Anti-HBV antibodies or fragments thereof and methods of their use

Abstract

The invention relates to antibodies or fragments thereof directed against HBV and uses thereof, such as their use in adoptive immunotherapy, and treating HBV infection or associated diseases.

Description

[0001] ANTI-HBV ANTIBODIES OR FRAGMENTS THEREOF AND METHODS OF THEIR USE

[0002] FIELD OF THE INVENTION

[0003]

[0001] The invention relates to antibodies or fragments thereof directed against HBV and uses thereof, such as their use in treating HBV infection or associated diseases.

[0004] BACKGROUND OF THE INVENTION

[0005]

[0002] Chronic HBV infection remains a global health problem. Despite the availability of an effective vaccine, approximately 296 million people are chronically infected worldwide and carry an increased risk of developing liver cirrhosis and hepatocellular carcinoma1. In contrast to other infectious diseases such as malaria, tuberculosis, or HIV, viral hepatitis-related mortality rates are still on the rise2, amounting to 820,000 HBV-associated deaths per year1. Current standard therapy using nucleotide analogs suppresses new virion production and HBV DNA and reduces liver inflammation but has little or no effect on the expression of the small HBV envelope (HBVenv) protein (S)3. Thus, viral eradication is rarely achieved, and a significant cancer risk remains4’5. This is why there is an ongoing need to research novel treatment options for the cure of HBV infection.

[0006]

[0003] A key characteristic of chronic HBV infection is a scarce and dysfunctional T-cell response6. In contrast, an acute and resolving course of infection is marked by a strong and polyfunctional virusspecific T-cell response7. Furthermore, clinical reports showed that transfer of pre-existing HBV immunity through bone marrow transplantation for lymphoma therapy cleared the infection in the receiving patients8. Therefore, reconstituting HBV-specific T cell immunity via adoptive transfer of genetically redirected T cells emerged as a promising therapeutic strategy in recent years9. To date, both HBV-specific T-cell receptors (TCRs) and chimeric antigen receptors (CARs) have shown encouraging results in vitro and in vivo9-12. While TCRs are therapeutically confined to a limited number of patients given their HLA restriction, CARs harbor the advantage of targeting surface-bound antigen on target cells in an MHC-independent fashion9.

[0007]

[0004] A HBV-specific S-CAR based on the single-chain variable fragment (scFv) C8 has been previously identified from a phage display library generated out of peripheral blood mononuclear cells of HBV-vaccinated donors13. This S-CAR was shown to recognize all HBVenv proteins on the surface of infected cells14as the S-domain is also contained in the middle and large HBVenv proteins. Furthermore, it proved functional in vitro and in mouse models of chronic HBV infection11’12. Still, it showed limitations in terms of sensitivity, in particular, compared to HBV-specific TCRs, and it induces a certain level of tonic signaling, a problem known for several CARs targeting a variety of antigens.

[0008]

[0005] Therefore, there is a need in the art to develop alternative antibodies and fragments thereof that specifically target HBV infection, and may be used in cell immunotherapy to treat HBV infection or HBV related diseases. SUMMARY OF THE INVENTION

[0009]

[0006] In one aspect, there is provided an antibody comprising a heavy chain variable domain and a light chain variable domain, wherein: the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 1 -3, respectively, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 4-6 , respectively; or the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 7-9, respectively, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 10-12, respectively, wherein the antibody binds to Hepatitis B virus (HBV), more preferably binds to an HBV envelope protein.

[0010]

[0007] In various embodiments, the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 1 -3, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 4-6, and wherein the heavy chain variable domain (VH) comprises or consists of an amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof, and a light chain variable domain (VL) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID Nos. 1 -6 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0011]

[0008] In various embodiments, the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 7-9, respectively, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 10-12, respectively, and wherein the heavy chain variable domain (VH) comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof, and a light chain variable domain (VL) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID Nos. 7-12 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0012]

[0009] In various embodiments, the antibody is an IgG immunoglobulin, preferably an lgG1 immunoglobulin.

[0013]

[0010] In various embodiments, the antibody comprises a IgG constant region within a heavy chain of the immunoglobulin and a IgG constant region within a light chain of the immunoglobulin.

[0014]

[0011] In various embodiments, the antibody is a human antibody, a monoclonal antibody, a purified antibody, or a single chain antibody, preferably a human monoclonal antibody.

[0012] In another aspect there is provided an antigen-binding fragment of the antibody disclosed herein.

[0015]

[0013] In various embodiments, the antigen-binding fragment is a Fv, a Fab, a Fab’, a F(ab’)2, scFv or a scFv2 fragment.

[0016]

[0014] In various embodiments, the antigen-binding fragment comprises: the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 1 -3, respectively, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 4-6, respectively; or the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 7-9, respectively, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 10-12, respectively.

[0017]

[0015] In another aspect there is provided a chimeric antigen receptor (CAR) comprising the antigenbinding fragment, preferably a scFv fragment, disclosed herein.

[0018]

[0016] In various embodiments, the CAR further comprises an intracellular domain and a transmembrane domain, wherein the antigen-binding fragment is comprised in an extracellular domain.

