Patent Application
20230340081
The present invention relates to antibodies against the hepatitis B virus (HBV) and methods of using the same.
Chronic hepatitis B virus (HBV) infection is a major healthcare problem affecting more than 250 million people worldwide despite the availability of effective vaccines (WHO, 2017). The infection accounts for approximately 1 million deaths per year due to HBV-associated cirrhosis, liver failure, and hepatocellular carcinoma (WHO, 2017). HBV is a DNA virus belonging to the Hepadnaviridae family, which is produced as infectious virions or Dane particles, but also as non-infectious subviral particles (Seeger et al., 2013). Virions and subviral particles display at their surface three forms of HBV envelope glycoproteins or HBV surface antigens (HBsAg): the L-HBs (large), the M-HBs (middle) and the S-HBs(small). Defective particles outnumbering infectious HBV virions are therefore acting as immune decoys (Seeger et al., 2013). Current therapies for treating chronic HBV rarely achieve functional cure as defined by HBsAg loss and anti-HBs antibody seroconversion. Yet, HBV infection can be successfully controlled by natural immune responses in more than 90% of the patients infected as adults, and in the ~1% of chronically infected patients who spontaneously clear the infection called HBV seroconverters or natural controllers (Bauer et al., 2011; Chu and Liaw, 2016; McMahon, 2009; Rehermann and Nascimbeni, 2005). Robust and multispecific HBV-specific CD4+ and CD8+ T cell responses are key immune effectors in controlling the infection (Bauer et al., 2011). However, B cells and the antibodies are also instrumental for the long-term clearance and protection from viral rebound after functional cure (Bertoletti and Ferrari, 2016; Corti et al., 2018; Rehermann and Nascimbeni, 2005). For instance, despite that HBsAg-/anti-HBs+ antibody seroconversion in patients is correlated with an undetectable level of HBV DNA (McMahon, 2009), functionally cured individuals undergoing B cell depletion therapy to treat Non-Hodgkin Lymphoma have a higher risk of HBV reactivation, which can quickly lead to severe liver dysfunction (Kusumoto et al., 2019; Perrillo et al., 2015). Hence, key immune components to control and eventually eliminate HBV infection likely include a broad and robust antigen-specific T cell response, as well as the development of neutralizing anti-HBs antibodies mediating HBsAg clearance and lifelong protective immunity (Bertoletti and Ferrari, 2016; Corti et al., 2018).
Neutralizing antibodies produced in response to HBV infection target all 3 HBsAg forms. They recognize either the S-HBs antigenic loop and interfere with the pre-attachment to heparane sulfate proteoglycans (HS) on hepatocytes, or the preS1 domain of the L-HBs and block the binding to the host cellular receptor, the sodium taurocholate co-transporting polypeptide (NTCP) (Corti et al., 2018). IgG antibodies against the “a determinant” part of the S-HBs loop induced by HBV vaccination (based on recombinant S-HBs immunogens), or administrated to individuals at risk of exposure by polyclonal HBV immunoglobulin infusions confer protection against HBV infection (Samuel et al., 1993; West and Calandra, 1996). Several neutralizing anti-preS and anti-S-HBs antibodies have been isolated from immunized mice and from few human immune donors (Corti et al., 2018). S-HBs neutralizing antibodies may contribute to the viral clearance and long-term suppression in HBV seroconverters as they do for protecting vaccinees from infection. However, no studies have been performed on the memory B-cell response to HBV in functionally cured HBV-infected individuals by cloning and charactering human HBsAg-specific antibodies.
The invention provides anti-S-HBs antibodies and methods of using the same.
(A) Average S-HBsAg-reactivity of serum IgGs from HBV vaccinees (HBVv, n=6, bottom curve) and controllers (HBVc, n=8, top curve). Shaded regions indicate value ranges. Representative flow-cytometry plots showing S-HBs-binding IgG+ memory B cells in HBV vaccinees and controllers (Bv4 and Bc3 are shown). nS-HBsAg and rS-HBsAg are human-derived native and recombinant S-HBs antigens, respectively.
(B) S-HBsAg-ELISA reactivities of S-HBsAg-captured IgG+ memory B-cell antibodies (left) and percentage of S-HBs-specific monoclonal antibodies (% S-HBsAg+) isolated from HBVv and HBVc (right). % S-HBsAg+according to S-HBs antibody titers in HBVc (< 150 and > 900 IU/ml) are indicated. capt-rHBAgs, rHBAgs capture ELISA.
(C) Bubble plot showing the level of clonal expansions according to percentages of somatic mutations in the IgH and IgL chain variable domains of S-HBs-specific IgG antibodies. The size of the expansions for each donor is indicated in the bar graph below.
(D) Volcano plot analysis comparing the immunoglobulin (Ig) gene repertoire of S-HBsAg-specific B cells from HBV immune donors and IgG memory B cells from healthy individuals (Top). Dots above the dashed line indicate statistically significant differences between both Ig gene repertoires. Comparison of the VH(DH)JH rearrangement frequencies are shown (Bottom). pV, p-value; FC, fold changes.
(E) Distribution of conformational-dependent vs non-conformational antibodies among S-HBs memory IgGs. Total number of tested antibodies is indicated in the center of the pie chart. Infrared immunoblot shows anti-S-HBs IgG memory B-cell antibodies reactive against denatured S-HBs protein (top right). ELISA binding curves of peptide-reactive S-HBs antibodies are shown (bottom right, mean of quadruplicates ± SD).
(A) Neutralizing activity of S-HBs IgG antibodies against in vitro infection of HepaRG cells by genotype D HBV. Fifty percent inhibitory concentration (IC50) values for each antibody (n=72) (Top left), and distribution of neutralizing vs non-active antibodies (Bottom left) are shown. (B) Neutralization capacity according to bound S-HBs antigens (Top right) and percentage of somatic hypermutations (%SHM) (Bottom right) are indicated.
(C) In vitro neutralizing activity of selected S-HBs IgG antibodies against HDV using HDV RNA quantification in HepaRG cells by northern blotting. ge, genome equivalents.
(D) In vivo neutralization activity of human S-HBs antibodies in AAV-HBV-transduced mice. Circulating blood S-HBs levels were monitored in AAV-HBV-transduced mice treated once i.v. with 0.5 mg of anti-S-HBs antibodies PIBv4.104 (n=9), Bc1.187 (n=9), Bc1.263 (n=6), Bc4.204 (n=4) or mGO53 isotype control (n=5). Thick lines represent the average values.
(E) Log10 changes of S-HBs titers at nadir (day post-injection 2, dpi2) upon antibody administration of 0.25 mg (white) and 0.5 mg (black) per mouse are shown. Means are indicated with lines.
(F) Circulating blood S-HBs and HBV DNA levels were monitored in AAV-HBV-transduced mice (n=6) injected once i.v. with 1 mg of the anti-S-HBs antibody Bc1.187. Average log10 changes overtime of S-HBs and HBV DNA levels are shown on the right.
(A) Heat map comparing the ELISA reactivity of HBV neutralizing antibodies against Adw and Ayw genotype D S-HBs proteins (measured as AUC values from
(B) Heat map comparing the reactivity of HBV neutralizing antibodies against S-HBs antigens from genotypes depicted in the phylogenic tree (Top left), as % of bound S-HBs-expressing cells determined by flow cytometry. Data represent one of two independent experiments. HB1 and mGO53 are positive and negative controls, respectively. Cytograms in the bottom left shows a representative reactivity profile of HB 1 antibody. ELISA graphs on the right show the reactivity of selected antibodies against recombinant S-HBs antigens from all genotypes but G. Error bars indicate the SD of assay duplicates.
(C) Same as in (B) but for S-HBs mutant proteins depicted on the diagram in the top left.
(D) Graph comparing the neutralizing activity of Bc1.187 against infection of primary human hepatocytes by HBV viruses from A to D genotypes. Error bars indicate the SEM of assay triplicates.
(E) Graphs show neutralization curves of HBV viruses from genotype A, C and D by PIBv4.104 and Bc1.187 as determined by the HepaRG neutralization assay. Error bars indicate the SEM of assay triplicates.
(A) Heat map shows the ELISA binding of selected HBV neutralizing antibodies to recombinant HBsAg mutant proteins. Color value is proportional to the reactivity level. (B) Heatmap showing competition for S-HBs binding of the HBV neutralizing antibodies. Lighter colors indicate stronger inhibition; black indicates no competition.
(C) Graphs comparing binding to S-HBs of the selected HBV neutralizing antibodies and their germline counterparts as measured by flow cytometry (top) and ELISA (bottom).
(D) Neutralizing activity of the germline versions of Bc1.187, Bc4.204 and PIBv4.104 against genotype D HBV viruses as determined by the HepaRG neutralization assay. Error bars indicate the SD of assay triplicates.
(E) Reactivity profile of selected S-HBs human antibodies on human protein microarrays. Each spot correspond to the z-scores given on a single protein by the reference (Ref: mGO53, y axis) and test antibody (x axis). Labels indicate immunoreactive proteins (z > 5).
(A) Evolution of HBV infection overtime in AAV-HBV-transduced mice (n=7 per group) treated every 2 days for 16 days with 0.5 mg i.v. of anti-S-HBs Bc1.187 or isotype control mGO53 chimeric antibody. Circulating blood HBsAg, HBeAg and HBV DNA levels are shown. Thick lines indicate the averages.
(B) Evolution of HBV infection overtime in HUHEP mice receiving weekly i.p. injections of human anti-S-HBs antibody Bc1.187 (20 mg/kg ~ 0.4 mg, n=7, straight lines; 50 mg/kg ~ 1 mg, n=5, dotted lines) for 3 weeks. Circulating blood HBsAg, HBeAg and HBV DNA levels (left) and Δlog10 values compared to baseline (right) are shown. Thick lines indicate the averages.
(A) Representative ELISA graphs showing the reactivity of purified serum IgG antibodies from HBV vaccinees (HBVv) and controllers (HBVc) against recombinant (rS-HBs) and human-derived native (nS-HBs) S-HBs particules. Error bars indicate the SEM of duplicate values. (B) Flow cytometric cytograms showing the gating strategy used to single-cell sort IgG+ memory B cells binding to fluorescently labeled rS-HBs and nS-HBs proteins used as baits. The S-HBs-reactive IgG+ memory B-cell population is shown for all donors.
(A) Heatmap showing the ELISA reactivity against nS-HBs and rS-HBs (immobilized and captured) of S-HBs-binding memory antibodies cloned from HBV vaccinees and seroconverters. Mean of triplicate optical density values are shown. (B) Violin plots showing the cumulative ELISA optical density (COD) values for the bindings shown in (A). The proportion of S-HBs-specific antibodies cloned from S-HBs-captured IgG+ memory B cells is shown per donor (Right).
(A) Pie charts comparing the distribution of VH / JH gene usage of blood S-HBs-specific IgG+ memory B cells and IgG+ memory B cells from healthy individuals (IgG.mB) (Prigent et al., 2016). The number of antibody sequences analyzed is indicated in the center of each pie chart. (B) Bar graph comparing the distribution of single immunoglobulin VH genes expressed by S-HBs-specific and control IgG+ memory B cells. (C) Amino acid alignment of the CDRH2 region (defined by Kabat) of VH1-69-expressing S-HBs antibodies. Residues in grey indicate substitutions compared to the germline VH gene (on the top). (D) Same as (B) but for IgG subtypes (Left) and κ- vs λ-Ig chain usage (Right). (E) Same as (B) but for the CDRH3 lengths and positive charge numbers. The average of CDRH3 lengths is indicated below each histogram. (F) Same as (A) but for Vκ / Jκ and Vλ / Jλ gene usages. (G) Violin plots comparing the number of mutations in VH, Vκ and Vλ genes in S-HBs-specific and control IgG+ memory B cells. The average number of mutations (mut.) is indicated below each dot plot. Numbers of mutations were compared across groups of antibodies using unpaired student t-test with Welch’s correction. (H) Graphs showing the Bayesian estimation of antigen-driven selection based on anti-S-HBs IgH and IgL sequences. Groups were compared (in A, B, D, E, and F) using 2 × 2 and 2 × 5 Fisher’s Exact tests.
(A) ELISA reactivity of anti-S-HBs antibodies against transmembrane domains-deleted S-HBsAg protein (ΔTM-rS-HBsAg). HB1 and mGO53 are positive and negative control, respectively. The dotted line indicates the cut-off OD405nm for positive reactivity. (B) Same as (A) but for cyclic peptides corresponding to putative S-HBsAg loops 122-137 and 139-148. (C) Heat mapped reactivity of anti-S-HBs antibodies against S-HBsAg overlapping linear peptides. Amino acid sequences (Right) and values of grand average of hydropathy (GRAVY) (Below) of S-HBsAg peptides are indicated.
Graphs show neutralization curves of genotype D HBV viruses by selected human S-HBs antibodies as measured in the in vitro HepaRG assay. The dotted horizontal line indicated 50% neutralization, from which the IC50 value can be derived from the antibody concentration on the x-axis.
(A) In vivo neutralization activity of human S-HBs antibodies in AAV-HBV-transduced mice. Circulating blood HBsAg levels were monitored in AAV-HBV-transduced mice treated once i.v. with 0.25 mg of anti-S-HBs antibodies Bv4.104 (n=6), Bc1.187 (n=6), Bc1.263 (n=6), Bc4.204 (n=6) or mGO53 isotype control (n=5). Thick lines indicate means. Log10 changes overtime of HBsAg titers (Δlog10 S-HBs) upon antibody administration of 0.25 mg i.v. per mouse are shown. (B) Graphs showing the evolution of human IgG titers overtime in mice treated once with 0.25 mg (left) and 0.5 mg (right) of S-HBs antibodies. Thick lines indicate means.
Representative ELISA graph showing the binding of selected HBV neutralizing antibodies to purified recombinant Adw (Straight lines) and Ayw (Dotted lines) S-HBs proteins. HB1 and mGO53 are positive and negative control, respectively. Mean values ± SEM of assay duplicates from one of two independent experiments are shown.
(A) Amino acid alignment of the consensus S-HBs protein sequences from different HBV genotypes used in (B). Residue variations are highlighted in grey. (B) Cytograms comparing the reactivity profiles of selected HBV neutralizing antibodies against genotype-specific S-HBs antigens. Data represent one of two independent experiments. HB1 and mGO53 are positive and negative control, respectively. Ctr, non-transfected cell control (first from the bottom); FI, fluorescence intensity.
Cytograms comparing the reactivity of selected HBV neutralizing antibodies against genotype D S-HBs mutant proteins displaying naturally occurring escape mutations (T126A, M133T, Y134V or G145R), or a mutation in the S-HBs N-glycosylation site (N126S). Data represent one of two independent experiments. HB1 and mGO53 are positive and negative control, respectively. Ctr, non-transfected cell control (first from the bottom); FI, fluorescence intensity.
profiles of selected human S-HBs antibodies (n=8) human protein microarrays. For each protein spot, the mean fluorescence intensity (MFI) given by the reference (Ref: mGO53) and test are depicted on the y and x axis, respectively. Each dot represents the average of duplicate array proteins. The diagonal lines indicate equal binding for reference and test antibodies. Labelled dots indicate immunoreactive proteins with a z-score > 5. (B) Frequency histograms showing the log10 protein displacement (σ) of the MFI signals for S-HBs antibodies compared to non-reactive antibody mGO53. The polyreactivity index (PI) corresponds to the Gaussian mean of all array protein displacements. (C) Binding of selected S-HBs antibodies to HEp2-expressing self antigens was assayed by IFA and ELISA. Ctr+, positive control of the kit. mGO53 and ED38 are negative and positive control antibodies, respectively. The scale bars represent 40 µM. Bar graph in the bottom right shows the HEp-2 reactivity as measured by ELISA. Means ± SD of values from two independent experiments performed in duplicate are shown.
