unaffected. As shown in Figure 4, the HBC34-v35 dimer (full-length IgG or Fab) has reduced binding for FIBsAg as determined by surface plasmon résonance (SPR; with sîmilar amounts (by mass) of the monomer and dimer antibody captured on the ] 5 surface), consistent with dimerization involving CDRs.
Next, crystallization of HBC34-v35 rFab dimer or monomer was performed. The rFab dimer was isolated by préparative size-exclusion chromatography (Figure 5A). 3 x 96 conditions were set up at RT (concentration: 5.5 mg/ml). Crystals were obtained in three different conditions but diffracted poorly and were multi-crystal. A 20 new round of incubation and crystallization optimization led to high quality diffraction (Figure 5B). For rFab monomer, préparative size-exclusion chromatography was used for purification (Figure 6A). Material obtained after the incubation at 40ûC did not yield crystals (3 trays at RT, 5 mg/mL or 9 mg/mL). A second batch of monomer was prepared without an incubation step; crystals formed at 4°C but not RT (4 trays each, 2 25 concentrations) (Figure 6B). Analysis of the Fab dimer crystal structure indicated that dimerization involves L-CDR2 (Figures 7-9), that the Fabs présent in dimer form hâve a sîmilar conformation, and that L-CDR2 undergoes a conformation change between monomer and dimer (Figure 10). Potential interactions between L-CDR2 and Framework residues were identifted.
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Example 2: An Engineered Antibody with Reduced Dimer Formation
HBC34-v35 includes several mutations in the light chain compared to the germline sequence, including in L-CDR2. Three adjacent amino acids présent in the LCDR2 and believed to be involved in the Fab-Fab interactions were reverted to germline to generate the further variant antibody HBC34-v36. In separate experiments, FIBC34-v35 and HBC34-v36 Fabs (>10 mg/mL) were incubated at 40 °C for 5-7 days and percent dimer was evaluated by absolute size-exclusion chromatography (aSEC). As shown in Figure l IA, reversion to germline sequence dramatically reduced dimerization. HBC34-v36 full-length IgG at 3 mg/mL did not dimerize after 2 weeks at 10 40°C (data not shown).
Example 3: Binding and In Vitro Neutralization by Antibodies
Ability of HBC34-v35 and HBC34-v36 to bind HBsAg and neutralize HBV infection was compared. Binding was assessed by ELISA and showed that HBC34-v36 has similar binding activity to HBC34-v35 (EC50 = 0.7 ng/mL vs. 0.6 ng/mL, respectively, Figure 12). Neutralization was assessed by measuring the levels of HBeAg (génotype D) in the cell culture supernatant of HBV-infected HepG2 cells expressing NTCP. Data are shown in Figure 13, and show that neutralization of HBV génotype D is approximately 3-fold weaker for HBC34-v36 than HBC34-v35. For these experiments, the antibodies included wild-type IgGl Fc.
Example 4: Design and Testing of Additional Engineered Antibodies
Additional engineered variant antibodies with mutations in L-CDR2 and/or in framework sequence relative to HBC34-v35 were generated, using HBC34-v36 as the starting point. These variants were termed HBC34-v37-FIBC34-v50. Light chain variable région sequences from the various antibodies, and a summary of the mutations 25 versus HBC34-v35, are provided in Table 6. CDR sequences and numberîng of amino acid residues as shown in Table 6 are per the system developed by the Chemical Computing Group (chemcomp.com).
219
Table 6. VL amino acid sequences of HBC34 antibodies
| HBC34 | VL amino acid sequence (L-CDR2 (CCG) underlined) | Mutation(s) vs. IIBC34-v35 |
| v35 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGOSPVLVIYEVKYRPSGIPERFSGSNSGNTATLT ISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.:57) | |
| v36 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLT ISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ IDNO.:58) | E49Q, V50D, K51S, Y52K |
| v37 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGOSPVLVIYEDSKRPSGIPERFSGSNSGNTATLT ISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ 1DNO.:59) | V50D, K51S, Y52K |
| v38 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGOSPVLVIYQVSKRPSGIPERFSGSNSGNTATLT ISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.:60) | E49Q, K5 ] S, Y52K |
| v39 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGOSPVLVIYQDKKRPSGIPERFSGSNSGNTATL TISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ IDNO.:6l) | E49Q, V50D, Y52K |
| v40 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGOSPVLVIYQDSYRPSGIPERFSGSNSGNTATLT ISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.:62) | E49Q, V50D, K5IS |
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| HBC34 | VL amino acid sequence (L-CDR2 (CCG) underlined) | Mutation(s) vs. HBC34-v35 |
| v41 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGQSPVLVIYQVSYRPSGIPERFSGSNSGNTATLT ISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.;63) | E49Q, K51S |
| v42 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGOSPVLVIYEVSYRPSGIPERFSGANSGNTATLT ISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ 1DNO.:64) | K51S, S 64A |
| v43 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGQSPVLVIYOVKYRPSGIPERFSGSNSGNTATL TISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.:65) | E49Q |
| v44 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HK.PGOSPVLV1YAVKYRPSGIPERFSGSNSGNTATL T1SGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.:66) | E49A |
| v45 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGOSPVLVIYEVKYRPSGIPENFSGSNSGNTATLT ISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ IDNO.:67) | R60N |
| v46 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGOSPVLVIYEVKYRPSGIPEAFSGSNSGNTATLT ISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.:68) | R60A |
| v47 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGOSPVLVIYEVSYRPSGIPENFSGANSGNTATLT ASGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.:69)’ | K51 S, S64A, R60N, 174A |
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| HBC34 | VL amino acid sequence (L-CDR2 (CCG) underlined) | Mutationl s) vs. HBC34-v35 |
| v48 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGQSPVLVIYEVKYRPSGIPENFSGANSGNTATL TASGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.:70) | R60N, S64A, 174A |
| v49 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPGQSPVLVIYEVSYRPSGIPERFSGSNSGNTATLT ISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.:7I) | K51S |
| v50 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQ HKPG0SPVLV1YEVKYRPSG1PEKFSGSNSGNTATL TISGTQAMDEAAYFCQTFDSTTVVFGGGTRLTVL (SEQ ID NO.:72) | R60K |
HBsAg binding and HBV neutralizing activity of HBC34-v37-HBC34-v50 was tested using assays as described herein. Results from the binding assays are provided in Figures 14A-14E, and show that all of the tested variant antibodies except for HBC345 v47 and HBC34-v48 had similar or even stronger binding as compared to HBC34-v35.
HBC34-v47 and HBC34-v48 also had low production yields, and were not selected for further testing. Results from the neutralîzation assay are provided in Figure 15, and show thaï several antibodies (HBC34-v40-FIBC34-v46, HBC34-v49, and HBC34-v50) had similar or even improved neutralizing activity (EC50) as compared to HBC34-v35.
HBC34-v36-HBC34-v39 had less potent neutralizing activity.
Example 5: Purification of Certain Engineered Antibodies
Engineered variant antibodies were evaluated for formation of aggregates following incubation at different températures over the course of 32 days. Nine HBC34-v35 antibody variants and parental HBC34-v35 were expressed as reconibinant 15 IgG (allotype Glml7, 1) in a host cell line and purified from supernatant. Antibodies
222 were received later than one week after production and concentrated to 25 mg/ml. Sizeexclusion chromatography (SEC) analysis was used to to monitor high molecular weight species (HMWS) corresponding to an antibody dîmer at day -l, day 0, day 5, day 15, and day 32. Day -1 samples were evaluated prior to concentration. Antibody compositions were incubated at 4°C (Figure 16A), 25°C (Figure 16B), or 40°C (Figure 16C) over the course of the 32-day analysis. A summary of the frequency of HMWS following a 32-day incubation at 40°C is shown in Figure 16D. Four variant antibodies (-v40, -v44, -v45, -v50) exhibited low génération of HMWS (Figure 16D) and were selected for further studies.
Example 6: Binding and Ζλ Vitro Neutralisation by certain Engineered Antibodies
Binding of HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50, to HBsAg from ten ((A)-(J)) génotypes was tested by FACS. HBC34-v35 was included as reference. Ail of the tested variants bound to HBsAg, with HBC34-v40 showing the most potent binding (Figures 17A-17J). Binding of HBC34-v40, HBC34-v44, HBC34v45, and HBC34-v50 to ten HBsAg-génotype D mutants was tested by FACS. HBC34v35 was included as reference. Ail of the engineered variants bound to HBsAg (Figures 18A-18K).
Example 7: Production of Certain Engineered Antibodies
Antibody titers for HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 were measured to evaluate productivity in host cells. Antibodies were expressed as recombinant IgG (allotype Glml7, I) in a host cell line and purified from supernatant. Both 5 ml- and 100 ml-scale transfection Systems were evaluated, with the 100 ml system tested in duplicate or triplicate. Antibody titers from individual 5 mland 100 ml-scale tests as well as average titer from 100 ml-scale tests are shown in Figure 19.
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Example 8: Thermostability of Certain Engineered Antibodies
HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 were expressed as recombinant IgG (allotype Glml7, l) in a host cell line and purified from supematant. Antibodies were concentrated to 25 mg/ml and incubated at 40°C for four days, Size-exclusion chromatography analysis was used to to quantify high molecular weight species (HMWS) corresponding to an antibody dimer at day 4, as shown in Figure 20, Only HBC34-v35 showed significant HMWS after 4 days.
Example 9: Analysis of Light Chain Amino Acids Involved in Forming Antibody Dimers
Structural studies identified of number of amino acid residues in the HBC34v35 VL région that were involved in forming antibody:antibody dimers. Interactions between light chain residues of two HBC34-v35 antibody molécules (herein, antibody molécule l and antibody molécule 2) are illustrated in Figures 21Λ, 22A, and 23 A, wherein: E49 (antibody molécule l) înteracts with S64 and K5l (antibody molécule 2); V50 (antibody molécule l) înteracts with V50 (antibody molécule 2); K5l (antibody molécule l) înteracts with E49 (antibody molécule 2); and S64 (antibody molécule l) înteracts with E49 (antibody molécule 2). Interactions between other light chain amino acids of two HBC34-v35 antibodies is shown in Figures 21 B, 22B, and 23B, wherein: R60 (antibody molécule l) înteracts with D8l and Q78 (antibody molécule 2); F6l (antibody l) înteracts with 174 (antibody molécule 2); 174 (antibody molécule 1) înteracts with F6I (antibody molécule 2); Q78 (antibody molécule 1) înteracts with R60 (antibody molécule 2); and D81 (antibody molécule 1) înteracts with R60 (antibody molécule 2).