[0019]

[0017] In various embodiments, the intracellular domain comprises a CD28 and a CD3zeta signalling domain, the transmembrane domain comprises a CD28 transmembrane domain, and the extracellular domain further comprises a hinge region, a linker and a tag.

[0020]

[0018] In various embodiments, the CAR disclosed herein is for use in adoptive immunotherapy.

[0021]

[0019] In another aspect there is provided a nucleic acid molecule encoding the antibody disclosed herein, or the antigen-binding fragment disclosed herein, or the chimeric antigen receptor disclosed herein.

[0022]

[0020] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy and / or light chain variable domain of the antibody disclosed herein.

[0023]

[0021] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable domain as set forth in SEQ ID Nos: 29 or 31 , or a variant thereof.

[0024]

[0022] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the light chain variable domain as set forth in SEQ ID Nos: 30 or 32 or a variant thereof.

[0023] In various embodiments, the nucleic acid molecule comprises (i) a nucleotide sequence encoding the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the nucleotide sequences set forth in SEQ ID Nos. 17-19, respectively, or degenerate variants thereof, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the nucleotide sequence set forth in SEQ ID Nos. 20-22, respectively, or degenerate variants thereof; or (ii) a nucleotide sequence encoding the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the nucleotide sequences set forth in SEQ ID Nos. 23-25, respectively, or degenerate variants thereof, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the nucleotide sequence set forth in SEQ ID Nos. 26- 28, respectively, or degenerate variants thereof.

[0025]

[0024] In various embodiments, the nucleic acid molecule is a vector.

[0026]

[0025] In another aspect there is provided a host cell comprising the nucleic acid molecule disclosed herein.

[0027]

[0026] In various embodiments, the host cell has been transfected, transduced or transformed by the nucleic acid molecule.

[0028]

[0027] In various embodiments, the nucleic acid molecule encodes the chimeric antigen receptor disclosed herein, wherein the host cell is engineered to express the chimeric antigen receptor.

[0029]

[0028] In various embodiments, the host cell is a T cell for use in adoptive immunotherapy.

[0030]

[0029] In another aspect there is provided a composition for preventing, treating or ameliorating an HBV infection or a disease associated with HBV infection, in a subject, comprising the antibody disclosed herein, or the antigen-binding fragment disclosed herein, the chimeric antigen receptor disclosed herein, or the nucleic acid molecule disclosed herein, or the host cell disclosed herein; and a pharmaceutically acceptable carrier and / or excipient.

[0031]

[0030] In another aspect there is provided a method of preventing, treating or ameliorating an HBV infection or a disease associated with HBV infection in a subject, comprising administering to the subject a therapeutically effective amount of the antibody disclosed herein, the antigen-binding fragment disclosed herein, the chimeric antigen receptor disclosed herein, the nucleic acid molecule disclosed herein, the host cell disclosed herein, or the composition disclosed herein.

[0032]

[0031] In another aspect there is provided a use of the antibody disclosed herein, the antigen-binding fragment disclosed herein, the chimeric antigen receptor disclosed herein, the nucleic acid molecule disclosed herein, the host cell disclosed herein, or the composition disclosed herein, in the manufacture of a medicament for preventing, treating or ameliorating an HBV infection or a disease associated with HBV infection in a subject.

[0032] In various embodiments, the disease associated with HBV infection comprises hepatitis, liver fibrosis, liver cirrhosis, and liver cancer, preferably HBV-induced hepatocellular carcinoma.

[0033]

[0033] In another aspect there is provided an in vitro method of inhibiting HBV in a sample, comprising contacting the sample with the antibody disclosed herein, the antigen-binding fragment disclosed herein, the chimeric antigen receptor disclosed herein or the host cell disclosed herein, under conditions suitable to form an immune complex.

[0034]

[0034] In another aspect there is provided a method of detecting a HBV infection or a disease associated with HBV, in a subject, comprising: contacting a biological sample that has been obtained from the subject with the antibody disclosed herein, or antigen binding fragment disclosed herein, or the chimeric antigen receptor disclosed herein or the host cell disclosed herein, under conditions sufficient to form an immune complex; and detecting the presence of the immune complex on the biological sample, wherein the presence of the immune complex on the biological sample indicates that the subject has an HBV infection or a disease associated with HBV.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036]

[0035] In the following description, various embodiments of the invention are described with reference to the following drawings.

[0037]

[0036] Figure 1. shows the isolation and identification of HBV-specific memory B cells. (A) Representative gating strategy to identify and isolate live CD19+ lgG+ HBsAg+ memory B cells of a donor with resolved acute HBV infection after magnetic B cell enrichment and incubation with HBsAg- biotin. (B) HBsAg+ gate of B cells of the donor with resolved acute infection. Comparison to a staining control using streptavidin-APC. Depicted cell populations were pre-gated on live CD19+ lgG+ B cells. Frequencies refer to lgG+ cells. (C) Representative ELISpot well images of HBsAg-specific and total IgG. ELISpot was performed after in vitro stimulation of PBMC for five days. (D) Transfection of HEK293 cells with corresponding antibody heavy and light chain plasmids. Secretion of total IgG (detected with polyclonal goat ant-human IgG HRP) and anti-HBs-specific antibodies (on protein G-coated plates, detected with HBsAg-biotin and Avidin-HRP) was determined via ELISA. Medium only served as negative control (neg. Ctrl). Polyclonal HBIg diluted in medium served as a positive control (pos. Ctrl). Mean and SD are shown. Data points represent mean values ± SD from triplicates.