(A) Circulating blood HBsAg levels overtime in AAV-HBV-transduced mice (n=7) treated every 3-4 days for 17 days with 0.5 mg i.p. of human anti-S-HBs antibody Bc1.187 or mGO53 isotype control. Averaged Δlog10 HBsAg values are shown (Right). Shaded area indicates the antibody therapy period. (B) Murine anti-human IgG antibody levels in the treated mice shown in (A) as measured by ELISA. (C) IgG concentrations of passively- administrated chimeric Bc1.187 antibody (0.5 mg i.v.) in B6 mice (n=4). Half-life in days (t½) of muBc1.187 antibody is indicated in the upper-right corner. (D) Δlog10 S-HBs levels overtime in C57BL/6J mice receiving weekly i.v. injections (0.5 mg) of chimeric anti-S-HBs antibody Bc1.187 and mGO53 isotype control. Thick lines indicate the averages. (E) Δlog10 HBsAg and HBV DNA levels overtime in AAV-HBV-transduced mice (n=7) treated every 2 days for 16 days with 0.5 mg i.v. of chimeric anti-S-HBs antibody Bc1.187 or mGO53 isotype control. Thick lines indicate the averages. Shaded area indicates the antibody therapy period.
(A) Blood HBsAg, HBeAg and HBV DNA levels overtime for each individual HUHEP mouse infected with genotype D HBV, and treated with anti-HBs Bc1.187 for 3 weeks with either 20 mg/kg or 50 mg/kg human antibody i.p. (B) Evolution of HBV infection overtime in HUHEP mice receiving a treatment with the non-HBV isotype control mGO53 (20 mg/kg i.p.) or Entecavir (ETV) every 3-4 days for 3 weeks. Blood levels of HBsAg, HBeAg and HBV DNA are shown. (C) Graphs showing the levels of human serum albumin overtime in infected HUHEP mice and treated i.p. with 20 mg/kg (straight lines) and 50 mg/kg (dotted lines) of Bc1.187 for 17 days (shaded area).
An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes are 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. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described in the following.
An “affinity matured” antibody refers to an antibody with one or more alterations in one or more complementary determining regions (CDRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
The terms “anti- S-HBs-antibody” and “an antibody that binds to S-HBs” refer to an antibody that is capable of binding S-HBs with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting S-HBs. In one aspect, the extent of binding of an anti- S-HBs antibody to an unrelated, non- S-HBs protein is less than about 10% of the binding of the antibody to S-HBs as measured, e.g., by surface plasmon resonance (SPR) or using ELISA or flow cytometry as disclosed herein. In certain aspects, an antibody that binds to S-HBs has a dissociation constant (KD) of ≤ 1 µM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10-8 M or less, e.g., from 10-8 M to 10-13 M, e.g., from 10-9 M to 10-13 M). An antibody is said to “specifically bind” to S-HBs when the antibody has a KD of 1 µM or less, and/or or if the extent of binding of an anti- S-HBs antibody to an unrelated, non- S-HBs protein is less than about 10% of the binding of the antibody to S-HBs as above, e.g., as measured by surface plasmon resonance (SPR) or by a flow cytometry or ELISA assay as described herein. In certain aspects, an anti- S-HBs antibody binds to an epitope of S-HBs that is conserved among S-HBs from different HBV genotypes.
The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), other antibody formats (e.g., comprising a VH domain, a VL domain and optionally an Fc domain, in a format different to a regular IgG) and antibody fragments so long as they exhibit the desired antigen-binding activity.
An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′ -SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).
The term “epitope” denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which an anti- S-HBs antibody binds. Epitopes can be formed both from contiguous amino acid stretches (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g., coming in spatial proximity due to the folding of the antigen, i.e. by the tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by an anti S-HBs antibody after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation.
Screening for antibodies binding to a particular epitope (i.e., those binding to the same epitope) can be done using methods routine in the art such as, e.g., without limitation, alanine scanning, peptide blots (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).
Antigen Structure-based Antibody Profiling (ASAP), also known as Modification-Assisted Profiling (MAP), allows to bin a multitude of monoclonal antibodies specifically binding to S-HBs based on the binding profile of each of the antibodies from the multitude to chemically or enzymatically modified antigen surfaces (see, e.g., US 2004/0101920). The antibodies in each bin bind to the same epitope which may be a unique epitope either distinctly different from or partially overlapping with epitope represented by another bin.
Also competitive binding can be used to easily determine whether an antibody binds to the same epitope of S-HBs as, or competes for binding with, a reference anti- S-HBs antibody. For example, an “antibody that binds to the same epitope” as a reference anti- S-HBs antibody refers to an antibody that blocks binding of the reference anti- S-HBs antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. Also for example, to determine if an antibody binds to the same epitope as a reference anti-S-HBs antibody, the reference antibody is allowed to bind to S-HBs under saturating conditions. After removal of the excess of the reference anti-S-HBs antibody, the ability of an anti-S-HBs antibody in question to bind to S-HBs is assessed. If the anti-S-HBs antibody is able to bind to S-HBs after saturation binding of the reference anti-S-HBs antibody, it can be concluded that the anti-S-HBs antibody in question binds to a different epitope than the reference anti-S-HBs antibody. But, if the anti-S-HBs antibody in question is not able to bind to S-HBs after saturation binding of the reference anti-S-HBs antibody, then the anti-S-HBs antibody in question may bind to the same epitope as the epitope bound by the reference anti-S-HBs antibody. To confirm whether the antibody in question binds to the same epitope or is just hampered from binding by steric reasons routine experimentation can be used (e.g., peptide mutation and binding analyses using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art). This assay should be carried out in two set-ups, i.e. with both of the antibodies being the saturating antibody. If, in both set-ups, only the first (saturating) antibody is capable of binding to S-HBs, then it can be concluded that the anti-S-HBs antibody in question and the reference anti-S-HBs antibody compete for binding to S-HBs.
In some aspects, two antibodies are deemed to bind to the same or an overlapping epitope if a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90% or even 99% or more as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).
In some aspects, two antibodies are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies are deemed to have “overlapping epitopes” if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain aspects, the antibody is of the IgG1 isotype. In certain aspects, the antibody is of the IgG1 isotype with the P329G, L234A and L235A mutation to reduce Fc-region effector function. In other aspects, the antibody is of the IgG2 isotype. In certain aspects, the antibody is of the IgG4 isotype with the S228P mutation in the hinge region to improve stability of IgG4 antibody. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and µ, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.
The terms “constant region derived from human origin” or “human constant region” as used in the current application denotes a constant heavy chain region of a human antibody of the subclass IgG1, IgG2, IgG3, or IgG4 and/or a constant light chain kappa or lambda region. Such constant regions are well known in the state of the art and e.g. described by Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also e.g. Johnson, G., and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, numbering of amino acid residues in the constant region is according to the EU numbering system, also called the EU index of Kabat, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.
“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.
An “effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including an Fc region are denoted herein with the C-terminal glycine-lysine dipeptide if not indicated otherwise. In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention, lacks the C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one aspect, a heavy chain including an Fc region as specified herein, comprised in an antibody according to the invention, lacks the C-terminal lysine residue (K447, numbering according to EU index). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
“Framework” or “FR” refers to variable domain residues other than complementary determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2- CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.
The terms “full length antibody”, “intact antibody”, and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
The terms “host cell”, “host cell line”, and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one aspect, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one aspect, for the VH, the subgroup is subgroup III as in Kabat et al., supra.
A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.
The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”).
Generally, antibodies comprise six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3), and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:
Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.
An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain aspects, the individual or subject is a human.
An “isolated” antibody is one which has been separated from a component of its natural environment. In some aspects, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). Any of the antibodies described herein may be isolated antibodies.
The term “nucleic acid molecule” or “polynucleotide” includes any compound and/or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5′ to 3′. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and/or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038/nm.4356 or EP 2 101 823 B1).
An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
“Isolated nucleic acid encoding an anti-S-HBs antibody” refers to one or more nucleic acid molecules encoding anti-S-HBs antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical composition.
“Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain.
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001/007611.
Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227-258; and Pearson et. al. (1997) Genomics 46:24-36 and is publicly available from www.fasta.bioch.virginia.edu/fasta www2/fasta down.shtml or www. ebi.ac.uk/Tools/sss/fasta. Alternatively, a public server accessible at fasta.bioch.virginia.edu/fasta_www2/index.cgi can be used to compare the sequences, using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.
The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
The terms “S-HBs” or “S-HBsAg” as used interchangeably herein (referring to small hepatitis B surface antigen) encompass “full-length”, unprocessed S-HBs as well as any form of S-HBs that results from processing in the cell. The term also encompasses naturally occurring variants of S-HBs, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary S-HBs is shown in SEQ ID NO: 253. Antibodies of the present invention may bind at least to S-HBs of SEQ ID NO: 253 e.g., with affinity or binding activity as discussed herein. Optionally, antibodies of the present invention may additionally or alternatively bind to one or more proteins comprising or consisting of consensus sequences selected from SEQ ID NO: 254-262, e.g., to a protein of SEQ ID NO: 257.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
Hepatitis B may be either chronic or acute, and the antibodies of the present invention may be used to treat either condition. Acute hepatitis generally refers to an infection within the first six months after exposure to the virus. Chronic hepatitis generally refers to an infection which is ongoing after six months. In some embodiments, treatment of hepatitis may refer to a reduction of HBV virus, an elimination of HBV virus, the reduction of symptoms due to HBV infection, the prevention or reduction of the progression of hepatitis to liver disease such as cirrhosis, liver fibrosis, liver failure and/or liver cancer, a reduction in detectable HBV surface antigen (HBsAg) in serum, and/or HBV surface antigen (HBsAg) seroconversion, i.e., an absence of detectable HBsAg in serum. Detection of HBsAg may be by any of the screening assays well established in the art, such as Elecsys HBsAg II (Roche Diagnostics), Auszyme Monoclonal [overnight incubation] version B, IMx HBsAg (Abbott) or Monolisa S-HBsAg ULTRA (Bio-Rad, France) ELISA.
The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
In one aspect, the invention is based, in part, on the identification by the inventors of antibodies from individuals that can naturally clear chronic hepatitis B virus (HBV) infection and acquire protection from re-infection (“controllers”) and from individuals vaccinated against HBV (“vaccinees”). The inventors have determined that such antibodies may have advantageous properties, such as high affinity or binding activity to the S-HBs antigen, potent neutralizing activity and/or cross-genotypic reactivity. In certain aspects, antibodies that bind to S-HBs are provided. Antibodies of the invention are useful, e.g., for the diagnosis or treatment of hepatitis B.
In one aspect, the invention provides antibodies that bind to S-HBs. In one aspect, provided are isolated antibodies that bind to S-HBs. In one aspect, the invention provides antibodies that specifically bind to S-HBs. In certain aspects, an anti-S-HBs antibody has one or more of the following properties:
In other exemplary embodiments, an antibody may have the following properties:
Said exemplary antibodies may preferably also avoid significant cross-reactivity against self-antigens such as galectin-3/-8 and E3 ubiquitin-protein ligase UBR2.
The antibodies may bind to S-HBs and/or to one or more S-HBs proteins having SEQ ID NO: 254 to 262 with a KD as discussed herein, e.g., ≤ 1 µM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM. In some embodiments, the antibody may bind to S-HBs and/or one or more S-HBs proteins having SEQ ID NO: 254 to 262 with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than the KD of a reference antibody against the same antigen, or which is less than or equal to the KD of said reference antibody, wherein the reference antibody is selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105.
Alternatively, the antibodies may show binding activity against S-HBs and/or to one or more S-HBs proteins having SEQ ID NO: 254 to 262 which is at least 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody to the same antigen, wherein the reference antibody is selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105 (when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay). In some embodiments, it may be preferred that the activity is at least 50% of the activity of the reference antibody. Optionally the reference antibody may be Bc1.187, i.e., the antibody may show at least 25%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of Bc1.187 to the reference antigen, when assessed in the same assay.
In other aspects, the antibodies may bind to S-HBs /or to one or more S-HBs proteins having SEQ ID NO: 254 to 262 with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of a reference antibody, wherein the EC50 is measured by ELISA or by flow cytometry, and wherein the reference antibody is selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105.
“Avoids significant cross reactivity with” means that the antibody does not show significant binding to the reference protein, e.g., has a KD greater than 1 µM, or has no detectable binding e.g., in an assay described herein.
In some embodiments antibodies according to the invention which are neutralizing against a HBV genotype, e.g., genotype D, may for instance have an IC50 value for viral infectivity in vitro of ≤ 50 ng/ml, or ≤10 ng/ml. It may be preferred that the antibody has an IC50 of ≤1 ng/ml, ≤500pg/ml or in some embodiments ≤100pg/ml, ≤50pg/ml, or ≤10pg/ml. In some embodiments, a neutralizing antibody may have an IC50 value of ≤1pg/ml, optionally ≤ 0.1pg/ml. Optionally the IC50 value may be greater than 0.01 pg/ml or 0.005pg/ml. In another embodiment, an antibody which is neutralizing against a particular HBV genotype, e.g., genotype D, may have an IC50 value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of a reference antibody against the same genotype, wherein the reference antibody is selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105 (when assessed in the same assay).
In another embodiment, an antibody which is neutralizing against a particular HBV genotype may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity or viremia suppressing activity of a reference antibody to the same genotype, e.g., a reference antibody selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105 when assessed using the same assay (e.g., an assay as described herein).
In some embodiments, an antibody may be cross reactive with each of the proteins of SEQ ID NO: 254 to 262 (having binding activity to each e.g., as described above) and have an IC50 for in vitro neutralization of HBV-genotype D of ≤ 100pg/ml. Optionally, the IC50 value may be ≤ 50pg/ml or ≤ 10pg/ml. In some embodiments, the IC50 value may be ≤ 1pg/ml, optionally ≤ 0.1pg/ml.
Various embodiments of particular antibodies according to the present invention are set out in sections A-N below.