Four engineered antibodies, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50, were determined to bave a low propensity for aggregation while maintaining potent binding.
HBC34-v40 comprises E49Q, V50D, and K51S mutations in L-CDR2 (CCG numberîng) compared to parental HBC34-v35, as shown in Figure 2 IC. These mutations change from hydrophobie interaction to electrostatic répulsion and the loss of
224 a sait bridge, though loss of the sait bridge alone was not sufficient to reduce aggregation (compare to HBC34-v4l comprising E49Q and K5IS mutations, but not V50D, and .sec Figure 16D).
HBC34-v44 comprises an E49A mutation in L-CDR2 as comparedto HBC345 v35, as shown in Figure 22C. This mutation results in loss of a sait bridge.
HBC34-v45 and HBC34-v50 comprise Framework mutations at R60 relative to HBC34-v35, as shown in Figure 23C. R60N and R60K mutations in HBC34-v45 and HBC34-v50, respectively, reduced dimer formation. A R60A mutation in HBC34-v46 was less effective in reducing dimer formation (see Figure 16D).
TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING):
| SEQ ID NO | Sequence | Remarks |
| l | XiX2X3 TC X4 X5 XüA XtG wherein Χι, X?, X3, X4, X?, Xs and X? may be any amino acid | epitope |
| 2 | XiX2 X3TC X4 Xs XsA X?G wherein Xj is P, T or S, X? is C or S, X3 is R, K, D or I, X4 is M or T, Xs is T, A or I, Xè is T, P or L, and X? is Q, H or L. | |
| 3 | MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNF LGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFI IFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRT CMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFL WEWASARFSWLSLLVPFVQWFVGLSPTVWLSVl WMMW Y WGPSLYSILSPFLPLLPIFFCLWVYI | S domain of HBsAg (GenBank acc. no. J02203) |
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| 4 | MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLN FLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRR FlIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTGTGPCR TCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFL WEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMW Y WGPSLYSTLSPFLPLLPIFFCL WV Yl | S domain of HBsAg (GenBank acc. no. FJ899792) |
| 5 | QGMLPVCPLiPGSSTTSTGPCRTCMTTAQGTSMYPSCCCT KPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | J02203 (D, ayw3) |
| 6 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCT K.PSDGNCTCIPIPSSWAFGKFLWEWASARFSW | FJ899792 (D, adw2) |
| 7 | QGMLPVCPLIPGTTTTSTGPCKTCTTPAQGNSMFPSCCCT KPSDGNCTCIPIPSSWAFAKYLWEWASVRFSW | AM282986 (Λ) |
| 8 | QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGTSMFPSCCCT KPTDGNCTCIPIPSSWAFAKYLWEWASVRFSW | D23678(Bl) |
| 9 | QGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCT K PSDGNCTCIPIPSSWAFARFLWE WAS VRFSW | ABU7758(Cl) |
| ΙΟ | QGMLPVCPLIPGSSTTSTGPCRTCTTLAQGTSMFPSCCCS KPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | AB205192 (E) |
| 11 | QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTSMFPSCCCS KPSDGNCTCIPIPSSWALGKYLWEWASARFSW | X69798(F4) |
| 12 | QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNSMYPSCCCT KPSDGNCTCIPIPSSWAFAKYLWEW AS VRFSW | AF160501 (G) |
| 13 | QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTSMFPSCCCT KPSDGNCTCIPIPSSWAFGKYLWEWASARFSW | AY090454 (H) |
| 14 | QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNSMYPSCCCT KPSDGNCTCIPIPSSWAFAKYLWEWASARFSW | AF241409 (I) |
| 15 | QGMLPVCPLLPGSTTTSTGPCRTCTITAQGTSMFPSCCCT KPSDGNCTCIPIPSSWAFAKFLWEWASVRFSW | AB486012 (J) |
| Ιό | CQGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCC TKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg Y100C/P120T |
| 17 | QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCT KPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg P120T |
| 18 | QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCT KPLDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg P120T/S143L |
| 19 | QGMLPVCPLIPGSSTTGTGPSRTCTTPAQGTSMYPSCCCT KPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg C121S |
| 20 | QGMLPVCPLIPGSSTTGTGPCDTCTTPAQGTSMYPSCCCT KPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg R122D |
| 21 | QGMLPVCPLIPGSSTTGTGPCITCTTPAQGTSMYPSCCCT KPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg R122I |
| 22 | QGMLPVCPLIPGSSTTGTGPCRNCTTPAQGTSMYPSCCCT KPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg T123N |
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| 23 | QGMLPVCPLÏPGSSTTGTGPCRTCTTPAHGTSMYPSCCCT K PSDGNCTCIPIPSS W AFGKFL WEWAS ARFS W | HBsAg Q129H |
| 24 | QGMLPVCPLIPGSSTTGTGPCRTCTTPALGTSMYPSCCCT KPSDGNCTCJPIPSSWAFGKFLWEWASARFSW | HBsAg Q129L |
| 25 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSHYPSCCCT KPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg M133H |
| 26 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSLYPSCCCT K PSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg Ml33L |
| 27 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSTYPSCCCT KPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg Ml33T |
| 28 | QGMLPVCPLJPGSSTTGTGPCRTCTTPAQGTSMYPSCCCT EPSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAgKl4lE |
| 29 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCT KSSDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg P142S |
| 30 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCT K PKDGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAgSl43K |
| 31 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCT KPSAGNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg DI44A |
| 32 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCT KPSDRNCTCIPIPSSWAFGKFLWEWASARFSW | HBsAg G145R |
| 33 | QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCT KPSDGACTCIPIPSSWAFGKFLWEWASARFSW | HBsAg NI 46A |
| 34 | GRJFRSFYMS | HBC34 Ab CDRHl aa (CCG numbering) |
| 35 | TINQDGSEK.LYVDSVKG | HBC34 Ab CDRH2 aa (CCG numbering) |
| 36 | NINQDGSEKLYVDSVKG | HBC34 AbCDR.H2_2 aa (CCG numbering) |
| 37 | WSGNSGGMDV | HBC34 Ab CDRH3 aa (CCG numbering) |
| 38 | ELQLVESGGGWVQPGGSQRLSCAASGRIFRSFYMSWVR QAPGKGLEWVATINQDGSEKLYVDSVKGRFTISRDNAK NSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQG TTVSVSS | HBC34 VH_l |
| 39 | EVQLVESGGGLVQPGGSLRLSCAASGRIFRSFYMSWVRQ APGKGLEWVANINQDGSEKLYVDSVKGRFTISRDNAKNS LFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTT VTVSS | HBC34 VH„2 |
| 40 | SGDKLGNKNVC | HBC34, -v7,-v3l,v32 CDRLl aa (CCG numbering) |
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| 41 | SGDKLGNKNVA | HBC34-v35 - v50 CDRL1 aa (CCG numbering) |
| 42 | SGDKLGNKNVS | HBC34-v34 CDRL1 aa (CCG numbering) |
| 43 | SGDKLGNKNAC | HBC34-v23, -v33 CDRL1 aa (CCG numbering) |
| 44 | EVKYRPS | HBC34, -v7, -v23, v31-v33,-v35, -v45, v46, -v48 CDRL2 aa (CCG numbering) |
| 45 | QDSKRPS | HBC34-v36 CDRL2 aa (CCG numbering) |
| 46 | EDSKRPS | HBC34-v37 CDRL2 aa (CCG numbering) |
| 47 | QVSKRPS | HBC34-V38 CDRL2 aa (CCG numbering) |
| 48 | QDDKRPS | ]-IBC34-v39 CDRL2 aa (CCG numbering) |
| 49 | QDSYRPS | HBC34-v40 CDRL2 aa (CCG numbering) |
| 50 | QVSYRPS | HBC34-V41 CDRL2 aa (CCG numbering) |
| 51 | EVSYRPS | HBC34-v42, -v47, v49, -v50 CDRL2 aa (CCG numbering) |
| 52 | QVKYRPS | HBC34-v43 CDRL2 aa (CCG numbering) |
| 53 | AVKYRPS | HBC34-V44 CDRL2 aa (CCG numbering) |
| 54 | [reserved] | |
| 55 | QTFDSTTVV | HBC34-v7, -v23, v32,-v33,-v35-v50 CDRL3 aa (CCG numbering) |
| 56 | QTWDSTTVV | HBC34,-v31 CDRL3 aa (CCG numbering) |
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| 57 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQS P VL V1YE VK YRPSGIPERF SG SNSGNTATLTl SGTQAM DEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-V35 VL aa |
| 58 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAM DEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v36 VL aa |