[0038]

[0037] Figure 2. shows the staining of live CD19+ lgG+ B cells with HBsAg on B cells from naTve donor. Frequencies refer to lgG+ cells.

[0039]

[0038] Figure 3. shows the assessment of purity of mAbs 4D06 and 4D08 after Protein G column affinity purification. (A) 5 pg of the mAbs 4D06 or 4D08 was separated with PAGE under reducing and non-reducing conditions and total protein was stained with Coomassie Blue. (B) 0.1 pg of the indicated antibody was separated by PAGE under reducing and non-reducing conditions and wet-blotted to a PVDF membrane. After membrane blocking, antibodies were detected with polyclonal goat anti-human IgG HRP antibodies. Purity was > 95% for both constructs.

[0040]

[0039] Figure 4. shows the characterization of mAbs 4D06 and 4D08. (A) Dot blot analysis of the interaction between mAbs and HBV envelope (HBV env) proteins blotted on a PVDF membrane under reducing or non-reducing conditions. 4D06 and 4D08 served as primary antibodies, goat anti-human IgG HRP was used for detection. (B) Western blot analysis of the interaction of HBV env proteins with mAbs. HBV env proteins were separated by SDS PAGE under reducing conditions and blotted on a PVDF membrane. Env protein detection was performed as described in (A). HB-1 served as a positive control for an antibody recognizing a linear epitope. (C) ELISA analysis of the interaction between mAbs and plate-bound HBsAg; polyclonal goat anti-human IgG HRP antibody was used for detection. ECso values were calculated by nonlinear regression. (D) ELISA analysis for the interaction between mAbs and HBsAg from several HBV geno- and serotypes. HBsAg was captured with HBsAg -specific antibodies from a commercial anti-HBs ELISA kit (DiaSorin, Italy). Binding of biotinylated mAbs 4D06 and 4D08 was detected with Avidin-HRP. (E) HBV uptake assay with differentiated HepG2-NTCP cells inoculated with HBV after pre-incubation with heparin (Hep), HBIg (0.3 IU / well), 4D06 or 4D08 (100 nM). No Ab served as neg. control. Absolute quantification of intracellular HBV cccDNA 24 hours post infection (p.i.). (F) Neutralization assay: Antibodies were pre-incubated with HBV, followed by inoculation of HepG2-NTCP cells with HBV. Absolute quantification of intracellular HBV cccDNA eight days p.i.; ICso determination with non-linear regression. (G) Level of HBeAg in the cell culture supernatant four and eight days p.i.. All data points represent mean values ± SD from triplicates.

[0041]

[0040] Figure 5. shows the neutralization capacity of 4D06 and 4D08 in the background of HDV. Huh7- NTCP cells were inoculated with HDV enveloped with HBV envelope proteins of HBV genotypes A-H after pre-incubation with serial dilutions of 4D06 and 4D08. 6-7 days post infection, cells were fixed and analyzed for HDAg with immunofluorescence assay. IC50 concentration of 4D06 and 4D08 to prevent HDV infection enveloped with HBV envelope proteins A-H were calculated with non-linear regression. Mean and SD of triplicates are shown.

[0042]

[0041] Figure 6. shows the expression and functional characterization of CARs using different S- binders. (A) Schematic representation of CAR constructs. (B) Retroviral transduction rates of primary human CD4+ and CD8+ T cells from two healthy donors for C8-, 4D06-, and 4D08-CARs determined by flow cytometry. (C) The expression levels of C8-, 4D06-, and 4D08-CARs are shown as mean fluorescence intensity (MFI) of HA-tag staining. (D) 1 x105CAR-transduced T cells were cultured on plate-bound HBsAg titrated from 0.01 -1 Ogg / ml. IFNg secretion was measured after 48h by ELISA. Data points represent mean values ± SD from triplicates.

[0042] Figure 7. shows the functional evaluation of CARs from novel binders on HBsAg-expressing target cells. (A) CAR-transduced T cells were co-cultured at indicated effector-to-target (E:T) ratios on HepG2, HepG2-SML, or HepG2.215 cells. IFNg secretion was measured via ELISA after 72h. Data points represent mean values ± SD from triplicates. (B) CAR-transduced T cells were pre-incubated with CellTrace Violet (CTV) and cultured at an E:T ratio of 2:1 on indicated target cells. Proliferation as a measure of CTV decrease was observed by flow cytometry after 72h. Histograms are representatives of duplicates. (C) Intracellular cytokine staining of CAR-transduced T cells co-cultured at an E:T ratio of 2:1 for 48h on indicated target cells (2pg / ml Brefeldin A added after 24h). (D) Polyfunctionality of CAR- transduced T cells derived from intracellular cytokine staining described in (C), indicating single (SP), double (DP), triple (TP), or quadruple positive (QP) populations of CD4+or CD8+CAR-transduced T cells incubated on HepG2-SML cells. (E) xCELLigence cytotoxicity assay of the different CAR- transduced T cells co-cultured at an E:T ratio of 1 :3 for 72h with the indicated target cells. Data points represent mean values from triplicate analyses.