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 7 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:8, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:9, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO: 10 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 16. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 16. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO: 16, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO: 1, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:2, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 3.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO: 16. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO: 16 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 16. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO: 11 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO: 12 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO: 13 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO: 14 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:15. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO: 15, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:4, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:5, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:6.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO: 15. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO: 15 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO: 15. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:3; and a VL domain comprising CDR-L3 of SEQ ID NO:6. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 2.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:3; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:6, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO: 16 and a VL sequence of SEQ ID NO: 15.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:16 and SEQ ID NO: 15, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 17 and/or the full length heavy chain of SEQ ID NO: 18 or 263.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO: 16 and a VL sequence of SEQ ID NO: 15. In certain aspects, an antibody is provided that binds to one or more of the following residues of S-HBs of SEQ ID NO: 253: C137; and optionally C138, K141, G145, and/or C149. In some embodiments the antibody may further bind to one or more of I152, N146, C147, and/or T148; and optionally W156. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc1.187 to S-HBs (e.g., of SEQ ID NO: 253), when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay. Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc1.187 to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bc1.187 is an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO: 16 and a VL sequence of SEQ ID NO: 15. It has the full length heavy chain of SEQ ID NO: 18 or 263 and the full length light chain of SEQ ID NO: 17. SEQ ID NO: 263 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc1.187 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody as described in this section may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc1.187 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc1.187, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254 to 262 with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc1.187, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D with an IC50 value of ≤1pg/ml, optionally ≤ 0.1pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bc1.187 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bc1.187 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo, e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bc1.187 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:20; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:21. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:20; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:21; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 25 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:26, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:27, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:28 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:34. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:34. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:34. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:34, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 19, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:20, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 21.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:34. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:34 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:34. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:22; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:24. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:22; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:24; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:29 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:30 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:31 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:32 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:33. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:33. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:33. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:33, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:22, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:23, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:24.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:33. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:33 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:33. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:21; and a VL domain comprising CDR-L3 of SEQ ID NO:24. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 20.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:19; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:20; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:21; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:22; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:23; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:24, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:34, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:33. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:34. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:33. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:34 and a VL sequence of SEQ ID NO:33.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:34 and SEQ ID NO:33, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 35 and/or the full length heavy chain of SEQ ID NO: 36 or 264.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:34 and a VL sequence of SEQ ID NO:33. In certain aspects, an antibody is provided that binds one or more of to the following residues of S-HBs of SEQ ID NO: 253: C138, C139 and/or C149, and optionally R169. Optionally the antibody may further bind to one or more of L109, R122, C147, T148, I152, W156, F161, and/or W165. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc1.180 to S-HBs (e.g., of SEQ ID NO: 253), when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc1.180 to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally, each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bc1.180 has a VH sequence of SEQ ID NO:34 and a VL sequence of SEQ ID NO:33. It has a full length heavy chain of SEQ ID NO: 36 or 264 and a full length light chain of SEQ ID NO: 35. SEQ ID NO: 264 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc1.180 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody as described in this section may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc1.180 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc1.180, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254 to 262 with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc1.180, as measured by ELISA or by flow cytometry. In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 1pg/ml, optionally ≤ 0.1pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bc1.180 against the same genotype, when assessed using the same assay (e.g., an assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bc1.180 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bc1.180 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:37; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:38; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:39. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:37; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:38; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:39; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 43 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:44, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:45, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:46 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:52. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:52. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:52. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:52, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 37, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:38, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 39.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:52. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:52 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:52. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:40; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:41; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:42. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:40; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:41; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:42; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:47 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:48 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:49 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:50 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:51. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:51. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:51. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:51, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:40, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:41, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:42.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:51. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:51 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:51. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:39; and a VL domain comprising CDR-L3 of SEQ ID NO:42. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 38.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:37; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:38; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:39; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:40; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:41; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:42, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:52, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:51. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:52. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:51. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:52 and a VL sequence of SEQ ID NO:51.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:52 and SEQ ID NO:51, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 53 and/or the full length heavy chain of SEQ ID NO: 54 or 265.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:52 and a VL sequence of SEQ ID NO:51. In certain aspects, an antibody is provided that binds to one or more of the following residues of S-HBs of SEQ ID NO: 253: L109, C138, I152, W156, and/or R169. Optionally the antibody further binds to P111 and/or F161. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc3.106 to S-HBs (e.g., of SEQ ID NO:253), when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc3.106 to one or more of the proteins SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally, to each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bc3.106 has the VH sequence of SEQ ID NO:52 and a VL sequence of SEQ ID NO:51. It has the full length heavy chain of SEQ ID NO: 54 or 265 and the full length light chain of SEQ ID NO: 53. SEQ ID NO: 265 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc3.106 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a
KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc3.106 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc3.106, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254 to 262 with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc3.106, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 10pg/ml, optionally ≤ 1pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bc3.106 against the same genotype, when assessed using the same assay (e.g., an assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bc3.106 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bc3.106 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:55; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:56; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:57. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:55; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:56; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:57; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 61 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:62, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:63, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:64 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:70. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:70. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:70. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:70, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 55, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:56, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 57.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:70. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:70 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:70. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:58; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:59; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:60. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:58; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:59; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:60; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:65 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:66 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:67 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:68 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:69. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:69. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:69. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:69, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:58, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:59, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:60.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:69. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:69 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:69. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:57; and a VL domain comprising CDR-L3 of SEQ ID NO:60. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 56.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:55; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:56; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:57; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:58; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:59; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:60, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:70, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:69. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:70. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:69. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:70 and a VL sequence of SEQ ID NO:69.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:70 and SEQ ID NO:69, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 71 and/or the full length heavy chain of SEQ ID NO: 72 or 266.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:70 and a VL sequence of SEQ ID NO:69. In certain aspects, an antibody is provided that binds to one or more of the following residues of S-HBs of SEQ ID NO: 253: C121, R122, C124, C137, C139, K141, N146, C147 and/or C149. In some embodiments the antibody may also bind to one or more of I110, T118, P120, C138, P142, D144, T148 and/or I152. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bv4.104 to S-HBs (e.g., of SEQ ID NO:253), when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bv4.104 to one or more, or to each of the proteins of SEQ ID NO: 257 and 258, optionally at least to a protein of SEQ ID NO: 257, when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bv4.104 has a VH sequence of SEQ ID NO:70 and a VL sequence of SEQ ID NO:69. It has a full length heavy chain of SEQ ID NO: 72 or 266 and a full length light chain of SEQ D NO: 71. SEQ ID NO: 266 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bv4.104 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody may bind to one or more, or to each of the proteins of SEQ ID NO: 257 and 258, optionally at least to a protein of SEQ ID NO: 257, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bv4.104 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bv4.104, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more, or to each of the proteins of SEQ ID NO: 257 and 258, optionally at least to a protein of SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bv4.104, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 100pg/ml, optionally ≤ 10pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype D which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bv4.104 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bv4.104 against HBV genotype D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bv4.104 against HBV genotype D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:73; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:74; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:75. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:73; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:74; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:75; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 79 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:80, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:81, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:82 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:88. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:88. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:88. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:88, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 73, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:74, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 75.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:88. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:88 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:88. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:76; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:77; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:78. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:76; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:77; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:78; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:83 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:84 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:85 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:86 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:87. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:87. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:87. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:87, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:76, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:77, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:78.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:87. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:87 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:87. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:75; and a VL domain comprising CDR-L3 of SEQ ID NO:78. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 74.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:73; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:74; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:75; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:76; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:77; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:78, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:88, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:87. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:88. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:87. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:88 and a VL sequence of SEQ ID NO:87.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:88 and SEQ ID NO:87, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 89 and/or the full length heavy chain of SEQ ID NO: 90 or 267.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:88 and a VL sequence of SEQ ID NO:87. In certain aspects, an antibody is provided that binds to one or more of the following residues of S-HBs of SEQ ID NO: 253: C121, and/or one or more of I110, P120, C124, C137, C139, C147, T148 and/or C149. Optionally the antibody may further bind to one or more of C138, K141, P142, D144, G145, P150, 1152, and/or W156. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc8.111 to S-HBs (e.g., of SEQ ID NO:253), when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of reference antibody Bc8.111 to one or more of the proteins of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally to each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bc8.111 has a VH sequence of SEQ ID NO:88 and a VL sequence of SEQ ID NO:87. It has a full length heavy chain of SEQ ID NO: 90 or 267 and a full length light chain of SEQ ID NO: 89. SEQ ID NO: 267 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.In some embodiments, the antibody described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc8.111 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc8.111 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc8.111, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more, or to each of the proteins of SEQ ID NO: 254-262, optionally at least to a protein of SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc8.111, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 100pg/ml, optionally ≤ 10pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bc8.111 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bc8.111 against HBV genotype A, B, C and/or D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bc8.111 against HBV genotype A, B, C and/or D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:91; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:92; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:93. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:91; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:92; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:93; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 97 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:98, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:99, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO: 100 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 106. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 106. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 106. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO: 106, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 91, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:92, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 93.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO: 106. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:106 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO: 106. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:94; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:95; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:96. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:94; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:95; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:96; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:101 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:102 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:103 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:104 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:105. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:105. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:105. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:105, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:94, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:95, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:96.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:105. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:105 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:105. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:93; and a VL domain comprising CDR-L3 of SEQ ID NO:96. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 92.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:91; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:92; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:93; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:94; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:95; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:96, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:106, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:105. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 106. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO 105. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:106 and a VL sequence of SEQ ID NO:105.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:106 and SEQ ID NO:105, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 107 and/or the full length heavy chain of SEQ ID NO: 108 or 268.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:106 and a VL sequence of SEQ ID NO:105. In certain aspects, an antibody is provided that binds to one or more of the following residues of S-HBs of SEQ ID NO: 253:W156 and/or R169. Optionally the antibody may additional bind to one or more of I152 and/or C138. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc8.104 to S-HBs (e.g., of SEQ ID NO:253), when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of reference antibody Bc8.104 to one or more of the proteins of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bc8.104 has a VH sequence of SEQ ID NO:106 and a VL sequence of SEQ ID NO:105. It has a full length heavy chain of SEQ ID NO: 108 or 268 and a full length light chain of SEQ ID NO: 107. SEQ ID NO: 268 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc8.104 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody described in this section may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc8.104 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc8.104, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more, or to each of the proteins of SEQ ID NO: 254-262, optionally at least to a protein of SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc8.104, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 100pg/ml, optionally ≤ 10pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bc8.104 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the neutralizing activity of reference antibody Bc8.104 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bc8.104 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:109; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:110; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:111. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:109; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:110; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:111; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 115 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:116, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:117, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:118 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:124. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:124. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:124. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:124, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 109, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:110, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 111.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:124. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:124 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:124. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:112; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 113; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:114. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:112; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:113; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:114; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:119 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:120 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:121 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:122 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:123. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:123. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:123. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:123, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:112, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:113, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:114.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:123. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:123 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:123. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:111; and a VL domain comprising CDR-L3 of SEQ ID NO:114. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 110.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:109; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:110; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:111; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:112; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:113; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:114, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:124, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:123. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:124. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO 123. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:124 and a VL sequence of SEQ ID NO:123.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:124 and SEQ ID NO:123, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 125 and/or the full length heavy chain of SEQ ID NO: 126 or 269.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:124 and a VL sequence of SEQ ID NO:123. In certain aspects, an antibody is provided that binds to one or more of the following residues of S-HBs of SEQ ID NO: 253: C137, C138 and /or D144. Optionally, the antibody may further bind to one or more of P142, N146, T148, C149, I152 and/or W156. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bv6.172 to S-HBs (e.g., of SEQ ID NO:253), when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of reference antibody Bv6.172 to one or more of the proteins of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bv6.172 has a VH sequence of SEQ ID NO:124 and a VL sequence of SEQ ID NO:123. It has a full length heavy chain of SEQ ID NO: 126 or 269 and a full length light chain of SEQ ID NO: 125. SEQ ID NO: 269 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bv6.172 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bv6.172 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bv6.172, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more, or to each of the proteins of SEQ ID NO: 254-262, optionally at least to a protein of SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bv6.172, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 100pg/ml, optionally ≤ 10pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bv6.172 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bv6.172 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bv6.172 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:127; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:128; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:129. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:127; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:128; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:129; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 133 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:134, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:135, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:136 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:142. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:142. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:142. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:142, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 127, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:128, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 129.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:142. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:142 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:142. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:130; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 131; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:132. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:130; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:131; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:132; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:137 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:138 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:139 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:140 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 141. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 141. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:141. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:141, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:130, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:131, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:132.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO: 141. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:141 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:141. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:129; and a VL domain comprising CDR-L3 of SEQ ID NO:132. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 128.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:127; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:128; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:129; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:130; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:131; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:132, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:142, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:141. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:142. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO 141. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO: 142 and a VL sequence of SEQ ID NO:141.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:142 and SEQ ID NO: 141, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 143 and/or the full length heavy chain of SEQ ID NO: 144 or 270.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:142 and a VL sequence of SEQ ID NO:141. In certain aspects, an antibody is provided that binds to one or more of the following residues of S-HBs of SEQ ID NO: 253: C137, C138 and/or C139. Optionally, the antibody also binds to C124, optionally further to W156, and optionally to one or more of N146, T148 and/or I152. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bv4.115 to S-HBs (e.g., of SEQ ID NO:253), when assessed in the same assay, e.g., an ELISA or flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of reference antibody Bv4.115 to one or more of the proteins of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay, e.g., an ELISA or flow cytometry assay.
Reference antibody Bv4.115 has a VH sequence of SEQ ID NO:142 and a VL sequence of SEQ ID NO:141. It has a full length heavy chain of SEQ ID NO: 144 or 270 and a full length light chain of SEQ ID NO: 143. SEQ ID NO: 270 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bv4.115 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bv4.115 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bv4.115, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more, or to each of the proteins of SEQ ID NO: 254-262, optionally at least to a protein of SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bv4.115, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 100pg/ml, optionally ≤ 10pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bv4.115 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bv4.115 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bv4.115 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:145; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:146; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:147. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:145; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:146; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:147; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 151 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO: 152, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:153, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:154 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:160. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:160. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:160. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:160, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 145, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:146, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 147.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:160. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:160 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:160. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:148; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 149; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:150. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:148; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:149; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:150; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:155 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO: 156 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:157 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:158 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:159. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:159. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:159. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:159, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:148, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:149, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:150.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:159. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:159 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:159. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:147; and a VL domain comprising CDR-L3 of SEQ ID NO:150. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 146.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:145; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:146; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:147; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:148; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:149; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:150, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:160, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:159. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:160. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO 159. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:160 and a VL sequence of SEQ ID NO:159.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:160 and SEQ ID NO: 159, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 161 and/or the full length heavy chain of SEQ ID NO: 162 or 271.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:160 and a VL sequence of SEQ ID NO:159. In certain aspects, an antibody is provided that binds to one or more the following residues of S-HBs of SEQ ID NO: 253: C121, K141, D144, and/or G145. Optionally the antibody may further bind one or more of C139, C147, C149, I152 and/or W156. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc1.229 to S-HBs (e.g., of SEQ ID NO:253), when assessed in the same assay, e.g., an ELISA or flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of reference antibody Bc1.229 to one or more of the proteins of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay, e.g., an ELISA or flow cytometry assay.