| 59 | SYELTQPPSVSVSPGQTVS1PCSGDKLGNKNVA WFQHKP GQSPVLVIYEDSKRPSGIPERFSGSNSGNTATLTISGTQAM DEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v37 VL aa |
| 60 | S YELTQPPS VSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQSPVLVIYQVSKRPSGIPERFSGSNSGNTATLTISGTQAM DEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v38 VL aa |
| 61 | S YELTQPPS VS VS PGQT VSIPCSGDKLGN KN VA WFQHKP GQSPVLVIYQDKKRPSGIPERFSGSNSGNTATLTISGTQA MDEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v39 VL aa |
| 62 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQSPVLVIYQDSYRPSGIPERFSGSNSGNTATLTISGTQAM DEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v40 VL aa |
| 63 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNV A WFQHKP GQSPVLVIYQVSYRPSGIPERFSGSNSGNTATLTI SGTQAM DEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v4l VL aa |
| 64 | S YELTQPPS VSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQSPVLVIYEVSYRPSGIPERFSGANSGNTATLTISGTQAM DEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v42 VL aa |
| 65 | SYELTQPPS VSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQSPVLVIYQVKYRPSGIPERFSGSNSGNTATLTISGTQA MDEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-V43 VL aa |
| 66 | S YELTQPPS VSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQSPVLVIYAVKYRPSGIPERFSGSNSGNTATLTISGTQA MDEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-V44 VL aa |
| 67 | S YELTQPPSVSVSPGQTVSJPCSGDKLGNKNVA WFQHKP GQSPVLVIYEVKYRPSGIPENFSGSNSGNTATLTISGTQA MDEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-V45 VL aa |
| 68 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVA WFQHKP GQSPVLVIYEVKYRPSGJPEAFSGSNSGNTATLTISGTQA MDEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v46 VL aa |
229
| 69 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQSPVLVIYEVSYRPSGIPENFSGANSGNTATLTASGTQA MDEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v47 VL aa |
| 70 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQSPVLVIYEVKYRPSGIPENFSGANSGNTATLTASGTQA MDEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v48 VL aa |
| 71 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQSPVLVIYEVSYRPSGIPERFSGSNSGNTATLTISGTQAM DEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-V49 VL aa |
| 72 | SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVAWFQHKP GQSPVLVIYEVKYRPSGIPEKFSGSNSGNTATLTISGTQA MDEAAYFCQTFDSTTVVFGGGTRLTVL | HBC34-v50 VL aa |
| 73 | APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK | WT hlgGl Fc |
| 74 | ESKYGPPCPPCPAPPVAGP | Chimeric hînge sequence |
| 75 | ELQLVESGGGWVQPGGSQRLSCAASGRIFRSFYMSWVR QAPGKGLEWVAT1NQDGSEKLYVDSVKGRFTISRDNAK NSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQG TTVSVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPLPEEKT1SKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVLHEALHSHYTQKSLSLSPGK | HC of HBC34-v35-50 MLNS-GAAL1E (glM17, 1) |
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| 76 | ELQLVESGGGWVQPGGSQRLSCAASGRIFRSFYMSWVR QAPGKGLEWVATINQDGSEKLYVDSVKGRFTISRDNAK NSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQG TTVSVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVLHEALHSHYTQKSLSLSPGK | HC ofHBC34-v35-50 MLNS (glMl7, l) |
| 77 | ELQL VESGGG WVQPGGSQRLSC AA SGRIFRSFYMS W VRQA PGKGLEWVATrNQDGSEKLYVDSVKGRFTISRDNAKNSLFL QMNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTTVSVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYÏC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPSVF LFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALH1MHYTQKSLSLSP GK | HBC34-V35-50 HC with GAALIE mutation inhlgGl Fc |
| 78 | ELQLVESGGGWVQPGGSQRLSCAASGRÎFRSFYMSWVR QAPGKGLEWVATINQDGSEKLYVDSVKGRFTISRDNAK NSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQG TTVSVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK | HBC34-V35-50 HC (wild-type) |
| 79 | GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS | HBC antibody light chain constant région |
| • | 231 | ||
| 80 | GAACTGCAGCTGGTGGAGTCTGGGGGAGGCTGGGTCC AGCCGGGGGGGTCCCAGAGACTGTCCTGTGCAGCCTC TGGACGCATCTTTAGAAGTTTTTACATGAGCTGGGTCC GCCAGGCCCCAGGGAAGGGGCTGGAGTGGGTGGCCAC TATAAACCAAGATGGAAGTGAGAAATTATATGTGGAC TCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACG CCAAGAACTCACTATTTCTGCAAATGAACAACCTGAG AGTCGAGGACACGGCCGTTTATTACTGCGCGGCTTGGA GCGGCAATAGTGGGGGTATGGACGTCTGGGGCCAGGG GACCACGGTCTCCGTCTCCTCA | HBC34 VH nue | |
| 81 | GAGGTGCAGCTGGTGGAATCCGGCGGGGGACTGGTGC AGCCTGGCGGCTCACTGAGACTGAGCTGTGCAGCTTCT GGAAGAATCTTCAGATCTTTTTACATGAGTTGGGTGAG ACAGGCTCCTGGGAAGGGACTGGAGTGGGTCGCAAAC ATCAATCAGGACGGATCAGAAAAGCTGTATGTGGATA GCGTCAAAGGCAGGTTCACTATTTCCCGCGACAACGCC AAAAATTCTCTGTTTCTGCAGATGAACAATCTGCGGGT GGAGGATACCGCTGTCTACTATTGTGCAGCCTGGTCTG GCAACAGTGGAGGCATGGACGTGTGGGGACAGGGAAC CACAGTGACAGTCAGCTCC | HBC34-v35-v50 VH (nue) | |
| 82 | GAACTGCAGCTGGTCGAATCAGGAGGAGGGTGGGTCC AGCCCGGAGGGAGCCAGAGACTGTCTTGTGCCGCATC AGGGAGGATCTTCAGGAGCTTCTACATGTCCTGGGTGC GCCAGGCACCAGGCAAGGGACTGGAGTGGGTCGCCAC CATCAACCAGGACGGATCTGAAAAGCTGTATGTGGAT AGTGTCAAAGGCCGGTTCACAATTAGCAGAGACAACG CTAAAAATTCTCTGTTTCTGCAGATGAACAATCTGCGA GTGGAGGATACCGCCGTCTACTATTGCGCCGCTTGGTC TGGCAACAGCGGCGGGATGGATGTCTGGGGGCAGGGC ACAACAGTGAGCGTCTCTTCC | HBC34 wt VH codon optimized |
232
| 83 | GCCTCCACAAAGGGCCCAAGCGTGTTTCCACTGGCTCCCTCT TCCAAGTCTACCTCCGGCGGCACAGCCGCTCTGGGATGTCTG GTGAAGGATTACTTCCCAGAGCCCGTGACCGTGTCTTGGAA CTCCGGCGCCCTGACCAGCGGAGTGCATACATTTCCAGCTGT GCTGCAGAGCTCTGGCCTGTACTCTCTGTCCAGCGTGGTGAC CGTGCCCTCTÏCCAGCCTGGGCACCCAGACATATATCTGCAA CGTGAATCACAAGCCAAGCAATACAAAGGTGGACAAGAAG GTGGAGCCCAAGTCTTGTGATAAGACCCATACATGCCCTCC ATGTCCAGCTCCAGAGCTGCTGGGCGGCCCAAGCGTGTTCCT GTTTCCACCCAAGCCTAAGGATACCCTGATGATCTCCAGAAC CCCCGAGGTGACATGCGTGGTGGTGGACGTGAGCCACGAGG ATCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAG GTGCATAATGCTAAGACCAAGCCCAGGGAGGAGCAGTACAA CTCTACCTATCGGGTGGTGTCCGTGCTGACAGTGCTGCACCA GGATTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGTCTA ATAAGGCCCTGCCCGCTCCTATCGAGAAGACCATCTCCAAG GCCAAGGGCCAGCCTAGAGAGCCACAGGTGTACACACTGCC TCCATCTCGCGATGAGCTGACCAAGAACCAGGTGTCCCTGA CATGTCTGGTGAAGGGCTTCTATCCTTCCGACATCGCTGTGG AGTGGGAGAGCAATGGCCAGCCAGAGAACAATTACAAGAC CACACCCCCTGTGCTGGACAGCGATGGCTCTTTCTTTCTGTA TAGCAAGCTGACCGTGGACAAGTCTCGCTGGCAGCAGGGCA ACGTGTTTAGCTGTTCTGTGATGCATGAGGCCCTGCACAATC ATTATACACAGAAGTCCCTGAGCCTGTCTCCTGGCAAG | HBC34-V35-50 CHl- hinge-CH2-CH3 (codon-optimized) |
233
| 84 | GAGCTGCAGCTGGTGGAGTCCGGCGGCGGCTGGGTGCAGCC TGGCGGCTCCCAGAGGCTGAGCTGTGCCGCTTCTGGCAGGA TCTTCCGGTCCTTTTACATGTCTTGGGTGCGGCAGGCTCCAG GCAAGGGCCTGGAGTGGGTGGCTACCATCAACCAGGACGGC TCCGAGAAGCTGTATGTGGATAGCGTGAAGGGCAGATTCAC AATCTCTCGCGACAACGCCAAGAACTCCCTGTTTCTGCAGAT GAACAATCTGAGGGTGGAGGATACCGCCGTGTACTATTGCG CCGCTTGGTCTGGCAATAGCGGCGGCATGGACGTGTGGGGA CAGGGCACCACCGTGTCCGTGTCCAGCGCCTCCACAAAGGG CCCAAGCGTGTTTCCACTGGCTCCCTCTTCCAAGTCTACCTC CGGCGGCACAGCCGCTCTGGGATGTCTGGTGAAGGATTACT TCCCAGAGCCCGTGACCGTGTCTTGGAACTCCGGCGCCCTGA CCAGCGGAGTGCATACATTTCCAGCTGTGCTGCAGAGCTCTG GCCTGTACTCTCTGTCCAGCGTGGTGACCGTGCCCTCTTCCA GCCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAG CCAAGCAATACAAAGGTGGACAAGAAGGTGGAGCCCAAGT CTTGTGATAAGACCCATACATGCCCTCCATGTCCAGCTCCAG AGCTGCTGGGCGGCCCAAGCGTGTTCCTGTTTCCACCCAAGC CTAAGGATACCCTGATGATCTCCAGAACCCCCGAGGTGACA TGCGTGGTGGTGGACGTGAGCCACGAGGATCCTGAGGTGAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCTA AGACCAAGCCCAGGGAGGAGCAGTACAACTCTACCTATCGG GTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAA CGGCAAGGAGTATAAGTGCAAGGTGTCTAATAAGGCCCTGC CCGCTCCTATCGAGAAGACCATCTCCAAGGCCAAGGGCCAG CCTAGAGAGCCACAGGTGTACACACTGCCTCCATCTCGCGA TGAGCTGACCAAGAACCAGGTGTCCCTGACATGTCTGGTGA AGGGCTTCTATCCTTCCGACATCGCTGTGGAGTGGGAGAGC AATGGCCAGCCAGAGAACAATTACAAGACCACACCCCCTGT GCTGGACAGCGATGGCTCTTTCTTTCTGTATAGCAAGCTGAC CGTGGACAAGTCTCGCTGGCAGCAGGGCAACGTGTTTAGCT GTTCTGTGATGCATGAGGCCCTGCACAATCATTATACACAGA AGTCCCTGAGCCTGTCTCCTGGCAAGTGATGAGGTACCGTGC GACGGCCGGCAAGCCCCCGCTCCCCGGGCTCTCGCGGTCGT ACGAGGAAAGCTT | HBC34-V35-50 VH- CHl-hinge-CH2-CH3 (codon-optimized) |