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044]

[0043] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural and logical changes may be made without departing from the scope of the invention. Embodiments described below in context of the adapter and drape component are analogously valid for the respective drape assembly and system, and vice versa. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0045]

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes." “About”, as used herein in connection with numerical values refers to the referenced numerical value ±10% or ±5%.

[0046]

[0045] Conditional language, such as, among others, “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features. Thus, such conditional language is not generally intended to imply that features are in any way required for one or more embodiments.

[0047]

[0046] By "comprising" it is meant including, but not limited to, whatever follows the word "comprising".

[0048] Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0049]

[0047] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0050]

[0048] The present inventors identified and isolated novel, recombinant, and fully human monoclonal antibodies (mAbs) against HBV from single B cells of a patient with a resolved infection recognizing the HBV envelope proteins. In particular, HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ lgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into lgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 which displayed high affinities in a nanomolar range, were found to be highly specific for HBsAg by ELISA, recognized a conformational and a linear epitope, respectively, and showed broad neutralization capacity and reactivity against all major HBV genotypes.

[0051]

[0049] Accordingly, the present invention provides an antibody that is directed against HBV. In various embodiments, the antibody binds to an HBV envelope protein (HBV env), or more particularly binds to a hepatitis B surface antigen (HBsAg). This antibody may be termed as a ‘‘HBV-specific antibody" or an “anti-HBV antibody", which may be interchangeably used, and refer to an antibody that binds to HBV and inhibits the biological activity of HBV.

[0052]

[0050] The term "Hepatitis B virus," as used herein, may be used interchangeably with the term "HBV" and refers to the well-known non-cytopathic, liver-tropic DNA virus belonging to the Hepadnaviridae family. The HBV genome is partially double-stranded, circular DNA with overlapping reading frames. There are four transcripts (that may be referred to herein as "genes" or "open reading frames") based on size, encoded by the HBV genome. These contain open reading frames called C, X, P, and S. The core protein is coded for by gene C (HBcAg). Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of the pre-core (pre-C) protein. The DNA polymerase is encoded by gene P. Gene S is the gene that codes for the surface antigens (HBsAg). The HBsAg gene is one long open reading frame which contains three in frame "start" (ATG) codons resulting in polypeptides of three different sizes called large, middle, and small S antigens, pre-SI + pre-S2 + S, pre-S2 + S, or S. Surface antigens in addition to decorating the envelope of HBV, are also part of subviral particles, which are produced at large excess as compared to virion particles, and play a role in immune tolerance and in sequestering anti-HBsAg antibodies, thereby allowing for infectious particles to escape immune detection.

[0053]

[0051] The term “HBsAg” refers to the hepatitis B surface antigen, more particularly the envelope antigen of hepatitis B virus displaying in the surface of viral infected cells. HBV S / L / M surface proteins are the small, medium, and large surface proteins, which are transcribed and translated from one reading frame and differ from each other by N-terminal part. Accordingly, the large surface antigen comprises a part which is neither present in the medium nor in the small surface antigen, and the medium surface antigen comprises a part which being comprised in the large antigen but not comprised in the small antigen. The small antigen consists of a sequence, which is comprised in the C-terminal part of both the medium and the large antigen. It’s presumed that this occurs although the virus is released into intracellular vesicles because numbers of HBV surface proteins (HBsAg) remain integrated into the intracellular membrane of the endoplasmic reticulum. During vesicle transport processes said intracellular membrane may fuse with the cellular membrane, the consequence being that HBV surface proteins are displayed on the surface of the infected cells. Therefore, HBsAg provides an ideal target for virus aimed immunotherapeutic intervention to treat hepatitis B virus infection and any associated conditions.

[0054]

[0052] In various embodiments, the antibody may bind to HBV. The term “bind", as used herein, means that an antibody binds to its target, i.e. HBV envelope, based on recognition of an antigen or epitope on the target molecule. The antibody recognizes and binds to the target molecule HBV with a binding affinity or avidity or specificity that is higher than that for other compounds that may be present. As used herein, the term “affinity” refers to the strength of binding of an antibody to an antigen, at a single site, whereas the term “avidity” refers to the combined strength of multiple binding sites between two structures, such as between multiple antigen binding sites of antibodies simultaneously interacting with a target or e.g. between an antibody and HBV. When more than one binding interactions are present, the two structures will only dissociate when all binding sites dissociate, and thus, the dissociation rate will be slower than for the individual binding sites, and thereby providing a greater effective total binding strength (avidity) compared to the strength of binding of the individual binding sites (affinity).