Reference antibody Bc1.229 has a VH sequence of SEQ ID NO:160 and a VL sequence of SEQ ID NO:159. It has a full length heavy chain of SEQ ID NO 162 or 271 and a full length light chain of SEQ ID NO: 161. SEQ ID NO: 271 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc1.229 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc1.229 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc1.229, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more, or to each of the proteins of SEQ ID NO: 254-262, optionally at least to a protein of SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc1.229, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 1 ng/ml, optionally ≤ 100pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bc1.229 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bc1.229 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bc1.229 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:163; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:164; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:165. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:163; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:164; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:165; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 169 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:170, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:171, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:172 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:178. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:178. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:178. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:178, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 163, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:164, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 165.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:178. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:178 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:178. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:166; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 167; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:168. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:166; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:167; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:168; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:173 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:174 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:175 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:176 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:177. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:177. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:177. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:177, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:166, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:167, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:168.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:177. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:177 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:177. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:165; and a VL domain comprising CDR-L3 of SEQ ID NO:168. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 164.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:163; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:164; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:165; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:166; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:167; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:168, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:178, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:177. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:178. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO 177. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:178 and a VL sequence of SEQ ID NO:177.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:178 and SEQ ID NO:177, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 179 and/or the full length heavy chain of SEQ ID NO: 180 or 272.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:178 and a VL sequence of SEQ ID NO:177. In certain aspects, an antibody is provided that binds to the following residues of S-HBs of SEQ ID NO: 253: C121, C138, C139, C147 and/or C149. Optionally the antibody may additionally bind to one or more of L109, R122, T123, C124, M133, Y134, S136, K141 and/or I152. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc8.159 to S-HBs (e.g., of SEQ ID NO:253), when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of reference antibody Bc8.159 to one or more of the proteins of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bc8.159 has a VH sequence of SEQ ID NO:178 and a VL sequence of SEQ ID NO:177. It has a full length heavy chain of SEQ ID NO: 180 or 272 and a full length light chain of SEQ ID NO: 179. SEQ ID NO: 272 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc8.159 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc8.159 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc8.159, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more, or to each of the proteins of SEQ ID NO: 254-262, optionally at least to a protein of SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc8.159, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 1 ng/ml, optionally ≤ 100pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bc8.159 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bc8.159 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bc8.159 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:181; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:182; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:183. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:181; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:182; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:183; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 187 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:188, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:189, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:190 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:196. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO:196. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:196. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:196, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 181, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:182, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 183.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:196. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:196 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:196. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:184; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 185; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:186. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:184; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:185; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:186; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:191 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:192 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:193 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:194 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:195. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:195. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:195. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:195, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:184, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:185, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:186.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:195. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:195 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:195. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:183; and a VL domain comprising CDR-L3 of SEQ ID NO:186. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 182.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:181; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:182; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:183; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:184; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:185; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:186, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:196, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:195. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:196. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO 195. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:196 and a VL sequence of SEQ ID NO:195.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:196 and SEQ ID NO: 195, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 197 and/or the full length heavy chain of SEQ ID NO: 198 or 273.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:196 and a VL sequence of SEQ ID NO:195. In certain aspects, an antibody is provided that binds to the following residues of S-HBs of SEQ ID NO: 253: C121, C124, C137, C138, C139, C147 and/or C149, optionally also to I152. Optionally the antibody further binds to one or more of V106, P108, W156 and/or F161. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc1.128 to S-HBs (e.g., of SEQ ID NO:253), when assessed in the same assay e.g., an ELISA assay or a flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of reference antibody Bc1.128 to one or more of the proteins of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bc1.128 has a VH sequence of SEQ ID NO:196 and a VL sequence of SEQ ID NO:195. It has a full length heavy chain of SEQ ID NO: 198 or 273 and a full length light chain of SEQ ID NO: 197. SEQ ID NO: 273 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc1.128 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc1.128 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc1.128, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more, or to each of the proteins of SEQ ID NO: 254-262, optionally at least to a protein of SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc1.128, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 10 ng/ml, optionally ≤ 1 ng/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bc1.128 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bc1.128 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bc1.128 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:199; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:200; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:201. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:199; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:200; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:201; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 205 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:206, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:207, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:208 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:214. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 214. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:214. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:214, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 199, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:200, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 201.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:214. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:214 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:214. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:202; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 203; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:204. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:202; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:203; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:204; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:209 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:210 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:211 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:212 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:213. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:213. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:213. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:213, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:202, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:203, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:204.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:213. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:213 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:213. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:201; and a VL domain comprising CDR-L3 of SEQ ID NO:204. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 200.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:199; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:200; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:201; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:202; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:203; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:204, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:214, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:213. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:214. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 213. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:214 and a VL sequence of SEQ ID NO:213.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:214 and SEQ ID NO:213, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 215 and/or the full length heavy chain of SEQ ID NO: 216 or 274.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:214 and a VL sequence of SEQ ID NO:213. In certain aspects, an antibody is provided that binds to residue F179 of S-HBs of SEQ ID NO:253. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc4.204 to S-HBs (e.g., of SEQ ID NO: 253), when assessed in the same assay e.g., an ELISA assay or a flow cytometry assay. Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of reference antibody Bc4.204 to proteins having the sequence of SEQ ID NO: 254, 255, 256, 257, 258, 260 and/or 262, when assessed in the same assay, optionally at least to SEQ ID NO: 257 e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bc4.204 has a VH sequence of SEQ ID NO:214 and a VL sequence of SEQ ID NO:213. It has a full length heavy chain of SEQ ID NO: 216 or 274 and a full length light chain of SEQ ID NO: 215. SEQ ID NO: 274 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc4.204 to the same antigen, when assessed in the same assay. Additionally or alternatively, the antibody may bind to one or more of SEQ ID NO: 254, 255, 256, 257, 258, 260 and/or 262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc4.204 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc4.204, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more, or to each of, the proteins having the sequence of SEQ ID NO: 254, 255, 256, 257, 258, 260 and/or 262, optionally at least to a protein of SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc4.204, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 10 ng/ml, optionally ≤ 1 ng/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bc4.204 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of reference antibody Bc4.204 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bc4.204 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:217; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:218; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:219. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:217; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:218; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:219; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 223 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:224, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:225, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:226 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:232. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 232. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:232. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:232, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 217, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:218, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 219.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:232. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:232 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:232. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:220; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 221; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:222. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:220; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:221; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:222; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:227 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:228 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:229 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:230 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:231. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:231. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:231. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:231, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:220, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:221, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:222.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:231. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:231 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:231. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:219; and a VL domain comprising CDR-L3 of SEQ ID NO:222. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 218.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:217; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:218; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:219; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:220; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:221; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:222, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:232, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:231. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:232. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 231. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:232 and a VL sequence of SEQ ID NO:231.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:232 and SEQ ID NO:231, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 233 and/or the full length heavy chain of SEQ ID NO: 234 or 275.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:232 and a VL sequence of SEQ ID NO:231. In certain aspects, an antibody is provided that binds to the following residues of S-HBs of SEQ ID NO: 253: W165 and/or R169, and optionally M103. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bc1.263 to S-HBs (e.g., of SEQ ID NO: 253), when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of reference antibody Bc1.263 to one or more of the proteins of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally to each of the proteins of SEQ ID NO: 254 to 262, when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bc1.263 has a VH sequence of SEQ ID NO:232 and a VL sequence of SEQ ID NO:231. It has a full length heavy chain of SEQ ID NO: 234 or 275 and a full length light chain of SEQ ID NO: 233. SEQ ID NO: 275 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc1.263 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody may bind to one or more of SEQ ID NO: 254-262 (e.g., to at least SEQ ID NO: 257), optionally each of the proteins of SEQ ID NO: 254-262, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bc1.263 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc1.263, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind one or more, or to each of the proteins of SEQ ID NO: 254-262, optionally at least to a protein of SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bc1.263, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype A, B, C and/or D, optionally all of A, B, C and D. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 100pg/ml, optionally ≤ 10pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bc1.263 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the neutralizing activity of reference antibody Bc1.263 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype A, B, C or D, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bc1.263 against HBV genotype A, B, C and/or D, optionally at least D, optionally all of A, B, C and D, when assessed using the same assay (e.g., an assay as described herein).
In one aspect, the invention provides an antibody comprising a VH domain comprising at least one, at least two, or all three VH CDR sequences selected from (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:235; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:236; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:237. In another aspect, the antibody comprises a VH domain comprising the following CDRs (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:235; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:236; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:237; or a variant thereof in which one, two or three amino acids in one or more of CDR-H1, CDR-H2 or CDR-H3 are substituted with another amino acid.
In one aspect, the VH domain may further comprise one or more heavy chain framework sequences selected from (a) a heavy chain frame work region 1 (HC-FR1) of SEQ ID NO: 241 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a heavy chain frame work region 2 (HC-FR2) of SEQ ID NO:242, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto (c) a heavy chain frame work region 3 (HC-FR3) SEQ ID NO:243, or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (d) a heavy chain frame work region 4 (HC-FR4) of SEQ ID NO:244 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VH domain comprises each of the heavy chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:250. In one aspect, an anti-S-HBs antibody comprises a heavy chain variable domain (VH) sequence having at least 95%, sequence identity to the amino acid sequence of SEQ ID NO: 250. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:250. In certain aspects, substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VH sequence in SEQ ID NO:250, including post-translational modifications of that sequence. In a particular aspect, the VH comprises one, two or three CDRs selected from: (a) CDR-H1, comprising the amino acid sequence of SEQ ID NO 235, (b) CDR-H2, comprising the amino acid sequence of SEQ ID NO:236, and (c) CDR-H3, comprising the amino acid sequence of SEQ ID NO: 237.
In another aspect, an anti-S-HBs antibody comprises one or more of the heavy chain CDR sequences of the VH of SEQ ID NO:250. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the heavy chain CDR amino acid sequences of the VH domain of SEQ ID NO:250 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VH domain of SEQ ID NO:250. Optionally the sequence identity is 95% or 98%.
In another aspect, the invention provides an antibody comprising a VL domain comprising at least one, at least two, or all three VL CDR sequences selected from (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:238; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 239; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:240. In another aspect, the antibody comprises a VL domain comprising the following CDRs (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:238; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:239; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:240; or a variant thereof in which one, two or three amino acids in one or more of CDR-L1, CDR-L2 or CDR-L3 are substituted with another amino acid.
In one aspect, the anti-S-HBs antibody VL domain may further comprise one or more light chain framework sequences selected from (a) a light chain frame work region 1 (LC-FR1) of SEQ ID NO:245 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (b) a light chain frame work region 2 (LC-FR2) of SEQ ID NO:246 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, (c) a light chain frame work region 3 (LC-FR3) of SEQ ID NO:247 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (d) a light chain frame work region 4 (LC-FR4) of SEQ ID NO:248 or a variant having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Optionally, the VL domain comprises each of the light chain framework sequences as defined in (a) to (d). Optionally the percentage sequence identity is 95%. Optionally the percentage sequence identity is 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:249. In one aspect, an anti-S-HBs antibody comprises a light chain variable domain (VL) sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:249. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-S-HBs antibody comprising that sequence retains the ability to bind to S-HBs. In certain aspects, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:249. In certain aspects, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). Optionally, the anti-S-HBs antibody comprises the VL sequence in SEQ ID NO:249, including post-translational modifications of that sequence. In a particular aspect, the VL comprises one, two or three CDRs selected from: (a) CDR-L1, comprising the amino acid sequence of SEQ ID NO:238, (b) CDR-L2, comprising the amino acid sequence of SEQ ID NO:239, and (c) CDR-L3, comprising the amino acid sequence of SEQ ID NO:240.
In another embodiment, an anti-S-HBs antibody comprises one or more of the CDR sequences of the VL of SEQ ID NO:249. In one aspect, an anti-S-HBs antibody comprises one or more (preferably all three) of the light chain CDR amino acid sequences of the VL domain of SEQ ID NO:249 and a framework of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the framework amino acid sequence of the VL domain of SEQ ID NO:249. Optionally the sequence identity is 95% or 98%.
In another aspect, an anti-S-HBs antibody is provided, wherein the antibody comprises a VH sequence as in any of the aspects provided above, and a VL sequence as in any of the aspects provided above.
For instance, in one exemplary embodiment, the antibody comprises a VH domain comprising CDR-H3 of SEQ ID NO:237; and a VL domain comprising CDR-L3 of SEQ ID NO:240. Optionally the VH domain further comprises CDR-H2 of SEQ ID NO: 236.
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the antibody comprises:
In another exemplary embodiment, the anti-S-HBs antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:235; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:236; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:237; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:238; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:239; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:240, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:250, and a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:249. In one aspect, the VH domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:250. In one aspect, the VL domain has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 249. In one embodiment, the antibody specifically binds to S-HBs. In another embodiment, the antibody binds to S-HBs having a dissociation constant (KD) that is up to 10 fold reduced or up to 10 fold increased when compared to the dissociation constant (KD) of an antibody comprising a VH sequence of SEQ ID NO:250 and a VL sequence of SEQ ID NO:249.
In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO:250 and SEQ ID NO:249, respectively, including post-translational modifications of those sequences.
In a further aspect, the antibody may comprise the full length light chain of SEQ ID NO: 251 and/or the full length heavy chain of SEQ ID NO: 252 or 276.
In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-S-HBs antibody provided herein. For example, in certain aspects, an antibody is provided that binds to the same epitope as an anti-S-HBs antibody comprising a VH sequence of SEQ ID NO:250 and a VL sequence of SEQ ID NO:249. In certain aspects, an antibody is provided that binds to the following residues of S-HBs of SEQ ID NO: 253:C124, C137, C138 and/or C139; optionally R122, C149, and/or I152; and optionally W156. The antibody may comprise any of the sequences as defined above.
In a further aspect, the invention provides an antibody that competes for binding to S-HBs with an anti-S-HBs antibody provided herein.
Antibodies as described in this section may, in some embodiments, have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of a reference antibody Bv4.105 to S-HBs (e.g., of SEQ ID NO:253), when assessed in the same assay, e.g., an ELISA assay or a flow cytometry assay.
Additionally or alternatively, antibodies as described in this section may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the binding activity of reference antibody Bv4.105 to one or more, optionally each of the proteins of SEQ ID NO: 257, 258, 259, 260, and/or 261, optionally at least to SEQ ID NO: 257, when assessed in the same assay e.g., an ELISA assay or a flow cytometry assay.
Reference antibody Bv4.105 has a VH sequence of SEQ ID NO:250 and a VL sequence of SEQ ID NO:249. It has a full length heavy chain of SEQ ID NO: 252 or 276 and a full length light chain of SEQ ID NO: 251. SEQ ID NO: 276 comprises the IgG1 constant region expressed by vector LT615368.1, as used in the examples.
In some embodiments, the antibody described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bv4.105 to the same antigen, when assessed in the same assay.
Additionally or alternatively, the antibody may bind to one or more, optionally each of the proteins of SEQ ID NO: 257, 258, 259, 260, and/or 261, optionally at least to SEQ ID NO: 257, with a KD value which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of reference antibody Bv4.105 to the same antigen, when assessed in the same assay.
In some embodiments, the antibody as described in this section may bind to S-HBs (e.g., of SEQ ID NO: 253) with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bv4.105, as measured by ELISA or by flow cytometry. Alternatively or additionally, the antibody may bind to one or more, optionally each, of the proteins of SEQ ID NO: 257, 258, 259, 260, and/or 261, optionally at least to SEQ ID NO: 257, with an EC50 that is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the EC50 of reference antibody Bv4.105, as measured by ELISA or by flow cytometry.
In some embodiments antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, E, F, and/or H, optionally all of genotypes D, E, F and H. For example, antibodies as described in this section may have or retain in vitro neutralizing activity against HBV genotype D, e.g., with an IC50 value of ≤ 100pg/ml, optionally ≤ 10pg/ml.
In another embodiment, the antibody may have an IC50 value for neutralization of HBV genotype D, E, F, G and/ or H, optionally at least D, optionally all of D, E, F, G and/ or H, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of reference antibody Bv4.105 against the same genotype, when assessed using the same assay (e.g., an in vitro neutralization assay as described herein).
In another embodiment, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the neutralizing activity of reference antibody Bv4.105 against HBV genotype D, E, F, G and/ or H, optionally at least D, optionally all of D, E, F, G and H, when assessed using the same assay (e.g., an assay as described herein).
Optionally the antibody can suppress virus viremia in vivo e.g., in an individual infected with HBV genotype D, E, F, G or H, optionally D. Optionally, the antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo viremia suppressing activity of reference antibody Bv4.105 against HBV genotype D, E, F, G and/ or H, optionally at least D, optionally all of D, E, F, G and H, when assessed using the same assay (e.g., an assay as described herein).
In general, an anti-S-HBs antibody according to any aspect of the present invention (including any of A-N above) may be a monoclonal antibody, including a chimeric, humanized or human antibody. In one aspect, an anti-S-HBs antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)2 fragment.
In another general aspect, the antibody may be a full-length antibody, e.g., an intact IgG1, IgG2, IgG3 antibody or other antibody class or isotype as defined herein.
The C-terminal lysine (Lys447) of the full-length antibody may be either present or absent. In another aspect, the C-terminal glycine (Gly446) and the C-terminal lysine (Lys447) may be either present or absent.
In a further aspect, an anti-S-HBs antibody according to any of the above aspects may incorporate any of the features, singly or in combination, as described in Sections 1-7 below:
In certain aspects, an antibody provided herein has a dissociation constant (KD) against the target protein of ≤ 1 µM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10-8 M or less, e.g., from 10-8 M to 10-13 M, e.g., from 10-9 M to 10-13 M).
In other aspects, an antibody provided herewith has or retains a KD which is which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the KD value of a reference antibody against the target protein when assessed in the same assay, wherein the reference antibody is selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105.
In one aspect, KD is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25° C. with immobilized antigen CM5 chips at ~10 response units (RU). In one aspect, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N′- (3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 µg/ml (~0.2 µM) before injection at a flow rate of 5 µl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately 25 µl/min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio koff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M-1 s-1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO ™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.
In another aspect, ELISA can be used to calculate KD values, e.g., as described in Friguet et al J Immunol. Methods (1985) 77 (2): 305-19.
In certain aspects, an antibody provided herein is an antibody fragment.
In one aspect, the antibody fragment is a Fab, Fab′, Fab′-SH, or F(ab′)2 fragment, in particular a Fab fragment. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called “Fab” fragments containing each the heavy- and light-chain variable domains (VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). The term “Fab fragment” thus refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain, and a heavy chain fragment comprising a VH domain and a CH1 domain. “Fab′ fragments” differ from Fab fragments by the addition of residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. Fab′ -SH are Fab′ fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-binding sites (two Fab fragments) and a part of the Fc region. For discussion of Fab and F(ab′)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.
In another aspect, the antibody fragment is a diabody, a triabody or a tetrabody. “Diabodies” are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
In a further aspect, the antibody fragment is a single chain Fab fragment. A “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CH1 domain. In addition, these single chain Fab fragments might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).
In another aspect, the antibody fragment is single-chain variable fragment (scFv). A “single-chain variable fragment” or “scFv” is a fusion protein of the variable domains of the heavy (VH) and light chains (VL) of an antibody, connected by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids and is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. For a review of scFv fragments, see, e.g., Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458.
In another aspect, the antibody fragment is a single-domain antibody. “Single-domain antibodies” are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain aspects, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B1 ).
Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as recombinant production by recombinant host cells (e.g., E. coli), as described herein.
In certain aspects, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
In certain aspects, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat′l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).
Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).
In certain aspects, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).
Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. Pat. Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.
Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).