| 85 | AGCTGACACAGCCCCCTTCCGTGTCCGTGTCCCCTGGA CAGACCGTGTCCATCCCATGCAGCGGCGACAAGCTGG GCAACAAGAACGTGGCCTGGTTTCAGCATAAGCCTGG CCAGTCCCCCGTGCTGGTCATCTACCAGGACTCCAAGA GGCCCAGCGGCATCCCTGAGCGGTTCTCTGGCTCCAAC AGCGGCAATACAGCCACCCTGACAATCTCTGGCACAC AGGCTATGGACGAGGCCGCTTATTTCTGCCAGACCTTT GATTCCACCACAGTGGTGTTCGGCGGCGGCACCAGAC TGACAGTGCTGGGTCAGCCCAAGGCTGCCCCCTCGGTC ACTCTG | HBC34-v36 VL nt |
234
| 86 | TCCTACGAGCTGACACAGCCACCTTCCGTGAGCGTGTCTCCA GGACAGACCGTGTCCATCCCTTGCAGCGGCGACAAGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACGAGGATTCTAAGAGGCCTTCCG GCATCCCAGAGCGGTTTTCCGGCAGCAACTCTGGCAATACC GCCACACTGACCATCAGCGGCACACAGGCTATGGACGAGGC CGCTTATTTCTGTCAGACCTTTGATTCTACCACAGTGGTGTTC GGCGGCGGCACAAGGCTGACCGTGCTG | HBC34-v37 VL nt |
| 87 | TCCTACGAGCTGACACAGCCACCTTCCGTGAGCGTGTCTCCA GGAC A GACCGTGTCCATCCCTTGCAGCGGCG ACA AGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACCAGGTGTCTAAGAGGCCTTCCG GCATCCCAGAGCGGTTTTCCGGCAGCAACTCTGGCAATACC GCCACACTGACCATCAGCGGCACACAGGCTATGGACGAGGC CGCTTATTTCTGTCAGACCTTTGATTCTACCACAGTGGTGTTC GGCGGCGGCACAAGGCTGACCGTGCTG | HBC34-v38 VL nt |
| 88 | TCCTACGAGCTGACACAGCCACCTTCCGTGAGCGTGTCTCCA GGACAGACCGTGTCCATCCCTTGCAGCGGCGACAAGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACCAGGATAAGAAGAGGCCTTCC GGCATCCCAGAGCGGTTTTCCGGCAGCAACTCTGGCAATAC CGCCACACTGACCATCAGCGGCACACAGGCTATGGACGAGG CCGCTTATTTCTGTCAGACCTTTGATTCTACCACAGTGGTGTT CGGCGGCGGCACAAGGCTGACCGTGCTG | HBC34-v39 VL nt |
| 89 | TCCTACGAGCTGACACAGCCACCTTCCGTGAGCGTGTCTCCA GGAC AGACCGTGTCCATCCCTTGC A GCGGCG ACA AGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACCAGGATTCTTATAGGCCTTCCG GCATCCCAGAGCGGTTTTCCGGCAGCAACTCTGGCAATACC GCCACACTGACCATCAGCGGCACACAGGCTATGGACGAGGC CGCTTATTTCTGTCAGACCTTTGATTCTACCACAGTGGTGTTC GGCGGCGGCACAAGGCTGACCGTGCTG | HBC34-v40 VL nt |
| 90 | TCCTA CGAG CTG ACACAGCC ACCTTCCGTG A GCGTGTCTCC A GGACAGACCGTGTCCATCCCTTGCAGCGGCGACAAGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACCAGGTGTCTTATAGGCCTTCCG GCATCCCAGAGCGGTTTTCCGGCAGCAACTCTGGCAATACC GCCACACTGACCATCAGCGGCACACAGGCTATGGACGAGGC CGCTTATTTCTGTCAGACCTTTGATTCTACCACAGTGGTGTTC GGCGGCGGCACAAGGCTGACCGTGCTG | HBC34-v41 VL nt |
235
| 91 | TCTTACG A GCTGAC A CA GCCACCTTCCGTG AGCGTGTCTCC A GGACAGACCGTGTCCATCCCTTGCAGCGGCGACAAGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACGAGGTGTCTTATAGGCCTTCCG GCATCCCAGAGCGGTTTAGCGGCGCCAACTCTGGCAATACC GCTACACTGACCATCTCCGGCACACAGGCTATGGACGAGGC CGCTTATTTCTGTCAGACCTTTGATAGCACCACAGTGGTGTT CGGCGGCGGCACAAGGCTGACCGTGCTG | HBC34-V42 VL nt |
| 92 | TCCTACGAGCTGACACAGCCACCTTCCGTGAGCGTGTCTCCA GGACAGACCGTGTCCATCCCTTGCAGCGGCGACAAGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACCAGGTGAAGTATAGGCCTTCCG GCATCCCAGAGCGGTTTTCCGGCAGCAACTCTGGCAATACC GCCACACTGACCATCAGCGGCACACAGGCTATGGACGAGGC CGCTTA TTT CTGTC AG ACCTTTGATTCTACCACAGTGGTG TTC GGCGGCGGCACAAGGCTGACCGTGCTG | HBC34-V43 VL nt |
| 93 | TCCTACGAGCTGACACAGCCACCTTCCGTGAGCGTGTCTCCA GGACAGACCGTGTCCATCCCTTGCAGCGGCGACAAGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACGCTGTGAAGTATAGGCCTTCCG GCATCCCAGAGCGGTTTTCCGGCAGCAACTCTGGCAATACC GCCACACTGACCATCAGCGGCACACAGGCTATGGACGAGGC CGCTTA TTTCTGTC AG ACCTTTG ATTCT ACC AC AGTGGTG TTC GGCGGCGGCACAAGGCTGACCGTGCTG | HBC34-V44 VL nt |
| 94 | TCCTACGAGCTGACACAGCCACCTTCCGTGAGCGTGTCTCCA GGACAGACCGTGTCCATCCCTTGCAGCGGCGACAAGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACGAGGTGAAGTATAGGCCTTCCG GCATCCCAGAGAACTTTTCCGGCAGCAACTCTGGCAATACC GCCACACTGACCATCAGCGGCACACAGGCTATGGACGAGGC CGCTTA TTTCTGTCAGACCTTTGATTCTACCACAGTGGTGTTC GGAGGAGGAACAAGGCTGACCGTGCTG | HBC34-v45 VL nt |
| 95 | TCCTACGAGCTGACACAGCCACCTTCCGTGAGCGTGTCTCCA GGACAGACCGTGTCCATCCCTTGCAGCGGCGACAAGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACGAGGTGAAGTATAGGCCTTCCG GCATCCCAGAGGCTTTTTCCGGCAGCAACTCTGGCAATACCG CCACACTGACCATCAGCGGCACACAGGCTATGGACGAGGCC GCTTATTTCTGTC AG ACCTTTG ATTCT ACC AC AGTGGTGTTC GGAGGAGGAACAAGGCTGACCGTGCTG | HBC34-V46 VL nt |
236
| 96 | TCCTACGAGCTGACACAGCCACCTTCCGTGAGCGTGTCTCCA GGACAGACCGTGTCCATCCCTTGCAGCGGCGACAAGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACGAGGTGTCTTATAGGCCTTCCG GCATCCCAGAGCGGTTTTCCGGCAGCAACTCTGGCAATACC GCCACACTGACCATCAGCGGCACACAGGCTATGGACGAGGC CGCTTATTTCTGTCAGACCTTTGATTCTACCACAGTGGTGTTC GGCGGCGGCACAAGGCTGACCGTGCTG | HBC34-v49 VL nt |
| 97 | TCCTACGAGCTGACACAGCCACCTTCCGTGAGCGTGTCTCCA GGACAGACCGTGTCCATCCCTTGCAGCGGCGACAAGCTGGG CAACAAGAATGTGGCCTGGTTCCAGCACAAGCCAGGCCAGT CCCCCGTGCTGGTCATCTACGAGGTGAAGTATAGGCCTTCCG GCATCCCAGAGAAGTTTTCCGGCAGCAACTCTGGCAATACC GCCACACTGACCATCAGCGGCACACAGGCTATGGACGAGGC CGCTTATTTCTGTCAGACCTTTGATTCTACCACAGTGGTGTTC GGAGGAGGAACAAGGCTGACCGTGCTG | HBC34-v50 VL nt |
| 98 | GGACAGCCAAAGGCTGCTCCATCTGTGACCCTGTTTCCACCC TCTTCCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTG CCTGATCTCTGACTTCTACCCTGGAGCTGTGACAGTGGCTTG GAAGGCTGATAGCTCTCCCGTGAAGGCTGGCGTGGAGACAA CAACCCCTAGCAAGCAGTCTAACAATAAGTACGCCGCTTCC AGCTATCTGTCTCTGACACCAGAGCAGTGGAAGTCCCACCG CTCTTATTCCTGCCAGGTGACCCATGAGGGCAGCACCGTGG AGAAGACAGTGGCCCCCACCGAGTGTTCT | HBC34-V35-50 CL (c o don- o pt i m ized)_ l |
| 99 | GGACAGCCAAAGGCTGCTCCATCTGTGACCCTGTTTCCACCC TCTTCCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTG CCTGATCTCTGACTTCTACCCTGGAGCTGTGACAGTGGCTTG GAAGGCTGATAGCTCTCCCGTGAAGGCTGGCGTGGAGACAA CAACCCCTAGCAAGCAGTCTAACAATAAGTACGCCGCTTCC AGCTATCTGTCTCTGACACCAGAGCAGTGGAAGTCCCACCG CTCTTATTCCTGCCAGGTGACCCATGAGGGCAGCACCGTGG AGAAGACAGTGGCCCCCACCGAGTGTTCT | HBC34-V35-50 CL (codonoptimized)_2 |
| ΙΟΟ | XGS STTSTG PCRTCMTXPSDGN ATA IPIPSS WX wherein the residues coded as X were substituted with Cysteines | peptide |
| ΙΟΙ | TSTGPC RTCMTTA QG | peptide |
| 102 | GMLPVCPLIPGSSTTSTGPCRTCMTT | peptide |
| 103 | XSMYPSASATKPSDGNXTGPCRTCMTTAQGTSX wherein the residues coded as X were substituted with Cysteines | peptide |
| 104 | PCRTCMTTAQG | amino acids 120 - 130 of the S domain of HBsAg (HBV-D J02203 |
237
| 105 | PCX1TCX2X3X4AQG, wherein Xi is R or K, X: is M or T, X3 is T or 1, and X4 is T, P or L | epitope |
| 106 | GGSGG | linker |
| 107 | TGPCRTC | epitope |
| 108 | GNCTCIP | epitope |
| 109 | CCIPIPSSWAFGCSTTSTGPCRTCC wherein in particular the cysteines at positions 2, 21, and 24 are coupled to acetamidomethyl. | discontinuons epitope mimic |
| 110 | ÇGNCTCIP1PSSWAFÇSTTSTGPCRTCÇ wherein in particular the cysteines at positions 4, 6, 24, and 27 are coupled to acetamidomethyl. | discontinuous epitope mimic |
| 111 | CGGGCSTTSTGPCRTCC wherein in particular the cysteines at positions 13 and 16 are coupled to acetamidomethyl. | looped epitope mimic |
| 112 | STTSTGPCRTC | epitope |
| 113 | GNCTC1PIPSSWAFC | epitope |
| 1 14 | GNCTCIPIPSSWAF | epitope |
| 115 | PCRXC | epitope |
238