[0055]

[0053] In various embodiments, the antibody binds to the target molecule with at least about a 106-f old greater affinity, preferably at least about a 107-fo Id greater affinity, more preferably at least about a 108- fold greater affinity, and most preferably at least about a 109-fold greater affinity than it binds molecules unrelated to the target molecule. In various embodiments, the antibody may •‘specifically bind” to HBV. Typically, specific binding refers to affinities in the range of about 106-fold to about 109-fold greater than non-specific binding. In various embodiments, specific binding may be characterized by affinities greater than 1 OMold over non-specific binding. The binding affinity may be determined by any suitable method. Such methods are known in the art and include, without limitation, surface plasmon resonance and isothermal titration calorimetry. In various embodiments, the antibody may uniquely recognize and bind to the target molecule, in particular, with regard to the representative antibodies disclosed herein, termed 4D06 and 4D08, a higher affinity was observed for 4D06 in comparison to 4D08. Interestingly, a higher affinity observed for 4D06-mAb in comparison to 4D08-mAb correlated with a stronger neutralization capacity in an HBV infection assay, where avidities for both constructs were very similar, and approximately 3-fold higher than C8-CAR (amino acid sequence set forth in SEQ ID NO: 41 and corresponding nucleotide sequence set forth in SEQ ID NO: 42 encoding the same). Accordingly, in various embodiments, the binding affinity of the antibody disclosed herein is at least equal, or superior to the antibody relating to C8-CAR, preferably tested according to the protocols described in the Examples section of the present application.

[0056]

[0054] The term "antibody", as used herein, refers to a protein consisting of one or more polypeptide chains substantially encoded by all or part of the immunoglobulin genes. Known immunoglobulin genes, for example in humans, include the kappa (K), lambda (X), and heavy chain genetic loci, which together comprise the multitude of variable region genes, and the constant region genes mu (p), delta (5), gamma (y), epsilon (c), and alpha (a) which encode the IgM, IgD, IgG (lgG1 , lgG2, lgG3, and lgG4), IgE, and IgA (lgA1 and lgA2) isotypes respectively. The term "antibody”, as used herein is meant to include full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes. The terms “antibody” and “immunoglobulin” are used interchangeably herein to relate to polypeptides encoded by immunoglobulin genes. By "IgG" as used herein is meant a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this class comprises lgG1 , lgG2, lgG3, and lgG4. In various embodiments the antibody of the invention is an IgG antibody, for example an IgG 1 antibody or an IgG 1 kappa antibody. Such an antibody typically comprises two identical heavy chains and two identical light chains, both having the Ig domains detailed below.

[0057]

[0055] By "immunoglobulin (Ig) domain" herein is meant a region of an immunoglobulin that exists as a distinct structural entity as ascertained by one skilled in the art of protein structure. Ig domains typically have a characteristic p-sandwich folding topology. The known Ig domains in the IgG class of antibodies are VH, Cy1 , Cy2, Cy3, VL, and CL. As detailed above, VH and VL refer to the variable regions of the heavy (VH) and light (VL) chain and are herein defined by reference to their amino acid sequence. Cy1 , Cy2, and Cy3 refer to the Ig domains of the constant part of the heavy chain, i.e. the domains more generally referred to as CH1 , CH2 and CH3. The N-terminus of the CH1 or Cy1 domain is linked to the C-terminus of the VH domain. CL relates to the constant part of the light chain and is linked to the C- terminus of the VL domain. The linkage is typically by a peptide bond. The full light chain thus comprises in N- to C-terminal orientation a VL and a CL domain. The full heavy chain thus comprises in N- to C- terminal orientation a VH, CH1 , CH2 and CH3 domain. An IgG antibody comprises two full light chains and two full heavy chains.

[0058]

[0056] In various embodiments, the antibody may be an IgG immunoglobulin, preferably an lgG1 immunoglobulin. In various embodiments, the antibody comprises a IgG constant region within a heavy chain of the immunoglobulin and a IgG constant region within a light chain of the immunoglobulin.

[0059]

[0057] In various embodiments, the antibody may be a human antibody, a monoclonal antibody, a recombinant antibody, a polyclonal antibody, a chimeric antibody, a humanised antibody, a bispecific antibody, a multispecific antibody, a domain antibody, a synthetic antibody, a hetero-conjugate antibody, or a purified antibody, preferably in various embodiments, the antibody may be a human monoclonal antibody. In various embodiments, the antibody may be a minibody. Minibodies are minimized antibodylike proteins comprising a scFv joined to a CH3 domain. In some cases, the scFv can be joined to the Fc region, and may include some or all of the hinge region, the antibodies may be a variety of structures, including, but not limited to antibody fragments.