Human antibodies may also be generated by isolating variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
In certain aspects, an antibody provided herein is derived from a library. Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. Methods for screening combinatorial libraries are reviewed, e.g., in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Frenzel et al. in mAbs 8:1177-1194 (2016); Bazan et al. in Human Vaccines and Immunotherapeutics 8:1817-1828 (2012) and Zhao et al. in Critical Reviews in Biotechnology 36:276-289 (2016) as well as in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O′Brien et al., ed., Human Press, Totowa, NJ, 2001) and in Marks and Bradbury in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).
In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. in Annual Review of Immunology 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al. in EMBO Journal 12: 725-734 (1993). Furthermore, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter in Journal of Molecular Biology 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490 as well as U.S. Pat. Publication Nos. 2005/0079574, 2007/0117126, 2007/0237764 and 2007/0292936.
Further examples of methods known in the art for screening combinatorial libraries for antibodies with a desired activity or activities include ribosome and mRNA display, as well as methods for antibody display and selection on bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are reviewed, e.g., in Scholler et al. in Methods in Molecular Biology 503:135-56 (2012) and in Cherf et al. in Methods in Molecular biology 1319:155-175 (2015) as well as in Zhao et al. in Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, e.g., in He et al. in Nucleic Acids Research 25:5132-5134 (1997) and in Hanes et al. in PNAS 94:4937-4942 (1997).
Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
In certain aspects, an antibody provided herein is a multispecific antibody, e.g., a bispecific antibody. “Multispecific antibodies” are monoclonal antibodies that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain aspects, the multispecific antibody has three or more binding specificities. In certain aspects, one of the binding specificities is for S-HBs and the other specificity is for any other antigen. In certain aspects, bispecific antibodies may bind to two (or more) different epitopes of S-HBs. Multispecific (e.g., bispecific) antibodies may also be used to localize cytotoxic agents or cells to cells which express S-HBs. Multispecific antibodies may be prepared as full length antibodies or antibody fragments.
Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011/034605); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98/50431); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147:60 (1991).
Engineered antibodies with three or more antigen binding sites, including for example, “Octopus antibodies”, or DVD-Ig are also included herein (see, e.g., WO 2001/77342 and WO 2008/024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792, and WO 2013/026831. The bispecific antibody or antigen binding fragment thereof also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to S-HBs as well as another different antigen, or two different epitopes of S-HBs (see, e.g., US 2008/0069820 and WO 2015/095539).
Multi-specific antibodies may also be provided in an asymmetric form with a domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging the VH/VL domains (see e.g., WO 2009/080252 and WO 2015/150447), the CH1/CL domains (see e.g., WO 2009/080253) or the complete Fab arms (see e.g., WO 2009/080251, WO 2016/016299, also see Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). In one aspect, the multispecific antibody comprises a cross-Fab fragment. The term “cross-Fab fragment” or “xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged. A cross-Fab fragment comprises a polypeptide chain composed of the light chain variable region (VL) and the heavy chain constant region 1 (CH1), and a polypeptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL). Asymmetrical Fab arms can also be engineered by introducing charged or non-charged amino acid mutations into domain interfaces to direct correct Fab pairing. See e.g., WO 2016/172485.
Various further molecular formats for multispecific antibodies are known in the art and are included herein (see e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).
A particular type of multispecific antibodies, also included herein, are bispecific antibodies designed to simultaneously bind to a surface antigen on a target cell, e.g., an infected cell and to an activating, invariant component of the T cell receptor (TCR) complex, such as CD3, for retargeting of T cells to kill target cells. Hence, in certain aspects, an antibody provided herein is a multispecific antibody, particularly a bispecific antibody, wherein one of the binding specificities is for S-HBs and the other is for CD3.
Examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, the so-called “BiTE” (bispecific T cell engager) molecules wherein two scFv molecules are fused by a flexible linker (see, e.g., WO 2004/106381, WO 2005/061547, WO 2007/042261, and WO 2008/119567, Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (dual affinity retargeting) molecules which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and so-called triomabs, which are whole hybrid mouse/rat IgG molecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)). Particular T cell bispecific antibody formats included herein are described in WO 2013/026833, WO 2013/026839, WO 2016/020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.
In certain aspects, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to alter the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
In certain aspects, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading of “preferred substitutions”. More substantial changes are provided in Table 1 under the heading of “exemplary substitutions”, and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
Amino acids may be grouped according to common side-chain properties:
Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.
One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more. CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).
Alterations (e.g., substitutions) may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR “hotspots”, i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O′Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some aspects of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
In certain aspects, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex may be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody directed enzyme prodrug therapy)) or a polypeptide which increases the serum half-life of the antibody.
In certain aspects, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
Where the antibody comprises an Fc region, the oligosaccharide attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some aspects, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
In one aspect, antibody variants are provided having a non-fucosylated oligosaccharide, i.e. an oligosaccharide structure that lacks fucose attached (directly or indirectly) to an Fc region. Such non-fucosylated oligosaccharide (also referred to as “afucosylated” oligosaccharide) particularly is an N-linked oligosaccharide which lacks a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one aspect, antibody variants are provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region as compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e. no fucosylated oligosaccharides are present). The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues, relative to the sum of all oligosaccharides attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2006/082515, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and/or improved effector function, in particular improved ADCC function. See, e.g., US 2003/0157108; US 2004/0093621.
Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003/085107), or cells with reduced or abolished activity of a GDP-fucose synthesis or transporter protein (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).
In a further aspect, antibody variants are provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function as described above. Examples of such antibody variants are described, e.g., in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO 2003/011878.
Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.
In certain aspects, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
In certain aspects, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat′l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’lAcad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. Proc. Nat′l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int′l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929 Al).
Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)
In certain aspects, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
In certain aspects, an antibody variant comprises an Fc region with one or more amino acid substitutions which diminish FcyR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In one aspect, the substitutions are L234A and L235A (LALA). In certain aspects, the antibody variant further comprises D265A and/or P329G in an Fc region derived from a human IgG1 Fc region. In one aspect, the substitutions are L234A, L235A and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012/130831). In another aspect, the substitutions are L234A, L235A and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region.
In some aspects, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (See, e.g., U.S. Pat. No. 7,371,826; Dall’Acqua, W.F., et al. J. Biol. Chem. 281 (2006) 23514-23524).
Fc region residues critical to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see e.g. Dall’Acqua, W.F., et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU numbering of residues) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, J.K., et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be critical for the interaction of human Fc with murine FcRn (Kim, J.K., et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are crucial for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, Y.A., et al. (J. Immunol. 182 (2009) 7667-7671) various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and examined.
In certain aspects, an antibody variant comprises an Fc region (in some embodiments an Fc region of IgG1) with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 253, and/or 310, and/or 435 of the Fc-region (EU numbering of residues). In certain aspects, the antibody variant comprises an Fc region with the amino acid substitutions at positions 253, 310 and 435. In one aspect, the substitutions are I253A, H310A and H435A in an Fc region derived from a human IgG1 Fc-region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.
In certain aspects, an antibody variant comprises an Fc region (in some embodiments an Fc region of IgG1) with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 310, and/or 433, and/or 436 of the Fc region (EU numbering of residues). In certain aspects, the antibody variant comprises an Fc region with the amino acid substitutions at positions 310, 433 and 436. In one aspect, the substitutions are H310A, H433A and Y436A in an Fc region derived from a human IgG1 Fc-region. (See, e.g., WO 2014/177460 Al).
In certain aspects, an antibody variant comprises an Fc region (in some embodiments an Fc region of IgG1) with one or more amino acid substitutions which increase FcRn binding, e.g., substitutions at positions 252, and/or 254, and/or 256 of the Fc region (EU numbering of residues). In certain aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 252, 254, and 256. In one aspect, the substitutions are M252Y, S254T and T256E in an Fc region derived from a human IgG1 Fc-region. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.
In certain further aspects, an antibody variant comprises an Fc region (in some embodiments an Fc region of IgG1) with one or more amino acid substitutions which increase FcRn binding, e.g., substitutions at positions 428 and/or 434 and/or 436 of the Fc region (EU numbering of residues). In certain aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 428, 434, and 436. In one aspect, the substitutions are M428L, N434A and Y436T in an Fc region derived from a human IgG1 Fc-region. In another aspect, the antibody variant comprises an Fc region with amino acid substitution at position 434, e.g., N434A.
In certain further aspect, an antibody variant comprises an Fc region (in some embodiments an Fc region of IgG1) with one or more amino acid substitutions which increase FcRn binding, e.g., a substitutions at position 307 and/or 434, e.g., T307H and/or N434H, e.g., T307H and N434H.
In certain specific embodiments the antibody variant comprises a heavy chain as set out herein, modified by the substitutions i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; or iv) T307H and N434H.
For instance, the antibody may comprise the light chain of SEQ ID NO: 17 and the heavy chain of SEQ ID NO: 18 or 263 modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:35 and a heavy chain of SEQ ID NO:36 or 264, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:53 and a heavy chain of SEQ ID NO: 54 or 265, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:71 and a heavy chain of SEQ ID NO: 72 or 266, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:89 and a heavy chain of SEQ ID NO: 90 or 267, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:107 and a heavy chain of SEQ ID NO: 108 or 268, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:125 and a heavy chain of SEQ ID NO: 126 or 269, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:143 and a heavy chain of SEQ ID NO: 144 or 270, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:161 and a heavy chain of SEQ ID NO: 162 or 271, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:179 and a heavy chain of SEQ ID NO: 180 or 272, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:197 and a heavy chain of SEQ ID NO: 198 or 273, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:215 and a heavy chain of SEQ ID NO: 216 or 274, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:233 and a heavy chain of SEQ ID NO: 234 or 275, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
In another specific embodiment, the antibody may comprise a light chain of SEQ ID NO:251 and a heavy chain of SEQ ID NO: 252 or 276, modified by substitutions selected from the group consisting of i) M252Y, S254T and T256E; ii) M428L, N434A and Y436T; iii) N434A; and iv) T307H and N434H.
The C-terminus of the heavy chain of the antibody as reported herein can be a complete C-terminus ending with the amino acid residues PGK. The C-terminus of the heavy chain can be a shortened C-terminus in which one or two of the C terminal amino acid residues have been removed. In one preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending PG. In one aspect of all aspects as reported herein, an antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein, comprises the C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid positions). In one aspect of all aspects as reported herein, an antibody comprising a heavy chain including a C-terminal CH3 domain, as specified herein, comprises a C-terminal glycine residue (G446, EU index numbering of amino acid positions).
In certain aspects, it may be desirable to create cysteine engineered antibodies, e.g., THIOMAB™ antibodies, in which one or more residues of an antibody are substituted with cysteine residues. In particular aspects, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO 2016040856.
In certain aspects, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
Antibodies may be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. For these methods one or more isolated nucleic acid(s) encoding an antibody are provided.
In case of a native antibody or native antibody fragment two nucleic acids are required, one for the light chain or a fragment thereof and one for the heavy chain or a fragment thereof. Such nucleic acid(s) encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chain(s) of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.
In case of a bispecific antibody with heterodimeric heavy chains four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising the first heteromonomeric Fc-region polypeptide, one for the second light chain, and one for the second heavy chain comprising the second heteromonomeric Fc-region polypeptide. The four nucleic acids can be comprised in one or more nucleic acid molecules or expression vectors. Such nucleic acid(s) encode an amino acid sequence comprising the first VL and/or an amino acid sequence comprising the first VH including the first heteromonomeric Fc-region and/or an amino acid sequence comprising the second VL and/or an amino acid sequence comprising the second VH including the second heteromonomeric Fc-region of the antibody (e.g., the first and/or second light and/or the first and/or second heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors, normally these nucleic acids are located on two or three expression vectors, i.e. one vector can comprise more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMabs (see, e.g., Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). For example, one of the heteromonomeric heavy chain comprises the so-called “knob mutations” (T366W and optionally one of S354C or Y349C) and the other comprises the so-called “hole mutations” (T366S, L368A and Y407V and optionally Y349C or S354C) (see, e.g., Carter, P. et al., Immunotechnol. 2 (1996) 73) according to EU index numbering.
In one aspect, isolated nucleic acids encoding an antibody as used in the methods as reported herein are provided.
In one aspect, a method of making an anti-S-HBs antibody is provided, wherein the method comprises culturing a host cell comprising nucleic acid(s) encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
For recombinant production of an anti-S-HBs antibody, nucleic acids encoding the antibody, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) or produced by recombinant methods or obtained by chemical synthesis.
Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. 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., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized”, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.
Suitable host cells for the expression of (glycosylated) antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.
Plant cell cultures can also be utilized as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIESTM technology for producing antibodies in transgenic plants).
Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, e.g., in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.
In one aspect, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
Anti-S-HBs antibodies provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art.
In one aspect, an antibody of the invention is tested for its antigen binding activity, e.g., by known methods such as ELISA, flow cytometry, Western blot, etc.
S-HBs antigen for use in ELISA may be in the form of particles. Particles for use in an ELISA protocol may be provided by recombinant expression of antigen, e.g., S-HBs, in host cells and self-assembly of the antigen into particles. For instance, the antigen may be expressed in a yeast host cell such as Pichia pastoris or in a mammalian cell such as a Chinese Hamster Ovary (CHO) cell.
The ELISA assay may comprise the steps of i) coating an ELISA plate with S-HBs antigen ii) blocking the plate with BSA; iii) washing; iv) incubating with serial dilutions of IgG antibody; v) washing; vi) incubating with goat-anti-human IgG-HRP antibody; vii) developing the plate with HRP chromogenic substrate; and viii) measuring optical density at 405 nm (OD405nM).
The binding activity measured by ELISA may be quantified as the area under the curve (AUC) as determined from the OD405nM-concentration curve. Thus, the method may comprise ix) determining AUC from the OD405nM-concentration curve. These values may be compared to compare the activity of a test antibody to a reference antibody, as a percentage. Alternatively, an EC50 value can be determined from the ELISA assay, as the concentration at which the half maximal value of OD450nm is obtained.
A flow-cytometry assay may comprise the steps of i) expressing S-SBs in a human cell line; ii) fixing and permeabilizing the cells; iii) incubating the cells with IgG antibody; iv) washing; v) incubating the cells with AF647-conjugated goat anti-human IgG antibody; vi) washing and resuspending in PBS; and vii) determining % of bound S-HBs-expressing cells or the mean fluorescence intensity (MFI) by flow cytometry.
A serial dilution of antibody can be used to determine an EC50 in the flow cytometry assay. Alternatively, the values obtained for the % of bound S-HBs-expressing cells or the MFI may be compared to express the activity of a test antibody as a percentage of the activity a reference antibody, when the test antibody and the reference antibody are used at the same concentration, e.g., at 10ug/ml or alternatively at the EC50 of the reference antibody (as determined in the flow cytometry assay),
Where relative binding activity is assessed, the antibodies should be tested using the same method. They may also be assessed in the same format, e.g., both as IgGs. In such embodiments, the reference antibody will have the full length sequences for said reference antibody as provided
herein. The binding activity of a test antibody comprising a VH and VL domain as defined herein (e.g., comprising a VH and/or VL domain which is a variant of that of the reference antibody) may be assessed in an IgG format comprising the constant domain and hinge sequences of the reference antibody.
Exemplary protocols are set out below.
High-binding 96-well ELISA plates (Costar, Corning) are coated overnight with purified antigen (e.g., 125 ng/well in PBS). After washings (e.g., with 0.05% Tween 20-PBS (PBST)), plates are blocked 2 h with 2% BSA, 1 mM EDTA-PBST (Blocking solution), washed, and incubated with serially diluted antibody in PBS. After washings, plates are revealed by addition of goat HRP-conjugated anti-human IgG (e.g., 0.8 µg/ml final in blocking solution, Immunology Jackson ImmunoReseach) and HRP chromogenic substrate (e.g., ABTS solution, Euromedex). Optical density measurements are made at 405 nm (OD405 nm). Binding can be quantified as the area under the curve (AUC) from the OD405 nm-concentration curve, and the AUC of two antibodies can be compared to assess relative activity as a percentage. Alternatively, the EC50 of an antibody can be determined as the concentration at which the half maximal value of OD450 nm is obtained.
Experiments may be performed using HydroSpeed™ microplate washer and Sunrise™ microplate absorbance reader (Tecan Männedorf). A negative control antibody mGO53 and a suitable positive control such as HB1 (Kucinskaite-Kodze et al., 2016) may be included in each experiment.