| 116 | aattccacaa ccttccacca aactctgcaa gatcccagag tgagaggcct gtatttccct gctggtggct ccagticagg aacagtaaac cctgttctga ctactgcctc tcccttatcg tcaatcttct cgaggattgg ggacccigcg ctgaacatgg agaacatcac atcaggattc ctaggacccc ttctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata ccgcagagtc tagactcgtg gtggacttct ctcaattttc tagggggaac taccgtgtgt cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcttg tcctccaact tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tcttcctctt catcctgctg ciatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct claattccag gatcctcaac aaccagcacg ggaccatgcc ggacctgcat gactactgct caaggaacct ctatgtatcc ctcctgttgc tgtaccaaac cttcggacgg aaattgcacc tgtattccca tcccatcatc ctgggctttc ggaaaattcc tatgggagtg ggcctcagcc cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc ctaacaaaac aaagagatgg ggttactctc taaattttat gggttatgtc attggatgtt atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc ctattaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggttttg ctgccccttt tacacaatgt ggttatcctg cgttgatgcc tttgtatgca tgtattcaat ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgcl gacgcaaccc ccactggctg gggcttggtc atgggccatc agcgcatgcg tggaaccttt tcggctcctc tgccgateca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa acattatcgg gactgataac tctgttgtcc tatcccgcaa atatacatcg tttccatggc tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac cgtgaacgcc caccaaatat tgcccaaggt cttacataag aggactcttg gactctcagc aatgtcaacg accgaccttg aggcatactt caaagaclgt ttgtttaaag actgggagga gttgggggag gagattaggt taaaggtctî tgtactagga ggctgtaggc ataaattggt ctgcgcacca gcaccatgca actttttcac ctctgcctaa tcatctcttg ttcatgtcct actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat cgacccttat aaagaatttg gagctactgt ggagttactc tcgtttttgc cttctgactt ctttccttca gtacgagatc ttctagatac cgcctcagct ctgtatcggg aagccttaga gtctcctgag cattgttcac ctcaccatac tgcactcagg caagcaattc tttgctgggg ggaactaatg actctagcta cctgggtggg tgttaatttg gaagatccag cgtctagaga cctagtagtc agttatgtca acactaatat gggcctaaag ttcaggcaac tcttgtggtt tcacatttct tgtctcactt ttggaagaga aacagttata gagtatttgg tgtctttcgg agtgtggatt cgcactcctc cagcttatag accaccaaat gcccctatcc tatcaacact tccggagact actgttgtta gacgacgagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga aggtctcaat cgccgcgtcg cagaagatct caatctcggg aatctcaatg ttagtattcc ttggactcat aaggtgggga actttactgg gctttattct tctactgtac ctgtctttaa rcctcattgg aaaacaccat cttttcciaa tatacattta caccaagaca ttatcaaaaa atgtgaacag tttgtaggcc cactcacagt taatgagaaa agaagattgc aattgattal gcctgccagg ttttatccaa aggttaccaa atatttacca ttggataagg gtattaaacc ttattatcca gaacatciag ttaatcatta cttccaaact agacactatt tacacactct atggaaggcg ggtatattat ataagagaga aacaacacat agcgcctcat tttgtgggtc accatattct tgggaacaag atctacagca tggggcagaa tctttccacc agcaatcctc tgggattctt tcccgaccac cagttggatc cagccttcag agcaaacacc gcaaatccag attgggactl caatcccaac aaggacacct ggccagacgc caacaaggta ggagctggag cattcgggct gggtttcacc ccaccgcacg gaggcctttt ggggtggagc cctcaggctc agggcatact acaaactttg ccagcaaatc cgcctcctgc ctccaccaat cgccagtcag gaaggcagcc taccccgctg tctccaccti tgagaaacac tcatcctcag gccatgcagt gg | NC_003977.2 |
239
| ] 17 | GTGTGCACTTCGCTTCAC | 1579-1597 of NC_003977.2 |
| 118 | GUGUGCACUUCGCUUCACA | HBV001 sense |
| 119 | UGUGAAGCGAAGUGCACACUU | HBV001 antisense |
| 120 | GGUGGACUUCUCUCAAUUUUA | HBV003 sense |
| 121 | UAAAAUUGAGAGAAGUCCACCAC | HBV003 antisense |
| 122 | gsusguGFcAFCFUFucgcuucacaL96 | HBV002v2 sense |
| 123 | usGFsugaAFgCFGFaaguGfcAfcacsusu | HBV002v2 antisense |
| 124 | gsusguGfcAfCfUFucgcuucacaL96 | HBV002vl sense |
| 125 | usGFsuga(Agn)gCFGFaaguGFcAFcacsusu | HBV002vl antisense |
| 126 | gsgsuggaCFuUFCFUFcucaAfUFuuuaL96 | No. 126 sense |
| 127 | usAFsaaaUFuGFAFgagaAFgUFccaccsasc | No. 127 antisense |
| 128 | AAVALLPAVLLALLAP | RFGF |
| 129 | AALLPVLLAAP | RFGF analogue |
| 130 | GRKKRRQRRRPPQ | HIV Tat protein |
| 131 | RQIK1WFQNRRMKWK | Drosophila Antennapedia protein |
| 132 | Gly Gin Ser Pro Val Leu Val Ile Tyr Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn | Synthetic sequence CDR Framework |
| 133 | Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr He Ser Gly Thr Gin Ala Met Asp Glu Ala Ala | Synthetic sequence CDR Framework |
| 134 | Gly Gin Ser Pro Val Leu Val Ile Tyr Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence HBC34 |
| 135 | Gly Gin Ser Pro Val Leu Val Ile Tyr Gin Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence GL L2 |
240
| ΐ 36 | Gly Gin Ser Pro Val Leu Val Ile Tyr Glu Asp Ser Lys Arg Pro Ser Gly lie Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence v36 + Q49E |
| 137 | Gly Gin Ser Pro Val Leu Val Ile Tyr Gin Va] Ser Lys Arg Pro Ser Giy Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Plie Cys | Synthetic sequence v36 + D50V |
| 138 | Gly Gin Ser Pro Val Leu Val Ile Tyr Gin Asp Lys Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr lie Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence v36 + S51K |
| 139 | Giy Gin Ser Pro Val Leu Val Ile Tyr Gin Asp Ser Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence v36 + K52Y |
| 140 | Gly Gin Ser Pro Val Leu Val Ile Tyr Gin Val Ser Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence v36 + D50V + K52Y |
| I4l | Gly Gin Ser Pro Val Leu Val Ile Tyr Glu Val Ser Tyr Arg Pro Ser Gly lie Pro Glu Arg Phe Ser Gly Ala Asn Ser Gly Asn Thr Ala Thi Leu Thr Ile Ser Gly Thr Gin Ala Met Asp Giu Ala Ala Tyr Phe Cys | Synthetic sequence HBC34 +K51S + S64A |
| 142 | Gly Gin Ser Pro Val Leu Va] Ile Tyr Gin Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Giy Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence HBC34 +E49Q |
| 143 | Gly Gin Ser Pro Val Leu Val Ile Tyr Ala Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gin Ala Met Asp Glu Ala Aia Tyr Phe Cys | Synthetic sequence HBC34 +E49A |
| 144 | Gly Gin Ser Pro Val Leu Val Ile Tyr Glu Val Lys Tyr Arg Pro Ser Gly Ile Pro Glu Asn Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gin Aia Met Asp G!u Ala Ala Tyr Phe Cys | Synthetic sequence HBC34 +R60N |
| 145 | Gly Gin Ser Pro Val Leu Val lie Tyr Glu Val Lys Tyr Arg Pro Ser Gly lie Pro Glu Ala Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr lie Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence HBC34 +R60A |
| 146 | Gly Gin Ser Pro Val Leu Val Ile Tyr Glu Val Ser Tyr Arg Pro Ser Gly Ile Pro Glu Asn Phe Ser Gly Ala Asn Ser Gly Asn Thr Ala Thr Leu Thr Ala Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence HBC34+K51S + S64A + R60N + I74A |
241
| 147 | Gly Gin Ser Pro Val Leu Val lie Tyr Glu Val Lys Tyr Arg Pro Set Gly Ile Pro Glu Asn Phe Ser Gly Ala Asn Ser Gly Asn Thr Ala Thr Leu Thr Ala Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence HBC34 +R60N + S64A -e- I74A |
| 148 | Gly Gin Ser Pro Val Leu Val Ile Tyr Glu Val Ser Tyr Arg Pro Set Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence HBC34 +K51S |
| 149 | Gly Gin Ser Pro Val Leu Val Ile Tyr Glu Val Lys Tyr Arg Pro Set Gly Ile Pro Glu Lys Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Tht Leu Thr Ile Ser Gly Thr Gin Ala Met Asp Glu Ala Ala Tyr Phe Cys | Synthetic sequence HBC34 + R60K |
| 150 | GRIFRSFY | CDRH1 (IMGT) |
| I5l | QDGSEK | CDRH2 — short (IMGT) |
| 152 | INQDGSEK | CDRH2 - long (IMGT) |
| 153 | AAWSGNSGGMDV | CDRH3 (IMGT) |
| 154 | KLGNKN | CDRL1 (IMGT) |
| 155 | EVK | HBC34-v35, -v45, -v46, -v48, -v50 CDRL2 - short |
| 156 | QDS | HBC34-v36, -v40 CDRL2 - short (IMGT) |
| 157 | EDS | HBC34-V37 CDRL2 short (IMGT) |
| 158 | QVS | HBC34-V38, -v41, CDRL2 - short (IMGT) |
| 159 | QDK | HBC34-v39 CDRL2 - short (IMGT) |
242
| 160 | EVS | HBC34-V42, -v47, -v49, -v50 CDRL2 - short (IMGT) |
| 161 | QVK | HBC34-v43 CDRL2 - short (IMGT) |
| 162 | AVK | HBC34-v44 CDRL2 - short (IMGT) |
| 163 | VIYEVKYRPS | HBC34-V35, -v45, -v46, -v48, -v50 CDRL2 - long |
| 164 | VIYQDSKRPS | HBC34-V36 CDRL2 - long (IMGT) |
| 165 | VIYEDSKRPS | HBC34-V37 CDRL2 - long (IMGT) |
| 166 | VIYQVSKRPS | HBC34-v38 CDRL2 - long (IMGT) |
| 167 | VIYQDKKRPS | HBC34-V39 CDRL2 - long (IMGT) |
| 168 | V1YQDSYRPS | HBC34-V40 CDRL2 - long (IMGT) |
| 169 | VIYQVSYRPS | HBC34-v41 CDRL2 - long (IMGT) |
| 170 | VTYEVSYRPS | HBC34-V42, -v47, v49 CDRL2 - long |
| 171 | VIYQVKYRPS | HBC34-v43 CDRL2 - long (IMGT) |
| 172 | VIYAVKYRPS | HBC34-v44 CDRL2 - long (IMGT) |
| 173 | QTFDSTTVV | HBC34-v35-v5O CDRL3 (IMGT) |
243
The various embodiments described above can be combined to provide further embodiments. Ail ofthe U.S. patents, U.S. patent application publications, U.S. patent applications, foreîgn patents, foreign patent applications and non-patent publications referred to in this spécification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
U.S. Provisional Application 63/043,692, ftled June 24, 2020 is incorporated herein by reference, in its entirety.