[0060]

[0058] In various embodiments, the antibodies of the invention may be antibody "fusion proteins", sometimes referred to herein as "antibody conjugates". The fusion partner or conjugate partner can be proteinaceous or non-proteinaceous; the latter generally being generated using functional groups on the antibody and on the conjugate partner. Conjugate and fusion partners may be any molecule, including small molecule chemical compounds and polypeptides. For example, a variety of antibody conjugates and methods are described in Trail et al., 1999, Curr. Opin. Immunol. 1 1 :584-588. Possible conjugate partners include but are not limited to cytokines, cytotoxic agents, toxins, radioisotopes, chemotherapeutic agent, anti-angiogenic agents, a tyrosine kinase inhibitors, and other therapeutically active agents. In various embodiments, conjugate partners may be considered payloads, that is to say that the goal of a conjugate is targeted delivery of the conjugate partner to a targeted cell, for example an immune cell, by the antibody. Thus, for example, the conjugation of a toxin to an antibody targets the delivery of the toxin to cells expressing the target antigen. As will be appreciated by one skilled in the art, in reality the concepts and definitions of fusion and conjugate are overlapping. The designation of an antibody as a fusion or conjugate is not meant to constrain it to any particular embodiment of the present invention. Rather, these terms are used loosely to convey the broad concept that any antibody of the present invention may be linked genetically, chemically, or otherwise, to one or more polypeptides or molecules to provide some desirable property. Suitable conjugates include, but are not limited to, labels as described below, drugs and cytotoxic agents including, but not limited to, cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or active fragments of such toxins.

[0061]

[0059] In various embodiments, an antibody of the present invention may be fused, conjugated, or operably linked to a radioisotope to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugate antibodies. Examples include, but are not limited to, At211 , 1131 , 1125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu.

[0062]

[0060] The variable region of an antibody contains the antigen binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The variable region is so named because it is the most distinct in sequence from other antibodies within the same class. In the variable region, three loops are gathered for each of the variable (V) domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a "CDR"), in which the variation in the amino acid sequence is most significant and which typically accounts for the binding specificity and affinity of the antibody. There are 6 CDRs total, three each per heavy and light chain, designated VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2, and VL CDR3. The region of the variable domain outside of the CDRs is referred to as the framework region (FR). Although not as diverse as the CDRs, sequence variability does occur in the FR region between different antibodies. Overall, this characteristic architecture of antibodies provides a stable scaffold (the FR region) upon which substantial antigen binding diversity (the CDRs) can be explored by the immune system to obtain specificity for a broad array of antigens. A number of high- resolution structures are available for a variety of variable region fragments from different organisms, some unbound and some in complex with antigen. Sequence and structural features of antibody variable regions are disclosed, for example, in Morea et al., 1997, Biophys Chem 68:9-16; Morea et al., 2000, Methods 20:267- 279, and the conserved features of antibodies are disclosed, for example, in Maynard et al., 2000, Annu Rev Biomed Eng 2:339-376.

[0063]

[0061] By "variable region" as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the VL (VK, VX) and / or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively. By ‘‘constant region” of an antibody as defined herein is meant the region of the antibody that is encoded by one of the light or heavy chain immunoglobulin constant region genes. By "constant light chain" or "light chain constant region" as used herein is meant the region of an antibody encoded by the kappa (C,) or lambda (CL) light chains. The constant light chain typically comprises a single domain, and as defined herein refers to positions 108-214 of C. or lambda CL, wherein numbering is according to the EU index. An lgG1 kappa antibody thus comprises the kappa light chain constant region. By "constant heavy chain" or "heavy chain constant region" as used herein is meant the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or Ig E, respectively For full length IgG antibodies, the constant heavy chain, as defined herein, refers to the N- terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 118-447, wherein numbering is according to the EU index.

[0064]

[0062] In various embodiments, the antibody of the invention comprises a heavy chain and a light chain. In various embodiments, the antibody comprises a constant region within a heavy chain of the antibody and a constant region within a light chain of the antibody. The antibody may comprise a framework region within the variable domain of said heavy chain and within the variable domain of said light chain as defined by the amino acid sequences set forth herein. The remaining parts of the antibody, namely the constant regions of the heavy and light chain can be selected by those skilled in the art based on their common general knowledge.

[0065]

[0063] The antibodies of the invention may comprise a heavy chain and a light chain variable region, the heavy chain variable region comprising a VH CDR1 , a VH CDR2, and a VH CDR3 region, and the light chain variable region comprising a VL CDR1 , a VL CDR2, and a VL CDR3.

[0066]

[0064] In various embodiments, the VH CDR1 comprises, or consists of the amino acid sequence set forth in SEQ ID NO:1 ; said VH CDR2 comprises, or consists of the amino acid sequences set forth in SEQ ID NO:2; and said VH CDR3 comprises, or consists of the amino acid sequence set forth in SEQ ID NO:3; and said VL CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:4; said VL CDR2 comprises, or consists of the amino acid sequence set forth in SEQ ID NO:5; said VL CDR3 comprises, or consists of the amino acid sequence set forth in SEQ ID NO:6.

[0067]

[0065] In various embodiments, the antibody may comprise a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID NOs. 1-3, respectively, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID NOs. 4-6 , respectively, wherein the antibody binds to Hepatitis B virus (HBV), more preferably binds to an HBV surface antigen (HBsAg).