In an exemplary protocol for measuring binding activity using flow cytometry, a human cell line (e.g., Freestyle™ 293-F) is transfected with S-HBs-encoding vectors (0.65 µg plasmid DNA per 106 cells), e.g., using the PEI-precipitation method as previously described (Lorin and Mouquet, 2015). Forty-eight hours post-transfection, transfected and non-transfected control cells are fixed and permeabilized e.g., using Cytofix/Cytoperm™ solution kit (BD Biosciences), and 0.5×106 cells are incubated with IgG antibodies for 45 min at 4° C. (e.g, in Perm/Wash™ solution; BD Biosciences). In one embodiment, the test and reference antibody are used at 10 ug/ml. In another embodiment, the test and reference antibody can be used at the EC50 of the reference antibody as determined in the flow cytometry assay. After washings, cells are incubated 20 min at 4° C. with AF647-conjugated goat anti-human IgG antibodies (1:1000 dilution; Thermo Fisher Scientific), washed and resuspended in PBS. The percentage of bound S-HBs-expressing cells and/or the mean fluorescence intensity (MFI) of the signals is assessed by flow cytometry, and the values obtained for two antibodies are compared to assess relative value as a percentage.. Data may be acquired using a CytoFLEX flow cytometer (Beckman Coulter), and analyzed using FlowJo software (v10.3; FlowJo LLC).
In an exemplary protocol for assessing EC50 using flow cytometry, a human cell line (e.g., Freestyle™ 293-F) is transfected with Ss-HBs-encoding vectors (0.65 µg plasmid DNA per 106 cells), e.g., using the PEI-precipitation method as previously described (Lorin and Mouquet, 2015). Forty-eight hours post-transfection, transfected and non-transfected control cells are fixed and permeabilized e.g., using Cytofix/Cytoperm™ solution kit (BD Biosciences), and 0.5×106 cells are incubated with serial dilutions of IgG antibodies in PBS for 45 min at 4° C. (e.g in Perm/Wash™ solution; BD Biosciences). After washings, cells are incubated 20 min at 4° C. with AF647-conjugated goat anti-human IgG antibodies (1:1000 dilution; Thermo Fisher Scientific), washed and resuspended in PBS. The percentage of bound S-HBs-expressing cells or the mean fluorescence intensity (MFI) of the signals is be assessed by flow cytometry, and the EC50 is determined as the concentration at which the half maximal percentage of bound cells or MFI is obtained.
In another aspect, competition assays may be used to identify an antibody that competes with a reference antibody selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104,Bc8.104, Bc4.204, Bc1.263 and Bv4.105 for binding to S-HBs. In certain aspects, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by a reference antibody selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols”, in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).
In an exemplary competition assay, immobilized S-HBs is incubated in a solution comprising a first labeled antibody that binds to S-HBs (e.g., a reference antibody selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to S-HBs. The second antibody may be present in a hybridoma supernatant. As a control, immobilized S-HBs is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to S-HBs, excess unbound antibody is removed, and the amount of label associated with immobilized S-HBs is measured. If the amount of label associated with immobilized S-HBs is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to S-HBs. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
A further exemplary competition assay is a competition ELISA. Purified antibodies (e.g., a reference antibody selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105) are biotinylated using the EZ-Link Sulfo-NHS-Biotin kit (Thermo Fisher Scientific). Antigen or antigen-particle (eg,. rS-HBs)-coated plates are blocked, washed, incubated for 2 h with the biotinylated antibody (at a concentration 0.33 nM) in 1:2 serially diluted solutions of antibody competitors in PBS (IgG concentration range from 0.83 to 106.7 nM), and revealed using HRP-conjugated streptavidin. Experiments are performed using HydroSpeed™ microplate washer and Sunrise™ microplate absorbance reader (Tecan Männedorf), with optical density measurements made at 405 nm (OD405 nm).
In one aspect, assays are provided for identifying anti-S-HBs antibodies thereof having biological activity. Biological activity may include, e.g., neutralizing activity in vitro or in vivo and/or the ability to reduce viremia in vivo. Antibodies having such biological activity in vivo and/or in vitro are also provided.
In vitro neutralizing activity may be inhibition of infection of primary human hepatocytes or human hepatocyte cell lines by HBV. This may be determined as a reduction in the supernatant S-HBs as compared to the absence of antibody. In vivo neutralizing activity may be determined as a reduction in circulating S-HBs animals infected with HBV, e.g., mice or humans.
Reduction of viral viremia can be determined as a reduction in HBV DNA in the serum of an HBV infected animal, e.g., mouse or human.
Antibodies may for instance have an IC50 value for in vitro inhibition of infectivity by HBV of a particular genotype e.g., genotype D, of ≤ 50 µg/ml, ≤ 10 µg/ml, ≤ 1 µg/ml, <500 ng/ml, <100 ng/ml, ≤ 50 ng/ml, <10 ng/ml, ≤1ng/ml, <500pg/ml, <100pg/ml, <50pg/ml, <10pg/ml or <1pg/ml. In some embodiments, preferred antibodies may have a IC50 of ≤ 50 ng/ml, or ≤ 10 ng/ml. It may be preferred that the antibody has an IC50 of ≤ 1 ng/ml, ≤ 500pg/ml or in some embodiments ≤ 100pg/ml, ≤ 50pg/ml, or ≤ 10pg/ml. In some embodiments, a neutralizing antibody may have an IC50 value of ≤ 1pg/ml, optionally ≤ 0.1pg/ml.
In another embodiment, an antibody may have an IC50 value against a particular genotype, e.g., genotype D, which is no more than 50, 10, 9, 8, 7, 6, 5, 4, 3, or 2 fold higher than, or is less than or equal to, the IC50 value of a reference antibody against the same genotype, wherein the reference antibody is selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105 (when assessed in the same in vitro assay).
In another embodiment, an antibody may have or retain at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the neutralizing activity of a reference antibody to a particular HBV genotype, when assessed using the same assay (e.g., an in vivo assay as described herein).
In some embodiments, an antibody of the invention has or retains at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the in vivo neutralizing activity of a reference antibody against an HBV genotype A, B, C and/or D virus, wherein the reference antibody is selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105.. In some embodiments, an antibodies of the invention has or retains at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the viremia- suppressing activity of a reference antibody against HBV genotype A, B, C and/or D viruses, wherein the reference antibody is selected from the group consisting of Bc1.187, Bv4.115, Bc8.159, Bv6.172, Bc1.229, Bc8.111, Bc1.128, Bc3.106, Bc1.180, Bv4.104, Bc8.104, Bc4.204, Bc1.263 and Bv4.105.
In certain aspects, an antibody of the invention is tested for such biological activity.
Exemplary in vitro neutralization assays are described below as protocol 4. An exemplary in vivo neutralization assay is described below as protocol 5. An exemplary assay for viremia suppression is described in protocol 6.
Where relative activities are assessed, the antibodies should be tested using the same method. The antibodies may both be assessed as IgGs: in such embodiments, the reference antibody will have the full length sequences for said reference antibody as provided herein. The binding activity of a test antibody comprising a VH and VL domain as defined herein (e.g., comprising a VH and/or VL domain which is a variant of that of the reference antibody) may be assessed in an IgG format comprising the constant domain and hinge sequences of the reference antibody.
In an exemplary protocol, the in vitro neutralizing activity of HBV antibodies is evaluated by incubating HBV virions (MOI 20-30) with serially diluted test antibodies for 1 h at room temperature. HBV-antibody mixtures are then added to hepatocytes in 96-well plates with a final concentration of 4% PEG 8000 (Sigma). Infected cells are incubated for 20 h at 37° C., and then washed 4 times with PBS to remove the HBV inoculum and refilled with complete media. Six days post-infection, in-supernatant S-HBs antigen content is quantified, e.g., using the S-HBs CLIA Kit (Autobio) according to the manufacturer’s instructions. Neutralization activity is determined as the reduction in the supernatant S-HBs as compared to a control in the absence of antibodies. The IC50 value is the half maximal inhibitory concentration, measuring the inhibition of infectivity.
Cells may be primary human hepatocytes or human hepatocyte cell lines. Exemplary hepatocytes which may be used in the protocol are primary human hepatocytes (PHH) isolated from chimeric uPA/SCID mice with humanized livers by a collagenase perfusion method (Tateno et al., 2015), obtainable from PhoenixBio (Hiroshima, Japan). Further exemplary hepatocytes are the HepaRG cell line obtainable from Biopredic International (Saint-Gregoire, France).
In an exemplary protocol for assessing in vivo neutralization activity, circulating blood S-HBs levels are monitored in AAV-HBV-transduced mice treated once i.v. with 0.5 mg of test antibody. Circulating blood S-HBs is measured by ELISA. Neutralizing activity is determined as a reduction in the amount of circulating blood S-HBs at the nadir (maximum decrease).
AAV-HBV mice carrying high levels of circulating S-HBsAg (> 104 IU/ml) are selected as subjects. A single intravenous (i.v.) injection of test antibodies is administered at 20 mg/kg. HBV DNA was purified from mouse sera using QIAamp Blood Mini kits (Qiagen, Germany), and quantified by quantitative PCR as previously described (Cougot et al., 2012). Viremia suppression activity is determined by assessing the amount of HBV DNA at the nadir (maximum decrease).
In certain aspects, any of the anti-S-HBs antibodies provided herein is useful for detecting the presence of S-HBs in a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. In certain aspects, a biological sample comprises a sample of blood, blood plasma or blood serum.
In one aspect, an anti-S-HBs antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of S-HBs in a biological sample is provided. In certain aspects, the method comprises contacting the biological sample with an anti-S-HBs antibody as described herein under conditions permissive for binding of the anti-S-HBs antibody to S-HBs, and detecting whether a complex is formed between the anti-S-HBs antibody and S-HBs. Such method may be an in vitro or in vivo method. In one aspect, an anti-S-HBs antibody is used to select subjects eligible for therapy with an anti-S-HBs antibody, e.g., where S-HBs is a biomarker for selection of patients.
Exemplary disorders that may be diagnosed using an antibody of the invention include hepatitis B, for instance chronic hepatitis B.
In certain aspects, labeled anti-S-HBs antibodies are provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
In a further aspect, provided are pharmaceutical compositions comprising any of the antibodies provided herein, e.g., for use in any of the below therapeutic methods. In one aspect, a pharmaceutical composition comprises any of the antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.
Pharmaceutical compositions of an anti-S-HBs antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized compositions or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as histidine, phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Pat. Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
Exemplary lyophilized antibody compositions are described in U.S. Pat. No. 6,267,958. Aqueous antibody compositions include those described in U.S. Pat. No. 6,171,586 and WO 2006/044908, the latter compositions including a histidine-acetate buffer.
The pharmaceutical composition herein may also contain more than one active ingredients as desired for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide one or more additional therapeutic agents selected from: an antiviral medication such as a nucleotide analog reverse-transcriptase inhibitor or a nucleoside analogue, e.g.,, entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, adefovir or telbivudine; an siRNA targeted to an HBV sequence; an agent aiming at restoring the host’s innate and adaptive immune responses, e.g., INFα or pegylated-IFNα; a therapeutic vaccine such as GS-4774, ABX-203, TG-1050, INO-1800; a TLR agonist such as an anti-TLR antibody, lipopeptide, lipopolysaccharide, Poly I:C (Polyriboinosinic-polyribocytidylic acid), Poly ICLC (Poly I:C-poly-l-lysine), imiquimod, GSK2245035, GSK2445053, RO6864018, RO7020531, GS-9620, GS-9688, 852A (Synthetic imidazoquinoline mimicking viral ssRNA), Resiquimod, VTX-2337 (Small-molecule selective TLR8 agonist mimicking viral ssRNA), Bacillus Calmette-Guérin (BCG), MPL (monophosphoryl lipid A) and/or CpG oligodeoxynucleotide; and/or a checkpoint inhibitor such as an antibody (e.g., an antagonist or blocking antibody), e.g., targeted against CTLA4 (e.g., ipilimumab, tremelimumab), PD-1 (e.g., nivolumab, pidilizumab), PD-L1 (e.g., MPDL3280A, MEDI4736, MSB0010718C), TIM-3, 2B4, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, VISTA, SIGLEC7 or SIGLEC9 (Fanning et al., 2019; Gehring and Protzer, 2019; Maini and Burton, 2019). Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Pharmaceutical compositions for sustained-release may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
The pharmaceutical compositions to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
Any of the anti-S-HBs antibodies provided herein may be used in therapeutic methods.
In one aspect, an anti-S-HBs antibody for use as a medicament is provided. In further aspects, an anti-S-HBs antibody for use in treating hepatitis B is provided, e.g., for use in treating chronic hepatitis B virus infection. In certain aspects, an anti-S-HBs antibody for use in a method of treatment is provided. In certain aspects, the invention provides an anti-S-HBs antibody for use in a method of treating an individual having hepatitis B, e.g., chronic hepatitis B virus infection, comprising administering to the individual an effective amount of the anti-S-HBs antibody. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents), e.g., as described below. In further aspects, the invention provides an anti-S-HBs antibody for use in reducing hepatitis B viral load, reducing detectable serum HBsAg or providing HBV functional cure. In certain aspects, the invention provides an anti-S-HBs antibody for use in a method of reducing hepatitis B viral load, reducing detectable serum HBsAg or providing HBV functional cure in an individual, comprising administering to the individual an effective amount of the anti-S-HBs antibody to reduce the viral load, reduce detectable serum HBsAg or provide HBV functional cure. HBV functional cure refers to seroclearance of hepatitis B surface antigen (HBsAg), i.e., the absence of detectable HBsAg in the serum. An “individual” according to any of the above aspects is preferably a human.
In a further aspect, the invention provides for the use of an anti-S-HBs antibody in the manufacture or preparation of a medicament. In one aspect, the medicament is for treatment of hepatitis B, e.g., chronic hepatitis B. In a further aspect, the medicament is for use in a method of treating hepatitis B, e.g., chronic hepatitis B, comprising administering to an individual having said condition an effective amount of the medicament. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In a further aspect, the medicament is for reducing hepatitis B viral load, reducing detectable serum HBsAg or providing HBV functional cure. In a further aspect, the medicament is for use in a method of reducing hepatitis B viral load, reducing detectable serum HBsAg or providing HBV functional cure in an individual comprising administering to the individual an effective amount of the medicament to reduce the viral load, reduce the detectable serum HBsAg or provide HBV functional cure. An “individual” according to any of the above aspects may be a human.
In a further aspect, the invention provides a method for treating hepatitis B, e.g., chronic hepatitis B. In one aspect, the method comprises administering to an individual having hepatitis B, e.g., chronic hepatitis B an effective amount of an anti-S-HBs antibody. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.
An “individual” according to any of the above aspects may be a human.
In a further aspect, the invention provides a method for reducing hepatitis B viral load, reducing detectable serum HBsAg or providing HBV functional cure. In one aspect, the method comprises administering to the individual an effective amount of an anti-S-HBs antibody to reduce the viral load/reduce the detectable serum HBsAg/provide a functional cure. In one aspect, an “individual” is a human.
In a further aspect, the invention provides pharmaceutical compositions comprising any of the anti-S-HBs antibodies provided herein, e.g., for use in any of the above therapeutic methods.
In one aspect, a pharmaceutical composition comprises any of the anti-S-HBs antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the anti-S-HBs antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.