] 0 These and other changes can be made to the embodiments in light of the above- detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the spécifie embodiments disclosed in the spécification and the claims, but should be construed to include ail possible embodiments along with the full scope of équivalents to which such claims are entitled. Accordingly, the claims 15 are not limited by the disclosure.
Claims (5)
- l. An antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable région (VH) comprising a CDRHl amino acid sequence, a CDRH2 amino acid5 sequence, and a CDRH3 amino acid sequence; and a light chain variable région (VL) comprising a CDRLl amino acid sequence, a CDRL2 amino acid sequence, and a CDRL3 amino acid sequence, wherein the CDRHl, CDRH2, CDRH3, CDRLl, CDRL2, and CDRL3 amino acid sequences are accordingto SEQ IDNOs.:(i)I0 (ii) (iii) (iv) (v) (vi)15 (vii) (viii) (ix) (x)34, 35, 37, 41, 49, and 55, respectively;34, 35, 37, 41,46, and 55, respectively;34, 35, 37, 41, 47, and 55, respectively;34, 35, 37, 41, 48, and 55, respectively;34, 35, 37, 41, 45, and 55, respectively;34, 35, 37, 41, 50, and 55, respectively;34, 35, 37, 41, 51, and 55, respectively;34, 35, 37, 41, 52, and 55, respectively;34, 35, 37, 41, 53, and 55, respectively; or34, 35, 37, 41, 44, and 55, respectively.wherein CDRs are defined according to the CCG numbering System,20 wherein, optionally, the VL comprises a R60N substitution mutation, a R60A substitution mutation, a R60K substitution mutation, a S64A substitution mutation, a I74A substitution mutation, or any combination thereof, relative to SEQ ID NO.:58 and wherein the amino acid numbering ofthe substitution mutation(s) is according to SEQ ID NO.: 5 8, and still further optionally wherein the VL does not comprise any further mutation(s) relative to SEQ ID NO.:58,25 and wherein the antibody or antigen-binding fragment thereof is capable of binding to the antigenic loop région of HBsAg and, optionally, neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof.245
- 2. The antibody or antigen-binding fragment of claim l, wherein the VH and the VL comprise or consist of amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequences set forth in SEQ ID NOs.: (i) 38 and 62, respectively; (ii) 38 and 59, respectively; (iii) 38 and 60, respectively; (iv) 38 and 61, 5 respectively; (v) 38 and 58, respectively; (vi) 38 and 63, respectively; (vii) 38 and 64, respectively; (viii) 38 and 65, respectively; (ix) 38 and 66, respectively; (x) 38 and 7 i, respectively; or (xi) 38 and 72, respectively.
- 3. The antibody or antigen-binding fragment of any one of claims l and 2, wherein the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.:I0 (i) 38 and 62, respectively;(ii) 38 and 59, respectively;(iii) 38 and 60, respectively;(iv) 38 and 61, respectively;(v) 38 and 58, respectively;15 (vi) (vii) 38 and 63, respectively; 38 and 64, respectively;(viii) 38 and 65, respectively;(ix) 38 and 66, respectively;(x) 38 and 71, respectively; or20 (xi) 38 and 72, respectively.
- 4. An antibody or antigen-binding fragment, comprising a heavy chain variable région (VH) and a light chain variable région (VL), wherein the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.:(i) 38 and 62, respectively;25 (ii) (iii) 38 and 66, respectively; 38 and 67, respectively;(iv) 38 and 68, respectively; or246 (ν) 38 and 72, respectively, wherein the antibody or antigen-binding fragment thereof is capable of binding to the antigenic loop région of HBsAg and neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, ï, or J, or any combination thereof.55. The antibody or antigen-binding fragment ofany one of claims l-4, which is capable of neutralizing infection by a hepatitis D virus (HDV).6. The antibody or antigen-binding fragment of any one of claims l-5, wherein, in a sample comprising a plurality of the antibody or antigen-binding fragment, less than 12%, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or 10 less, or 2% or less of the plurality is comprised in a dimer when the sample has been incubated for about ] 20 to about 168 hours at about 40°C, wherein the presence of dimer is determined by absolute size-exclusion chromatography; and/or wherein incubation of a plurality' of the antibody or antigen-binding fragment results in reduced formation of a dimer as compared to incubation of a plurality of a reference antibody or 15 antigen-binding fragment, wherein the reference antibody or antigen-binding fragment comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.:57, and wherein the presence of antibody dimer is determined by absolute size-exclusion 20 chromatography.7. The antibody or antigen-binding fragment of any one of claims l -6, which forms a lower amount of dimer, and/or forms dimers at a reduced frequency and/or as a lower percentage of total antibody or antigen-binding fragment molécules in a sample or composition as compared to a reference antibody:25 (i) in a 5-day, a 15-day, and/or a 32-day incubation at 4°C;(ii) in a 5-day, a 15-day, and/or a 32-day incubation at 25°C; and/or247 (iii) in a 5-day, a 15-day, and/or a 32-day incubation at 40’C, wherein the reference antibody or antigen-binding fragment comprises the VH amino acid sequence set forth in SEQ ID NO.:38 and the VL amino acid sequence set forth in SEQ ID NO.: 57.58. The antibody or antigen-binding fragment of any one of claims l -7, wherein the antibody or antigen-binding fragment is capable of binding to a HBsAg (adw) with an EC50 (ng/ml) of about 3.2 or less, less than 3.0, less than 2.5, less than 2.0, less than 1.5, or less than l.O.9. The antibody or antigen-binding fragment of any one of claims 1-8, wherein the 10 antibody, or the antigen-binding fragment thereof, comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, a Fab, a Fab’, a F(ab’)2, a Fv, or a scFv.10. The antibody or antigen-binding fragment of any one of claims 1-9, wherein the antibody or antigen-binding fragment is a multi-specific or bispecific antibody or antigenbinding fragment.1511. The antibody of any one of claims 1-10, or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment comprises a Fc moiety.12. The antibody or antigen-binding fragment of claim 11, wherein the Fc moiety comprises a mutation that enhances binding to FcRn as compared to a reference Fc moiety that does not comprise the mutation.2013. The antibody or antigen-binding fragment of claim 11 or 12, wherein the Fc moiety comprises a mutation that enhances binding to a FcyR as compared to a reference Fc moiety that does not comprise the mutation.24814. The antibody or antigen-binding fragment of any one of claims 11-13, wherein the Fc moiety is an IgG isotype, or is derived from an IgG isotype.15. The antibody or antigen-binding fragment of any one of claims 11-14, which comprises or is derived from ig Glml7, 1 (IgHGl *01).16. The antibody or antigen-binding fragment of any one of claims 12-15, wherein the mutation that enhances binding to FcRn comprises:(i) M428L/N434S;(ii) M252Y/S254T/T256E;(iii) T250Q/M428E;(iv) P257I/Q31II;(v) P257I/N434H;(vi) D376WN434H;(v ii) T307A/E380A/N434A; or (viîi) any combination of (i)-(vii), wherein amino acid numbering of the Fc moiety is according to the EU numbering system.17. The antibody or antigen-binding fragment of claim 16, wherein the mutation that enhances binding to FcRn comprises M428L/N434S.18. The antibody or antigen-binding fragment of any one of claims 13-17, wherein the mutation that enhances binding to a FcyR comprises S239D; I332E; A330L; G236A; or any combination thereof, wherein amino acid numbering of the Fc moiety is according to the EU numbering system.19. The antibody or antigen-binding fragment of claim 18, wherein the mutation that enhances binding to a FcyR comprises:(i) S239D/I332E;249 (ii) S239D/A330L/I332E;(iii) G236A/S239D/I332E; or (iv) G236A/A330L/I332E.20. The antibody or antigen-binding fragment of daim 18 or 19, wherein the mutation that enhances binding to a FcyR comprises or consists of G236A/A330L/I332E, and optionally wherein the antibody or antigen-binding fragment does not comprise S239D, and wherein the antibody or antigen-binding fragment further optionally comprises a native S at position 239.21. The antibody or antigen-binding fragment ofany one of claims l I-20, wherein the Fc moiety comprises the amino acid substitution mutations: M428L; N434S; G236A; A330L; and I332E, and optionally does not comprise S239D.22. The antibody or antigen-binding fragment of any one of daims l -21, comprising a light chain constant région (CL) that comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ IDNO.:79.23, The antibody or antigen-binding fragment of any one of daims l-22, comprising a CH 1-CH2-CH3 that comprises or consists of an amino acid sequence having 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.:73, or a variant thereof that comprises one or more of the following amino acid substitutions (EU numbering): G236A; A330L; I332E; M428L; N434S.24. The antibody or antigen-binding fragment of daim 23, wherein the CH1-CH2-CH3 has a C-terminal lysine removed.25. An antibody comprising; a heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:75, optionally with the C-terminal lysine removed:and a light chain (LC), wherein the LC comprises or consists of (i) the VL amino acid sequence250 set forth in any one of SEQ ID NOs.:62, 58-61, and 63-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.26. The antibody of claim 25, wherein the LC comprises the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 66, 67, and 72.27. An antibody comprising: a heavy chain (HC) comprising or consisting ofthe amino acid sequence set forth in SEQ ID NO.:76, optionally with the C-terminal lysine removed, and a light chain (LC), wherein the LC comprises or consists of (i) the VL amino acid sequence set forth in any one of SEQ ID NOs.;62, 58-61, and 63-72 and (ii) the CL amino acid sequence set forth in SEQ IDNO.:79.28. The antibody of claim 27, wherein the LC comprises the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 66, 67, and 72.29. An antibody comprising: a heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:77, optionally with the C-terminal lysine removed, and a light chain (LC), wherein the LC comprises or consists of (î) the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 58-61, and 63-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.30. The antibody of claim 29, wherein