[0068]

[0066] In various embodiments, the heavy chain variable domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:13 or a variant thereof, and the light chain variable domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:14 or a variant thereof.

[0069]

[0067] In various embodiments, the VH CDR1 comprises, or consists of the amino acid sequence set forth in SEQ ID NO:7; said VH CDR2 comprises, or consists of the amino acid sequences set forth in SEQ ID NO:8; and said VH CDR3 comprises, or consists of the amino acid sequence set forth in SEQ ID NO:9; and said VL CDR1 comprises, or consists of the amino acid sequence set forth in SEQ ID NO:10 said VL CDR2 comprises, or consists of the amino acid sequence set forth in SEQ ID NO:11 ; said VL CDR3 comprises, or consists of the amino acid sequence set forth in SEQ ID NO:12.

[0070]

[0068] In various embodiments, the antibody may comprise a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID NOs. 7-9, respectively, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID NOs. 10-12, respectively, wherein the antibody binds to Hepatitis B virus (HBV), more preferably binds to an HBV surface antigen (HBsAg).

[0071]

[0069] In various embodiments, the heavy chain variable domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:15 or a variant thereof, and the light chain variable domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:16 or a variant thereof.

[0072]

[0070] The variant may represent a functional variant of the VL or VH of the antibody disclosed herein, and will still allow the antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity / avidity and / or the specificity / selectivity of the parent antibody (i.e. 4D06 or 4D08 antibody) and in some cases such an antibody may be associated with greater affinity, selectivity and / or specificity than the parent Ab.

[0073]

[0071] In various embodiments, the variants of the amino acid sequences of the heavy and light chain variable domains as defined by the amino acid sequences set forth in SEQ ID Nos. 13-16 are contemplated. Said variants may be invariable with respect to the CDR regions as defined above, i.e. any variation occurs in the framework region. Said variants, in various embodiments, share at least 80%, preferably at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences set forth in SEQ ID Nos. 13-16. The variations, typically amino acid substitutions or deletions, occur in the framework region and not in the CDRs. Possible are for example short truncations on the C- or N-terminus of the variable domain, typically 1 , 2, 3 or 4 amino acids in length and / or single amino acid substitutions.

[0074]

[0072] By "amino acid" and "amino acid identity" as used herein is meant one of the 20 naturally occurring amino acids or any non-natural analogues that may be present at a specific, defined position. Thus "amino acid" as used herein is both naturally occurring and synthetic amino acids. For example, homophenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chain may be in either the (R) or the (S) configuration. In preferred embodiments, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, such non-amino acid substituents may be used, for example, to prevent or retard in vivo degradation. Amino acid or nucleic acid identity can easily be determined using alignments and methods well established in the field. For example, determination of the sequence identity of nucleic acid or amino acid sequences can be done by a sequence alignment based on well-established and commonly used BLAST algorithms (See, e.g. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410, and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): "Gapped BLAST and PSI- BLAST : a new generation of protein database search programs"; Nucleic Acids Res., 25, S.3389-3402). Such an alignment is based on aligning similar nucleotide or amino acid sequences stretches with each other. Another algorithm known in the art for said purpose is the FASTA algorithm. Alignments, in particular multiple sequence comparisons, are typically done by using computer programs. Commonly used are the Clustal series (See, e.g., Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31 , 3497-3500), T-Coffee (See, e.g., Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217) or programs based on these known programs or algorithms. Also possible are sequence alignments using the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA, USA) with the set standard parameters, with the AlignX module for sequence comparisons being based on the ClustalW. If not indicated otherwise, the sequence identity is determined using the BLAST algorithm. Such a comparison allows determination of the similarity of the compared sequences. Said similarity is typically expressed in percent identify, i.e. the portion of identical nucleotides / amino acids at the same or corresponding (in an alignment) sequence positions relative to the total number of the aligned nucleotides / amino acids. For example, if in an alignment 90 amino acids of a 100 aa long query sequence are identical to the amino acids in corresponding positions of a template sequence, the sequence identity is 90%. If not indicated otherwise, the “sequence identity” relates to the entire length of the aligned sequence.

[0075]

[0073] In various embodiments, the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID NOs. 1-3, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID NOs. 4-6, and wherein the heavy chain variable domain (VH) comprises or consists of an amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof, and a light chain variable domain (VL) comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID NOs. 1-6 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0076]

[0074] In various embodiments, the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID NOs. 7-9, respectively, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID NOs. 10-12, respectively, and wherein the heavy chain variable domain (VH) comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof, and a light chain variable region (VL) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID Nos. 7-12 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0077]

[0075] In the above embodiments, “comprises" means that the heavy and / or light chain variable domain can comprise additional amino acids on their C- or N-terminus. Typically, these extensions are however only 1 -10 amino acids in length, for example 1 , 2, 3, 4, ,5, 6, 7 or 8 amino acids in length. In various embodiments, the above heavy chain variable domains consist of the indicated amino acid sequence, i.e. do not comprise any C- or N-terminal extensions. In various embodiments, this similarly or alternatively applies to the light chain variable domain.