Antibodies of the invention can be administered alone or used in a combination therapy. For instance, the combination therapy includes administering an antibody of the invention and administering at least one additional therapeutic agent (e.g. one, two, three, four, five, or six additional therapeutic agents). In certain aspects, the combination therapy comprises administering an antibody of the invention and administering at least one additional therapeutic agent, such as: an antiviral medication such as a nucleotide analog reverse-transcriptase inhibitor or a nucleoside analogue, e.g.,, entecavir, tenofovir disoproxil fumarate, tenofovir alafenamide, lamivudine, adefovir or telbivudine; an siRNA targeted to the HBV sequence; an agent aiming at restoring the host’s innate and adaptive immune responses, e.g., INFα or pegylated-IFNα; a therapeutic vaccine such as GS-4774, ABX-203, TG-1050, INO-1800; a TLR agonist such as an anti-TLR antibody, lipopeptide, lipopolysaccharide, Poly I:C (Polyriboinosinic-polyribocytidylic acid), Poly ICLC (Poly I:C-poly-l-lysine), imiquimod, GSK2245035, GSK2445053, RO6864018, RO7020531, GS-9620, GS-9688, 852A (Synthetic imidazoquinoline mimicking viral ssRNA), Resiquimod, VTX-2337 (Small-molecule selective TLR8 agonist mimicking viral ssRNA), Bacillus Calmette-Guérin (BCG), MPL (monophosphoryl lipid A) and/or CpG oligodeoxynucleotide; and/or a checkpoint inhibitor such as an antibody (e.g., an antagonist or blocking antibody), e.g., targeted against CTLA4 (e.g., ipilimumab, tremelimumab), PD-1 (e.g., nivolumab, pidilizumab), PD-L1 (e.g., MPDL3280A, MEDI4736, MSB0010718C), TIM-3, 2B4, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, VISTA, SIGLEC7 or SIGLEC9 (Fanning et al., 2019; Gehring and Protzer, 2019; Maini and Burton, 2019).
Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate pharmaceutical compositions), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents. In one aspect, administration of the anti-S-HBs antibody and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other. In one aspect, the antibody and additional therapeutic agent are administered to the patient on Day 1 of the treatment.
An antibody of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 µg/kg to 15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 µg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or, e.g., about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. The progress of this therapy is easily monitored by conventional techniques and assays.
In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this aspect of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
Amino acids shown in parentheses in the below table can be present or absent.
The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.
Blood samples from HBV vaccinees and HBV seroconverters were obtained from the healthy donors cohort of the ICAReB biobanking platform (Institut Pasteur) under the CoSimmGen protocol approved by the French Agence Nationale de Sécurité du Médicament (ANSM) on May 24th 2012, and the Comité de Protection des Personnes (CPP) on January 17th 2014. The main clinical-biological characteristics of the donors are summarized in
Native S-HBsAg purified from infected subjects and recombinant S-HBsAg particles (ayw subtype) were prepared and biotinylated by Roche. Recombinant HBsAg particles (ayw) were produced in Chinese hamster ovary cells (Aventis Pasteur, Val de Reuil, France) (Michel et al., 1984). Adw-subtype HBsAg particles (HBV genotype A) were produced in the yeast Pichia pastoris expression system, and purified (<95% purity) at the Center for Genetic Engineering and Biotechnology production facilities (CIGB, Havana, Cuba; a kind gift from Dr. J. Aguilar). Consensus genotype-specific S-HBs fragments were constructed by multiple amino acid alignment of individual HBV genotypic sequences (A, n=205; B, n=495; C, n=1322; D, n=382; E, n=314; F, n=271; G, n=6; H, n=40; I, n=7) using CLC Main Workbench 7 software (v7.5.3, QIAGEN Aarhus A/S). Codon-optimized nucleotide fragments encoding the consensus S-HBs, a G3S linker, 10xHis- and Avi-tags were cloned into pcDNA™3.1/Zeo(+) expression vector (Thermo Fisher Scientific) using Anza 5 and 11 restriction enzymes (Thermo Fisher Scientific). For the production of the transmembrane (TM) domains-deleted S-HBs protein (ΔTM-S-HBs), the same construct but with the S-HBs DNA fragment lacking TM1, TM3, TM4 domains, and having the TM2 replaced by a (G3S)5 linker was generated. Disulfide-bridged S-HBs/D 122-137 (RTCTTTAQGTSMYPSC) and 139-148 (CTKPSDGNCT) loop peptides, and 3 amino acids-overlapping 12-mer peptides (n=24) encompassing the entire ConsD S-HBs region but one transmembrane domain 2 peptide with high hydrophobicity (IFLFILLLCLIF) were synthetized and desalted (GenScript HK Limited). Non-HBV antibody mGO53 (Wardemann and Nussenzweig, 2007) was used as isotype control. As positive control, neutralizing murine HB1 antibody specific to the 119-GPCRTCT-125 linear epitope of the “a” determinant of S-HBsAg (Kucinskaite-Kodze et al., 2016), was produced as a chimeric recombinant IgG1 with a human Fc using the expression-cloning system described below. Murine anti-pre-S2 antibody 1F6 (Kuttner et al., 1999) was produced as chimeric human Fab fragments to be used as capture molecules in the alanine scan mapping described below.
Peripheral blood human B cells were isolated from donors’ PBMCs by CD19 MACS (Miltenyi Biotec), and stained with LIVEDEAD fixable dead cell stain kit (Thermo Fisher Scientific). Purified B cells were then incubated for 30 min at 4° C. with biotinylated recombinant or native S-HBs antigens, washed with 1% FBS-PBS (FACS buffer), and incubated for 30 min at 4° C. with a cocktail of mouse anti-human antibodies (CD19 A700 (HIB19), IgG BV786 (G18-145), CD27 PE-CF594 (M-T271) (BD Biosciences), IgA FITC (IS11-8E10, Miltenyi Biotec)), and streptavidin R-PE conjugate (Thermo Fisher Scientific). Stained cells were washed and resuspended in 1 mM EDTA FACS buffer. Single S-HBs+CD19+IgG+ B cells were sorted into 96-well PCR plates using a FACS Aria III sorter (Becton Dickinson, Franklin Lakes, NJ) as previously described (Tiller et al., 2008). Single-cell cDNA synthesis using SuperScript IV reverse transcriptase (Thermo Fisher Scientific) followed by nested-PCR amplifications of IgH, Igκ and Igλ genes, and sequences analyses for Ig gene features were performed as previously described (Tiller et al., 2008). For the reversion to germline (GL) of the selected antibodies, sequences were constructed by replacing the mutated VH-(DH)-JH and VL-JL gene segments with their GL counterparts as previously described (Mouquet et al., 2012). Purified digested PCR products were cloned into human Igγ1-, Igκ- or Igλ-expressing vectors (GenBank# LT615368.1, LT615369.1 and LT615370.1, respectively) as previously described (Tiller et al., 2008). Murine Igγ2- and Igκ-expression vectors were generated from the original IgG1-expression vectors (Tiller et al., 2008) by substituting the DNA sequences coding for the human Igγ1/Igκ constant regions of by the ones of the mouse Igγ2- and Igκ (synthetic DNA fragment, GeneArt, Thermo Fisher Scientific), and then used for the cloning of chimeric mGO53 and Bc1.187 antibodies. Recombinant antibodies were produced by transient co-transfection of Freestyle™ 293-F suspension cells (Thermo Fisher Scientific) using PEI-precipitation method as previously described (Lorin and Mouquet, 2015). Recombinant human IgG antibodies were purified by protein G affinity chromatography (Protein G Sepharose® 4 Fast Flow, GE Healthcare, Chicago, IL). Purified antibodies were dialyzed against PBS. Preparations for in vivo infusions were micro-filtered (Ultrafree®-CL devices - 0.1 µm PVDF membrane, Merck-Millipore, Darmstadt, Germany), and checked for endotoxins levels using the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GenScript).
ELISAs were performed as previously described (Planchais et al., 2019). Briefly, high-binding 96-well ELISA plates (Costar, Corning) were coated overnight with purified rS-HBs and nS-HBs (125 ng/well in PBS). After washings with 0.05% Tween 20-PBS (PBST), plates were blocked 2 h with 2% BSA, 1 mM EDTA-PBST (Blocking solution), washed, and incubated with serially diluted purified serum IgG and recombinant monoclonal antibodies in PBS. For sandwich ELISAs, plates were coated overnight with purified IgG antibodies (250 ng/well in PBS), and treated as afore-mentioned prior to incubation with biotylinated-rS-HBs (100 ng/well in PBS) in PBS. After washings, plates were revealed by addition of goat HRP-conjugated anti-human IgG or HRP-conjugated streptavidin (0.8 µg/ml final in blocking solution, Immunology Jackson ImmunoReseach) and HRP chromogenic substrate (ABTS solution, Euromedex). For competition ELISAs, purified antibodies were biotinylated using the EZ-Link Sulfo-NHS-Biotin kit (Thermo Fisher Scientific). rS-HBs-coated plates were blocked, washed, incubated for 2 h with biotinylated antibodies (at a concentration 0.33 nM) in 1:2 serially diluted solutions of antibody competitors in PBS (IgG concentration range from 0.83 to 106.7 nM), and revealed using HRP-conjugated streptavidin. Experiments were performed using HydroSpeed™ microplate washer and Sunrise™ microplate absorbance reader (Tecan Männedorf), with optical density measurements made at 405 nm (OD405nm). Binding of anti-S-HBs antibodies to cyclic and overlapping linear peptides was tested using the same procedure as previously described (Mouquet et al., 2006). All antibodies were tested in duplicate or triplicate in at least two independent experiments, which included mGO53 negative and appropriate positive controls.
Mutant HBV envelope proteins were produced by co-transfection of Huh-7 cells with the psVLD3 and pT7HB2.7 plasmids using FuGENE 6 reagent (Roche) as previously described (Salisse and Sureau, 2009). High-binding 96-well ELISA plates (Costar, Corning) were coated overnight with purified human S-HBs antibodies (0.5 µg/well in PBS). After PBST washings, and a 2 h-blocking step, plates were incubated 2 h with culture supernatants containing HBsAg wildtype and mutants proteins. After washings, plates were incubated 1h with purified His-tagged 1F6 Fab fragments (125 ng/well), washed, and revealed by addition of rabbit HRP-conjugated anti-6xHis-tag antibodies (1:4,000-diluted in blocking solution, ab1187, Abcam) and HRP chromogenic substrate (ABTS solution, Euromedex) as described above. Percentage of binding was calculated following the formula: ([OD]mutant/ [OD]wildtype) × 100.
Freestyle™ 293-F were transfected with S-HBs-encoding vectors (0.65 µg plasmid DNA per 106 cells) using PEI-precipitation method as previously described (Lorin and Mouquet, 2015). Forty-eight hours post-transfection, transfected and non-transfected control cells were fixed and permeabilized using Cytofix/Cytoperm™ solution kit (BD Biosciences), and 0.5×106 cells were incubated with IgG antibodies for 45 min at 4° C. (10 ug/ml, excepted if specified otherwise, in Perm/Wash™ solution; BD Biosciences). After washings, cells were incubated 20 min at 4° C. with AF647-conjugated goat anti-human IgG antibodies (1:1000 dilution; Thermo Fisher Scientific), washed and resuspended in PBS. Data were acquired using a CytoFLEX flow cytometer (Beckman Coulter), and analyzed using FlowJo software (v10.3; FlowJo LLC).
All experiments were performed at 4° C. using ProtoArray Human Protein Microarrays (Thermo Fisher Scientific). Microarrays were blocked for 1h in blocking solution (Thermo Fisher), washed and incubated for 1 h30 with IgG antibodies at 2.5 µg/ml as previously described (Planchais et al., 2019). After washings, arrays were incubated for 1 h30 with AF647-conjugated goat anti-human IgG antibodies (at 1 µg/ml in PBS; Thermo Fisher Scientific), and revealed using GenePix 4000B microarray scanner (Molecular Devices) and GenePix Pro 6.0 software (Molecular Devices) as previously described(Planchais et al., 2019). Fluorescence intensities were quantified using Spotxel® software (SICASYS Software GmbH, Germany), and mean fluorescence intensity (MFI) signals for each antibody (from duplicate protein spots) was plotted against the reference antibody mGO53 (non polyreactive isotype control) using GraphPad Prism software (v8.1.2, GraphPad Prism Inc.). For each antibody, Z-scores were calculated using ProtoArray® Prospector software (v5.2.3, Thermo Fisher Scientific), and deviation (σ) to the diagonal, and polyreactivity index (PI) values were calculated as previously described (Planchais et al., 2019). Antibodies were defined as polyreactive when PI > 0.21.
Binding of human anti-S-HBs and control IgG antibodies (mGO53 and ED38 (Meffre et al., 2004; Wardemann et al., 2003)) to HEp-2 cell-expressing autoantigens were analyzed at 50 µg/ml by ELISA (AESKULISA® ANA-HEp-2, Aesku.Diagnostics, Wendelsheim, Germany), and indirect immunofluorescence assay (IFA) (ANA HEp-2 AeskuSlides®, Aesku.Diagnostics) following the manufacturer’ instructions. IFA sections were examined using the fluorescence microscope Axio Imager 2 (Zeiss, Jena, Germany), and pictures were taken at magnification × 40 with 5000 ms-acquisition using ZEN imaging software (Zen 2.0 blue version, Zeiss) at the Imagopole platform (Institut Pasteur).
HepaRG and HepG2.2.15 cell lines were obtained respectively from Biopredic International (Saint-Gregoire, France), and Dr. Michael Nassal (University Hospital Freiburg, Germany). HepaRG cells were cultured in Williams E medium (Gibco®, Thermo Fisher Scientific) supplemented with 10% HepaRG growth supplement (Biopredic), and differentiated using 1.8% DMSO for at least 2 weeks before infection. Primary human hepatocytes (PHH) isolated from chimeric uPA/SCID mice with humanized livers by a collagenase perfusion method (Tateno et al., 2015) were obtained from PhoenixBio (Hiroshima, Japan). PHH were plated on type I collagen coated 96-well plates at a concentration of 7 × 104 cells per well in culture media provided by Phoenix Bio. HBV genotype D viruses were produced in HepG2.2.15 cell culture supernatant, and concentrated using polyethylene glycol precipitation (Hantz et al., 2009). HBV viruses from genotypes A to C were purified from HBV-containing serum (American Red Cross) by gradient ultracentrifugation. Briefly, 1.5 ml of serum was applied on an OptiPrep™ gradient (10-50%, Sigma), and centrifuged at 32,000 rpm for 3 h at 4° C. Fractions (2ml) were collected and analyzed for HBs antigen expression using S-HBs CLIA Kit (Autobio) and qPCR quantification. Whole genome sequences of all purified virus isolates were obtained by ultra-deep sequencing (DDL Diagnostic Laboratory, Netherlands), and HBV genotypes were determined using the Hepatitis B Virus database HBVdb (http://hbvdb.ibcp.fr) (Hayer et al., 2013). The neutralizing activity of HBV antibodies was evaluated by incubating HBV virions (MOI 20-30) with serially diluted antibodies in HepaRG or PHH complete media for 1 h at room temperature. HBV-antibody mixtures were then added to the cells in 96-well plates with a final concentration of 4% PEG 8000 (Sigma). Infected cells were incubated for 20 h at 37° C., and then washed 4 times with PBS to remove the HBV inoculum and refilled with complete media. Six days post-infection, in-supernatant S-HBs antigen content was quantified using the S-HBs CLIA Kit (Autobio) according to the manufacturer’s instructions.
In vitro neutralization assays were conducted using the HDV model as previously described (Sureau, 2010), except that NTCP-expressing Huh-106 cells substituted for HepaRG cells (Verrier et al., 2016). At day-1 post seeding, Huh-106 cells (1 × 105 cells/20 mm diameter well) were exposed to 5×107 genome equivalents (ge) of HDV virions for 16 h, in the presence of 4% polyethylene glycol (PEG) 8000. To assay neutralization, the inoculums were mixed with 1:2 dilutions of 500 ng/ml of monoclonal antibodies, and the mixture was incubated for 1 h at 37° C. prior to inoculation. Cells were harvested at 9 days post-inoculation (dpi) for measurement of intracellular HDV RNA that served as a marker of infection. HDV RNA signals were detected by Northern blot analysis using a 32P-labeled RNA probe and quantified using a phosphorimager instrument r (BAS-1800 II; Fuji).