the LC comprises the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 66, 67, and 72.31. An antibody comprising: a heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:78, optionally with the C-terminal lysine removed, and a light chain (LC), wherein the LC comprises or consists of (i) the VL amino acid sequence set forth in any one of SEQ lDNOs.:62, 58-61, and 63-72 and (ii) the CL amino acid sequence set forth in SEQ ID NO.:79.25132. The antibody of claim 31, wherein the LC comprises the VL amino acid sequence set forth in any one of SEQ ID NOs.:62, 66, 67, and 72.33. A poiynucleotide comprising a nucléotide sequence that encodes the antibody, or the antigen-binding fragment, of any one of claims l -32.534. A poiynucleotide encodîng a light chain variable région (VL) and, optionaliy, a light chain constant domain (CL) of the antibody, or the antigen-binding fragment, of any one of claims 1-32.35 . The poiynucleotide of claim 33 or 34, wherein the nucléotide sequence that encodes the antibody or the antigen-binding fragment is codon optimized for expression in a host10 cell.36 The poiynucleotide of claim 35, comprising a nucléotide sequence having at least 50% identity to the nucléotide sequence according to any one of SEQ ID Nos.:89, 85-88, and 9099.37 The poiynucleotide of any one of claims 33-36, comprising (i) the poiynucleotide15 sequence set forth in SEQ ID NO.:81 or SEQ ID NO.:82, and (ii) the poiynucleotide sequence set forth in any one or more of SEQ ID NOs.:89, 85-88, and 90-99.38 The poiynucleotide of any one of claims 33-36, comprising (i) the poiynucleotide sequence set forth in SEQ ID NO.:83, and (ii) the poiynucleotide sequence set forth in any one or more of SEQ ID NOs.:89, 85-88, and 90-99.20 39 The poiynucleotide of any one of claims 33-36, comprising (i) the poiynucleotide sequence set forth in SEQ ID NO.:84, and (ii) the poiynucleotide sequence set forth in any one or more of SEQ ID NOs.:89, 85-88, and 90-99.25240. A vector comprising the polynucleotide of any one of claims 33-39.41 The vector of claim 40, wherein the vector comprises a lentiviral vector or a retroviral vector.42. A host cell comprising the polynucleotide of any one of claims 33-39 and/or the5 vector of claim 40 or 41.43. A pharmaceutical composition comprising:(i) the antibody or antigen binding fragment of any one of claims 1-32;(ii) the polynucleotide according to any one of claims 33-39;(iii) the vector according to claim 40 or 41 ;10 (iv) the host cell of claim 42; or (v) any combination of (i)-(iv), and a pharmaceuticaliy acceptable excipient, diluent or carrier.44. A kit comprising:(a) a component selected from:(i) the antibody or antigen-binding fragment of any one of claims 1-32;(ii) the polynucleotide according to any one of claims 33-39;(iii) the vector according to claim 40 or 41;(iv) the host cell of claim 42;(v) the pharmaceutical composition of claim 43; or (vi) any combination of (i)-(vi); and (b) (1) instructions for using the component to prevent, treat, atténuaie, and/or diagnose a hepatitis B virus infection and/or a hepatitis D virus infection and/or (2) a means for administering the component to the subject.45.The composition of claim 43 or the kit of daim 44, further comprising:253 (i) a polymerase inhibitor, wherein the polymerase inhibitor optionally comprises Lamivudine, Adefovir, Entecavir, Telbivudine, Tenofovir, or any combination thereof;<ü) an interferon, wherein the interferon optionally comprises IFNbeta and/or IFNalpha;(iii) a checkpoint inhibitor, wherein the checkpoint inhibitor optionally comprises an anti-PD-l antibody or antigen binding fragment thereof, an anti-PD-Ll antibody or antigen binding fragment thereof, and/or an antî-CTLA4 antibody or antigen binding fragment thereof;(iv) an agonist of a stimulatory immune checkpoint molécule; or (v) any combination of (i)-(îv).50. A method of producing the antibody or antigen binding fragment of any one of claims l-32, comprising culturing the host cell of claim 42 under conditions and for a time sufficient to produce the antibody or antigen-binding fragment.51. Use of: (i) the antibody or antigen-binding fragment of any one of claims I -32; (ii) the polynucleotide of any one of claims 33-39; (iii) the vector of claim 40 or 41 ; (iv) the host cell of claim 42; and/or (v) the pharmaceutical composition of claim 43 or 45, in the manufacture of a médicament to prevent, treat, attenuate, and/or diagnose a hepatitis B virus infection and/or a hepatitis D virus infection in a subject.52. An antibody or antigen-binding fragment of any one of claims l-32, the polynucleotide of any one of claims 33-39, the vector of claim 40 or 41, the host cell of claim 42, and/or the pharmaceutical composition of any one of claims 43 or 44 for use in a method of treating, preventing, and/or attenuating a hepatitis B virus and/or hepatitis D virus infection in a subject.53. The antibody or antigen-binding fragment for use, the polynucleotide for use, the vector for use, the host cell for use, and/or the pharmaceutical composition for use of claim 52, wherein the method comprises administering to the subject a single dose of a pharmaceutical composition comprising the antibody or antigen-binding fragment; and/or254 the single dose of the pharmaceutical composition comprises the antibody in a range from 2 to 18 mg/kg (subject body weight); and/or the single dose of the pharmaceutical composition comprises up to 6 mg, up to I0 mg, up to 15 mg, up to 18 mg. up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up to 50 mg, up to 55 mg, up to 60 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of the antibody, or wherein the single dose of the pharinaceutical composition comprises the antibody in an amount that is in a range from t mg to 3000 mg, or in a range from 5 mg to 3000 mg, or in a range from 10 mg to 3000 mg, or in a range from 25 mg to 3000 mg, or in a range from 30 mg to 3000 mg, or in a range from 50 mg to 3000 mg, or in a range from 60 mg to 3000 mg, or in a range from 75 mg to 3000 mg, or in a range from 90 mg to 3000 mg, or in a range from 100 mg to 3000 mg, or in a range from 150 mg to 3000 mg, or in a range from 200 mg to 3000 mg, or in a range from 300 mg to 3000 mg, or in a range from 500 mg to 3000 mg, or in a range from 750 mg to 3000 mg, or in a range from 900 mg to 3000 mg, or in a range from 1500 mg to 3000 mg, or in a range from 2000 mg to 3000 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 900 mg, or in a range from 5 mg to 900 mg, or in a range from 10 mg to 900 mg, or in a range from 25 mg to 900 mg, or in a range from 30 mg to 900 mg, or în a range from 50 mg to 900 mg, or in a range from 60 mg to 900 mg, or in a range from 75 mg to 900 mg, or in a range from 90 mg to 900 mg, or in a range from 100 mg to 900 mg, or in a range from 150 mg to 900 mg, or in a range from 200 mg to 900 mg, or in a range from 300 mg to 900 mg, or in a range from 500 mg to 900 mg, or in a range from 750 mg to 900 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from I mg to 500 mg, or in a range from 5 mg to 500 mg, or in a range from 10 mg to 500 mg, or in a range from 25 mg to 500 mg, or in a range from 30 mg to 500 mg, or in a range from 50 mg to 500 mg, or in a range from 60 mg to 500 mg, or in a range from 75 mg to 500 mg, or in a range from 90 mg to 500 mg, or in a range from 100 mg255 to 500 mg. or in a range from 150 mg to 500 mg, or in a range from 200 mg to 500 mg, or in a range from 300 mg to 500 mg. or in a range from 400 mg to 500 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from l mg to 300 mg, or in a range from 5 mg to 300 mg, or in a5 range from 10 mg to 300 mg, or in a range from 25 mg to 300 mg, or in a range from 30 mg to 300 mg, or in a range from 50 mg to 300 mg, or in a range from 60 mg to 300 mg, or in a range from 75 mg to 300 mg, or in a range from 90 mg to 300 mg, or in a range from 100 mg to 300 mg, or in a range from 150 mg to 300 mg, or in a range from 200 mg to 300 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in 10 an amount that is in a range from l mg to 200 mg, or in a range from 5 mg to 200 mg, or in a range from 10 mg to 200 mg, or in a range from 25 mg to 200 mg, or in a range from 30 mg to 200 mg, or in a range from 50 mg to 200 mg, or in a range from 60 mg to 200 mg, or in a range from 75 mg to 200 mg, or in a range from 90 mg to 200 mg, or in a range from 100 mg to 200 mg, or in a range from 150 mg to 200 mg,15 or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from l mg to 100 mg, or in a range from 5 mg to 100 mg, or in a range from 10 mg to 100 mg, or in a range from 25 mg to 100 mg, or in a range from 30 mg to 100 mg, or in a range from 50 mg to 100 mg, or in a range from 60 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 75 mg to 100 mg, or in a range from 90 mg to 100 mg, 20 or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 25 mg, or in a range from 5 mg to 25 mg, or in a range from 10 mg to 25 mg, or in a range from 15 mg to 25 mg, or in a range from 20 mg to 25 mg, or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is in a range from 1 mg to 50 mg, or in a range from 1 mg to 25 mg, or in a range 25 from 5 mg to 50 mg, or in a range from 5 mg to 25 mg, or in a range from 10 to 50 mg, or in a range from 10 to 25 mg, or in a range from 1 to 15 mg, or in a range from 5 mg to 1 5 mg, or in a range from 10 mg to 15 mg,or wherein the single dose of the pharmaceutical composition comprises 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20,25,30,35,40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 30 160, 165, 170, 175, 180, 185, 190, 195,200,205,210,215,220,225,230,235,240,245,250,256255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 3 ΙΟ, 315, 320, 325, 330, 335, 340,345,350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435,440,445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530,535,540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610,615, 620, 625,630,5 635, 640, 645, 650, 655,660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725,730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815,820,825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910,915,920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 mg, or more, of the antibody,10 or wherein the single dose of the pharmaceutical composition comprises the antibody in an amount that is less than 3000 mg, less than 2500 mg, less than 2000 mg, less than 1500 mg, less than 1000 mg, less than 900 mg, less than 500 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 90 mg, less than 75 mg, less than 50 mg, less than 25 mg, or less than 10 mg, but is more than 1 mg, more than 2 mg, more than 3 mg, more than 4 mg, or more than 515 mg; and/or the single dose of the pharmaceutical composition comprises the antibody at a concentration in a range from 100 mg/mL to 200 mg/mL.54. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-32 at a concentration ranging from 100 mg/mL to 200 mg/mL, 20 and a pharmaceuticaliy acceptable carrier, excipient, or diluent.55. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition comprises water, optionally USP water; and/or the pharmaceutical composition comprises histidine, optionally at a concentration from 10 mM to 40 mM in the pharmaceutical composition; and/or257 the pharmaceutical composition comprises a disaccharide, optionally at 5%, 6%, 7%, 8%, or 9%; and/or the pharmaceutical composition comprises a surfactant, optionally a polysorbate, wherein, optionally, the polysorbate is présent in a range from 0.0!% to û.05% (w/v); and/or the pharmaceutical composition has a pH ranging from 5.8 to 6.2, ranging from 5.9 to6.1, or of 5.8, of 5.9, of 6.0, of 6.1, or of6.2.56. The pharmaceutical composition of any one of claims 54 or 55, wherein the pharmaceutical composition comprises:(i) the antibody at 150 mg/mL;(ii) U SP w ater;(iii) 20 mM histidine;(iv) 7% sucrose; and (v) 0.02% PS80, wherein the pharmaceutical composition comprises a pH of 6.57. A method for in vitro diagnosis of a hepatîtis B virus infection, the method comprising:(i) contacting a sample from a subject with an antibody or antigen-binding fragment of any one of claitns 1-32; and (ii) detecting a complex comprising an antigen and the antibody, or comprising an antigen and the antigen-binding fragment.58. A method for detecting the presence or absence of an epitope in a correct conformation in an anti-hepatîtis-B and/or an anti-hepatitis-D vaccine, the method comprising:(i) contacting the vaccine with an antibody or antigen-binding fragment of any one of claims 1-32; and258 (ii) determining whether a complex comprising an antigen and the antibody, or comprising an antigen and the antigen binding fragment, has been formed.59. An anti-HBV antibody from any one ofclaims l-32 for use in a method of5 treatîng chronic HBV infection in a subject in need thereof, wherein the anti-HBV antibody is for administration to the subject with an agent that reduces HBV antigenic load and/or an inhibitor of HBV gene expression; wherein the agent that reduces HBV antigenic load and/or the inhibitor of HBV gene expression is an RNAi agent that inhibits expression of an HBV transcript.60. The anti-HBV antibody for use according to claim 59, wherein the RNAi agent comprises a sense strand and an antisense strand forming a double-stranded région, wherein the sense strand comprises at least 15 contîguous nucléotides differing by no more than 3 nucléotides from nucléotides 1579-1597 of SEQ ID NO;l 16.15 61. The anti-HBV antibody for use according to any one ofclaims 59 or 60, wherein the double-stranded région of the RNAi agent is 15-30 nucléotide pairs in length, or wherein each strand of the RNAi agent has 15-30 nucléotides.62. The anti-HBV antibody for use according to any one of the claims 59-61, wherein 20 the RNAi agent is an siRNA.63. The anti-HBV antibody for use according to claim 62, wherein (a) the siRNA inhibits expression of an HBV transcript that encodes an HBsAg protein, an HBcAg protein, and HBx protein, or an HBV DNA polymerase protein; (b) the siRNA bînds to at least 15 contîguous nucléotides of a target encoded by: P gene, nucléotides 2309-3182 and 1-1625 of NC_003977.2;259S gene (encoding L, M, and S proteins), nucléotides 2850-3182 and 1-837 of NC_003977.2; HBx, nucléotides 1376-1840 of NC_003977.2; orC gene, nucléotides 1816-2454 ol NC_003977.2; (c) siRNA that comprises an antisense strand, wherein the antisense strand ofthe siRNA comprises at least 15 contiguous nucléotides of the nucléotide sequence of 5'5 UGUGAAGCGAAGUGCACACUU -3' (SEQ IDNO:119); and/or (d) siRNA that comprises an antisense strand, wherein the antisense strand of the siRNA comprises at least 15 contiguous nucléotides of the nucléotide sequence of 5'- UAAAAUUGAGAGAAGUCCACCAC -3' (SEQ IDNO:121).64. The anti-HBV antibody for use according to any one of claims 62-63, wherein10 substantially ail of the nucléotides of said sense strand and substantially ail of the nucléotides of said antisense strand are modified nucléotides, and wherein said sense strand is conjugated to a ligand attached at the 3'-terminus.65. The anti-HBV antibody for use according to claim 64, wherein (a) the ligand is one or more GalNAc dérivatives attached through a monovalent linker, bivalent branched linker, 15 or trivalent branched linker; or (b)the ligand isthe siRNA is conjugated to the ligand as shown in the following structure:260wherein X is O or S.66. The anti-HBV antibody for use according to any one of claims 62-65, wherein at least one nucléotide of the siRNA is a modified nucléotide comprising a deoxy-nucleotide, a 3’-5 terminal deoxy-thymîne (dT) nucléotide, a 2'-O-methyl modified nucléotide, a 2'-fluoro modified nucléotide, a 2'-deoxy-modifîed nucléotide, a locked nucléotide, an uniocked nucléotide, a conformationally restricted nucléotide, a constrained ethyl nucléotide, an abasic nucléotide, a 2'amino-modified nucléotide, a2’-O-allyl-modified nucléotide, 2'-C-alkyl-modified nucléotide, 2hydroxyl-modifîed nucléotide, a 2'-methoxyethyl modified nucléotide, a 2'-O-alkyl-modified10 nucléotide, a morpholino nucléotide, a phosphoramidate, a non-natural base comprising nucléotide, a tetrahydropyran modified nucléotide, a l,5-anhydrohexitol modified nucléotide, a cyclohexenyl modified nucléotide, a nucléotide comprising a phosphorothioate group, a nucléotide comprising a methylphosphonate group, a nucléotide comprising a 5’-phosphate, an adenosine-giycol nucleic acid, or a nucléotide comprising a 5'-phosphate mimic; and/or15 the siRNA comprises a phosphate backbone modification, a 2' ribose modification, 5' triphosphate modification, or a GalNAc conjugation modification; and/or the phosphate backbone modification comprises a phosphorothioate bond; and/or261 the 2' ribose modification comprises a fiuoro or -O-methyl substitution.67. The anti-HBV antibody for use according to any one of claims 62-67, wherein (a) the siRNA has a sense strand comprising 5'- gsusguGfcAfCfUfucgcuucacaL96 -3' (SEQ ID NO:l22) and an antisense strand comprising 5'- usGfsugaAfgCfGfaaguGfcAfcacsusu 3'(SEQ ID NO:l23), (b) the siRNA has a sense strand comprising 5'- gsusguGfcAfCfUfucgcuucacaL96 -3' (SEQ ID NO:l24) and an antisense strand comprising 5’usGfsuga(Agn)gCIGfaaguGfcAfcacsusu -3' (SEQ ID NO: 125), and/or (c) the siRNA has a sense strand comprising 5'- gsgsuggaCfu(JfCfUfcucaAfUfuuuaL96 -3' (SEQ IDNO:l26) and an antisense strand comprising 5'- usAfsaaaUfuGfAfgagaAfgLJfccaccsasc -3' (SEQ ID NO: 127); and wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'phosphate, 2'-O-methylguanosine-3'-phosphate, and l'-O-methyluridine-S'-phosphate, respectively;Af. Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2,-fiuoroguanosine-3’-phosphate, and 2'-fluorouridine-3'-phosphate, respectively;s is a phosphorothioate linkage; andL96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinoL68. An antibody or antigen-binding fragment, comprising a heavy chain variable région (VH) and a light chain variable région (VL), wherein the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: 38 and 62, respectively;wherein the antibody or antigen-binding fragment thereof is capable of binding to the antigenic loop région of HBsAg and neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof and/or capable of neutralizing infection by a hepatitis D virus (HDV).26269. An antibody or antigen-binding fragment, comprising a heavy chain variable région (VH) and a light chain variable région (VL), wherein the VH and the VL comprise or consist of the amino acid sequences set forth in SEQ ID NOs.: 38 and 66, respectively;wherein the antibody or antigen-binding fragment thereof is capable of binding to the amigenic loop région of HBsAg and neutralizing infection by a hepatitis B virus (HBV) of génotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof and/or capable of neutralizing infection by a hepatitis D virus (HDV).70 The antibody of claim 68 or 69, wherein the antibody comprises;a heavy chain (HC) comprising or consisting ofthe amino acid sequence set forth in SEQ 1DNO.:76, and a light chain constant région (CL) that comprises or consists of an amino acid sequence set forth in SEQ ID NO.:79.71. The antibody of claim 68 or 69, wherein the antibody comprises:a heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ IDNO.:77, and a light chain constant région (CL) that comprises or consists of an amino acid sequence set forth in SEQ ID NO.:79.72. The antibody of claim 68 or 69, wherein the antibody comprises:a heavy chain (HC) comprising or consisting of the amino acid sequence set forth in SEQ IDNO.:78,and a light chain constant région (CL) that comprises or consists of an amino acid sequence set forth in SEQ ID NO.:79.73. An anti-HBV antibody from any one of claims 68-72 for use in a method of treating chronic HBV infection and/or HDV infection in a subject in need thereof.26374. An anti-HBV antibody from any one of claims 68-72 for use in a method of treating chronic HBV infection and/or HDV infection in a subject in need thereof, wherein hte method comprises administering to the subject an siRNA, wherein the siRNA has a sense strand comprising 5'- gsusguGfcAfCfUfucgcuucacaL96 -3' (SEQ ID NO: 124) and an antisense strand
- 5 comprising 5'- usGfsuga(Agn)gCfGfaaguGfcAfcacsusu -3' (SEQ ID NO: ] 25);wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2-0-methylcytidine-3 phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively;Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 10 2'-fluoroguanosine-3'-phosphate, and 2'-fluorourîdine-3'-phosphate, respectively;s is a phosphorothioate linkage; andL96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63/043,692 | 2020-06-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA21025A true OA21025A (en) | 2023-08-24 |
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