Chronic HBV infection was established in 6-8-week-old C57BL/6 mice (Janvier Labs, Le Genest-Saint-Isle, France) by a single intravenous injection (retro-orbital venous sinus) of 5 × 1010 viral genome of an adeno-associated-virus serotype 2/8 carrying a replication competent HBV-DNA genome (Dion et al., 2013). Virus stocks were produced and titrated as virus genome equivalents (GE Healthcare) and focus-forming units per milliliter by the Plateforme de Thérapie Génique (INSERM U1089, Nantes, France). Six weeks post-transduction, antibodies (0.25, 0.5 or 1 mg per injection per mouse) were administrated intravenously to HBV-carrier mice. Blood samples were collected and stored at -20° C. Serum S-HBsAg and HBeAg levels were determined using Monolisa S-HBsAg ULTRA (Bio-Rad, France) ELISA kits and ETI-EBK Plus NO140 (Diasorin SA, Italy), respectively. Concentrations were calculated by reference to standard curves established with known concentrations of S-HBsAg (Architect S-HBsAg Calibrators, Bio-Rad, France) and of the Paul-Ehrlich-Institut standard and are expressed in IU/mL and PEI U/mL, respectively. HBV DNA was purified from mouse sera using QIAamp Blood Mini kits (Qiagen, Germany), and quantified by quantitative PCR as previously described (Cougot et al., 2012). Serial dilutions of the payw1.2 plasmid containing 1.2 copies of HBV genome were used as quantification standards. Results are expressed in IU/ml with a detection threshold at 1000 IU/ml. All experimental animal protocols have been reviewed and approved by the institutional animal care committee of Institut Pasteur for compliance with the French and European regulations on Animal Welfare and with Public Health Service recommendations (APAFIS#15408-2018060517005070 v1). All experiments with HBV infections were performed in an A3 animal facility.
To generate the HUHEP model BALB/c Rag2-/-IL-2Rγc-/-NOD.sirpa uPatg/tg (BRGS-uPA) mice were intrasplenically injected with 7×105 freshly thawed human hepatocytes (BD Biosciences, Corning) (Strick-Marchand et al., 2015). Liver chimerism was determined on plasma samples with a species-specific human albumin (hAlb) ELISA (Bethyl Laboratories) and HUHEP mice with ≥100 microg/ml hAlb were intraperitoneally infected with 1×107 HBV genome equivalents (Dusseaux et al., 2017). HBV-infected mice with >106 HBV DNA copies/ml were given an intraperitoneal injection of either bNAb CH1-187 or an isotype control (mGO53) IgG every 3-4 days at 20 mg/kg mouse, or every week at 1 mg/mouse, or Entecavir (ETV) 0.3 mg/kg/day (Baraclude, BMS) delivered in MediDrop Sucralose (Clear H2O) per os. For the rebound phase mice were returned to drinking water. Animals were housed in isolators under pathogen-free conditions with humane care. Experiments were approved by an institutional ethical committee at the Institut Pasteur (Paris, France) and validated by the French Ministry of Education and Research (MENESR # 02162.02).
For Ig gene repertoire analyses, groups were compared using two-sided 2 × 2 and 2 × 5 Fisher’s Exact tests. The numbers of VH, Vκ and Vλ mutations were compared across groups of antibodies using unpaired student t-test with Welch’s correction. The volcano plot analysis was performed by comparing 206 individual antibody gene features between groups, and reporting for each parameter the Δmean (x axis) and the -log10 p-values given by the two-sided 2 × 2 Fisher’s Exact test (y axis). Statistical analyses were performed using GraphPad Prism software (v8.1.2, GraphPad Prism Inc.), and SISA online tools for 2 × 5 Fisher test (http://www.quantitativeskills.com/sisa).
To characterize the memory B-cell antibody response to the HBV surface glycoproteins, peripheral blood B cells from six vaccinees and eight seroconverters with high serum anti-HBsAg IgG antibody titers (
To determine whether the S-HBs memory B-cell antibodies neutralize HBV, we measured their in vitro neutralizing activity against genotype D viruses in the HepaRG cell-based assay. Overall, 61% of the S-HBs antibodies blocked HBV infection with 50% inhibitory concentrations (IC50) ranging from 50 µg/ml down to 0.05 pg/ml (
To evaluate the in vivo activity of neutralizing anti-S-HBs antibodies, we generated HBV-persistent mice based on the liver-targeted transduction of a recombinant HBV-encoding adeno-associated virus (AAV) (Dion et al., 2013). Four potent HBV neutralizers were selected and passively transferred into AAV-HBV mice carrying high levels of circulating S-HBsAg (> 104 IU/ml). A single intravenous (i.v.) injection of antibodies at ~20 mg/kg led to a marked viremia drop 2 days post-injection (dpi), whereas treatments with a lower antibody dose had milder effects due to the rapid exhaustion of antibodies by outnumbering HBV particles (
HBV is classified into four major serotypes based on specific amino acid variations in the “a” determinant (adr, adw, ayr, ayw), and ten genotypes according to viral genome-based phylogeny (A to J) (Kato et al., 2016). Binding analyses showed that most potent HBV neutralizing antibodies (71%) are able to recognize both adw and ayw HBsAg particles used in GenHevac-B and Engerix-B vaccines, respectively (
In humans, several HBV escape mutations have been described, of which G145R is the most prevalent substitution in the S protein region (Chotiyaputta and Lok, 2009; Huang et al., 2012). We thus evaluated whether some of these mutations could affect HBV recognition by the potent neutralizers (
To map the epitopes targeted by potent neutralizing HBV antibodies, we performed alanine-scanning ELISA experiments using a library of mutant HBsAg proteins with substitutions covering the major hydrophilic region of HBsAg. Although all tested antibodies presented a unique recognition pattern, common binding profiles could be identified based on mutation sensitivity (
To examine whether somatic mutation contributes to the activity of HBV neutralizing antibodies, we reverted mutations in three representative potent neutralizers to produce their putative germline precursors (GL). Representative GL versions showed heterogeneous HBV recognition profiles: Bc4.204-GL failed reacting with S-HBs, whereas Bc1.187-GL and Bv4.104-GL still bound but with much lower relative affinities compared to their matured counterparts (
Human class-switched memory B cells including high affinity B-cell clones against viral antigens can cross-react with self-antigens (Andrews et al., 2015; Prigent et al., 2018; Prigent et al., 2016; Tiller et al., 2007). We thus evaluated the self-reactivity of 10 selected potent HBV neutralizers using clinical autoantibody assays (HEp-2 cells IFA and ELISA), and microarray immunoblotting (> 9,000 human proteins). Apart from Bc1.263, none of the HBV neutralizers showed polyreactivity as measured by a global shift of the microarray fluorescence signals compared to the isotype control (
To determine whether potent HBV neutralizers from natural controllers can stably suppress HBV viremia in vivo, AAV-HBV mice were treated for 17 days with Bc1.187 antibody (0.5 mg i.v., ~20 mg/kg, twice a week) (
We next wonder whether Bc1.187 could alter the natural course of HBV infection in vivo. BALB/c Rag2-/-SirpaNODAlb-uPAtg/tg mice stably engrafted with human hepatocytes (HUHEP) were infected with genotype D HBV. Once infection was established, mice received injections of Bc1.187 for 3 weeks, either bi-weekly (20 mg/kg per mouse) or weekly (50 mg/kg per mouse). In agreement with the AAV-HBV in vivo model, treatment with Bc1.187, but not non-HBV antibody and nucleoside reverse transcriptase inhibitor controls, induced a decrease of circulating HBsAg levels in viremic HUHEP mice by an average 2.1 and 2 log10 fold at dpi21 for the 20 mg/kg and 50 mg/kg dosing regimen, respectively (
Rare individuals can naturally clear chronic hepatitis B virus (HBV) infection and acquire protection from re-infection as conferred by vaccination. To examine the protective humoral response against HBV, we cloned human antibodies to the viral surface glycoprotein S-SHBs from memory B cells of HBV vaccinees and controllers. We find that S-SHBs memory B cells from natural controllers produce mainly neutralizing antibodies able to cross-react with several viral genotypes. Human neutralizing HBV antibodies are encoded by a diverse set of immunoglobulin genes and recognize various conformational epitopes on the S-SHBs antigen. Strikingly, monotherapy in HBV mouse models with a potent cross-neutralizer isolated from a controller, Bc1.187, suppress viremia in vivo and lead to post-therapy control of the infection in some animals. Thus, neutralizing S-SHBs antibodies may play a key role in the spontaneous control of HBV and represent promising immunotherapeutics for achieving HBV functional cure in chronically infected humans.
Results for certain antibodies described herein are summarized below, in table 2. This summarizes results from
In chronic HBV infection, most HBsAg-specific B cells are atypical memory lymphocytes presenting several B-cell dysfunctions such as an altered capacity to differentiate into antibody-producing cells and thus, to potentially produce HBV neutralizing antibodies (Burton et al., 2018; Salimzadeh et al., 2018). In contrast, circulating B cells in individuals who resolved the infection, both during the acute and chronic phase, secrete HBsAg antibodies likely participating in the seroconversion (Salimzadeh et al., 2018; Xu et al., 2015). To characterize memory B-cells directed against the HBV surface antigen in immune donors, we investigated the molecular and functional properties of recombinant human antibodies cloned from single S-HBs-specific IgG+ circulating in the blood of HBV controllers and vaccinees. Our study reveals that despite a moderate enrichment in VH1-JH4-encoding clones, circulating S-HBs-specific memory B cells from HBV controllers, but also from vaccinees, express a diverse gene repertoire of immunoglobulins recognizing mostly conformational epitopes on the HBV surface glycoproteins. The majority of anti-S-HBs IgGs cloned from controllers could neutralize HBV in vitro, with some being active at very low concentrations, including against HDV infection. Potent neutralizers evaluated in vivo also showed antiviral effects by decreasing both serum HBsAg level and HBV viremia, suggesting that in infected humans these antibodies may be functionally important. The majority of HBV neutralizing antibodies were broadly reactive against various viral genotypes and recognized epitopes located in the “a” determinant although others were also mapped outside this region. Several neutralizing epitopes were identified including a main region involving the S-HBs second loop recognized by the potent cross-neutralizing antibody Bc1.187. A single passive infusion of Bc1.187 was particularly effective in vivo in a mouse model of chronic HBV infection to completely suppress for a few days serum HBsAg and HBV viremia. Interestingly, we showed here that the germline version of Bc1.187 could still bind and neutralize HBV with an IC50 < 0.1 µg/ml. This suggests that B-cell precursors expressing such immunoglobulins could rapidly affinity matured and thus, be readily active in neutralizing HBV.
Current therapies for treating chronic HBV infection include potent direct antivirals and PEG-IFNα. However, antiviral treatments have no significant impact on the serum HBsAg levels due principally to defective sub-viral particles outnumbering infectious HBV virions. To bypass the immune tolerance observed in chronically infected individuals, and induce effective anti-HBV antibody responses is therefore still a major challenge. One promising strategy is the development of immunotherapies based on the use of potent HBV neutralizing antibodies (Corti et al., 2018; Gao et al., 2017; Tu and Urban, 2018). Preclinical models in mice and monkeys, as well as several phase I clinical trials in chronically-infected humans have shown the in vivo efficacy of neutralizing HBV monoclonal antibodies, which when administrated in infected recipients altered the course of infection by suppressing HBsAg and reducing HBV DNA content (Eren et al., 2000; Galun et al., 2002; Lee et al., 2019; Lever et al., 1990; Li et al., 2017; van Nunen et al., 2001; Zhang et al., 2016; Zhu et al., 2016). In this study, we showed using two different mouse models that treating viremic animals with a potent human cross-neutralizing antibody had profound effects on HBV infection. Bc1.187 therapy induced a rapid loss and/or substantial decrease of circulating HBsAg and HBV DNA that persisted in most mice for several days to few weeks post-treatment. In vivo experiments also demonstrated that low viremia-bearing mice could clear HBV infection upon Bc1.187 antibody treatment. Antibodies are versatile immune effectors. Besides neutralization, they can exert a variety of immunological effector functions such as antibody dependent cellular cytotoxicity (ADCC), which allow the killing of infected cells by innate immune cells such as Natural Killer cells (NK). The ADCC activity is a key component of the therapeutic property of human neutralizing antibodies to viruses such as HIV-1 and influenza (Bruel et al., 2016; DiLillo et al., 2014). Early on, complement-dependent lysis and ADCC have been involved in the hepatocytes killing activity of murine anti-S-HBs antibodies using in vitro and in vivo systems (Shouval et al., 1982a; Shouval et al., 1982b). More recently, a human neutralizing HBV antibody against the pre-S1 region has been shown to exert therapeutic activity with sustained virological suppression in part, by eliciting Fc-dependent effector functions (Li et al., 2017). Finally, antibody therapy by engaging the host immune system can lead to a stimulation of immune responses, a property better appreciated and known as vaccine-like effects (Pelegrin et al., 2015), a property that could be essential in breaking HBV-induced immune tolerance. Hence, we propose that in addition to blocking de novo infection and possibly eliminating infected hepatocytes via Fc-dependent mechanisms, some of the potent human cross-neutralizing HBV antibodies described here could be used in chronically infected patients to greatly reduce serum HBsAg level. Such antibodies could indeed act as a powerful “antigenic sink”, which when combined with therapies aiming at restoring the host’s innate and adaptive immune responses, i.e., INFα, therapeutic vaccines, TLR agonists, checkpoint inhibitors (Fanning et al., 2019; Gehring and Protzer, 2019; Maini and Burton, 2019) could facilitate viral clearance and eventually lead to a long-term control of HBV infection.
An in vitro pulse-chase assay to measure antibody recycling and transcytosis (Antibody Recycling and Clearance (ARC) Assay) was used to flag potential clearance liabilities driven by two known mechanisms: 1) non-specific clearance - driven by non-specific binding/internalization into cells (ARC-score at pH=7.4), and 2) FcRn-mediated effects (ARC-score at pH=6 and ARC-fold shift). This assay provides an assessment of these parameters relative to controls that have shown IgG-like pharmacokinetics in human (for a further description of the assay, see MAbs. 2017 Jul;9(5):781-791. doi: 10.1080/19420862.2017.1320008). Wild-type IgG1 Bc1.187 displayed an ARC-score of 0.28 and 4.97 at pH 7.4 and pH 6, respectively, leading to an ARC-fold shift of 17.8. These values indicate a low potential for non-specific binding/internalization into cells, and an FcRn-recycling in line with IgG-molecules. Similar constructs that are based on Bcl.187 IgG, but with additional FcRn-modifications to enhance FcRn-binding (and recycling) were also assessed in the assay, and the results are summarized in Table 4. The data show that FcRn-engineering can increase the ARC-fold shift, which is predictive for lower clearance in vivo.
A second in vitro assay, the Large molecule Unspecific Clearance Assay (LUCA) was used to assess antibody clearance in human primary endothelial cells. This assay relates the rate of mAb uptake into endothelial cells expressing endogenous amounts of FcRn, and provides a relative LUCA rate that is resulting from unspecific uptake, FcRn-recycling, and protein degradation. Bcl.187 has a relative LUCA rate of 0.05, indicating a rate of cellular accumulation in between standardization compounds, motavizumab-YTE (rel. LUCA rate of 0) and CD20-TCB (rel. LUCA rate of 1). This also points to predicted IgG-like properties for Bc1.187 in human. Additionally FcRn-engineered constructs based on Bc1.187 were tested in the assay, and the results are summarized in Table 4. As for the ARC-assay, it is predicted that FcRn-engineered constructs have a lower clearance in vivo.
Lastly, the in vivo pharmacokinetics of wild-type Bc1.187 IgG and FcRn-engineered Bc1.187 IgG constructs were assessed in transgenic mice expressing human neonatal Fc receptor (huFcRn-Tg mice), as these mice have shown to be predictive for human PK. (For further description of the mouse models, see 28.Proetzel et al. Methods. 2014;65:148-53; Roopenian et al. Methods Mol Biol. 2016;1438:103-14; Avery LB et al. MAbs. 2016;8:1064-78). All constructs were administered intravenously at a dose of 5 mg/kg, and the clearance parameters are summarized in Table 4. For all tested constructs, a clearance in the typical range for IgGs in these mice was observed. Taken together these in vitro and in vivo pharmacokinetics benchmarking studies indicate that neutralizing antibodies based on the Bc1.187 sequence are likely to display IgG-like pharmacokinetics in human. Protein engineering can lead to lower predicted clearance, which would confer significant advantages for neutralizing antibodies, as it is expected to translate to longer duration of HBsAg-neutralization, or longer dosing intervals in the clinic (i.e. increased convenience and compliance).
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20305612.2 | Jun 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/065260 | 6/8/2021 | WO |