[0078]

[0076] In various embodiments, the antibody disclosed herein may be a neutralising antibody, which refers to the antibody disclosed herein having the capability of viral neutralization and inhibiting uptake of HBV. Virus neutralization and uptake inhibition can efficiently prevent HBV infection. The term “neutralizes” as used throughout the present specification in the context of HBV neutralisation means that the biological activity and expression of HBV is reduced or inhibited in the presence of the antibody as described herein in comparison to the activity and expression of HBV in the absence of the antibody , in vitro or in vivo. The reduction or inhibition in biological activity may be partial or total. A neutralising antibody may neutralise the activity of HBV by at least 20%, 30% 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% relative to HBV activity in the absence of the antibody. Neutralisation may be determined or measured using one or more assays known to the skilled person or as described herein.

[0079]

[0077] Both exemplified mAbs 4D06 and 4D08 disclosed herein showed a concentration-dependent neutralization capacity and recognized various HBV geno- and serotypes. 4D06 was slightly superior to 4D08, as a 10-fold lower concentration was needed to achieve complete neutralization. This may be due to the recognition of a conformational epitope since it was reported that mAbs that recognize conformational epitopes are more potent in neutralization than antibodies that detect linear epitopes24. In this regard, the antibody disclosed herein may possess a broad neutralization property in that they are capable of neutralizing more than one HBV geno- and serotype. Broadly neutralizing antibodies recognize epitopes in conserved regions of HBsAg, which are less affected by amino acid substitutions introduced upon virus mutation but rather by the virus origin. As HBV comes in nine genotypes (A-l) and four serotypes (adw, ayw, adr, ayr)21, broadly neutralizing antibodies, such as 4D06 and 4D08, are advantageous to combat HBV around the globe.

[0080]

[0078] Accordingly, in various embodiments, the antibody disclosed herein may have the capability to neutralize at least one HBV genotype and / or serotype. In various embodiments, the antibody disclosed herein may have the capability to neutralize at least two, three, four, five, six, seven or eight HBV genotype and / or at least two or three serotypes. In various embodiments, the antibody disclosed herein may have the capability to neutralize HBV genotypes A-H and / or serotypes adw, ayw, and adr. In various embodiments, the antibody disclosed herein may have the capability to neutralize all nine HBV genotypes (A-l) and / or all HBV serotypes (adw, ayw, adr, ayn.

[0081]

[0079] The present inventors also found that the higher binding affinity of the representative antibodies disclosed herein, termed 4D06 and 4D08, also correlated with a stronger neutralization capacity in an HBV infection assay. Accordingly, in various embodiments, the antibody disclosed herein relating to 4D06 is at least equal, or superior in neutralization capacity to the antibody disclosed herein relating to 4D08.

[0080] In various embodiments, the antibody disclosed herein binds to the HBV envelope protein. In various embodiments, the antibody disclosed herein binds to a HBsAg within the HBV envelope protein. In various embodiments, the antibody disclosed herein is capable of recognizing and binding to one or all of the the large (L), middle(M), or small (S) HBsAg antigens, and more particularly the pre-SI + pre- S2 + S, pre-S2 + S, or S. In various embodiments, the antibody disclosed herein is capable of recognizing and binding to the S domain in the HBV envelope, and as a consequence the antibody disclosed herein is capable of recognizing and binding to the L, M and S domains of the HBV envelope.

[0082]

[0081] The detection of a particular epitope is important for the potential of antibodies to neutralize HBV infection. Most published neutralizing HBsAg-specific mAbs recognize conformational epitopes in the a-determinant of HBsAg, which is the most important antigenic region in the S-domain of the HBVenv proteins19,23. Epitope characteristics of the exemplified antibodies 4D06 and 4D08 varied, where 4D06 only recognized the native protein, suggesting a conformational epitope formed by different amino acids across the protein. In contrast, 4D08 could recognize both the native and the reduced form of the small HBVenv protein, pointing towards a linear epitope.

[0083]

[0082] Accordingly, in various embodiments, the antibody disclosed herein binds to an a-determinant epitope, a confirmational epitope, or a linear epitope in the HBV envelope protein, more particularly the HBsAg. In various embodiments, the antibody disclosed herein binds to a confirmational epitope, or a linear epitope in the HBsAg.

[0084]

[0083] References to "VH" or "VH" refer to the variable region or domain of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab. References to "VL" or "VL" refer to the variable region or domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

[0085]

[0084] Accordingly, another aspect of the invention therefore also provides an antigen-binding fragment of the antibody disclosed herein.

[0086]

[0085] The term “antigen-binding fragment” refers to one or more portions of a full-length antibody, said portion retaining the ability to bind an antigen (e.g., HBsAg) in competition with the intact antibody for specific binding to the antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments can be generated by recombinant DNA technology or by enzymatic or chemical breakage of intact antibodies. In various embodiments, an antigen-binding fragment includes Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies comprising at least a portion of an antibody sufficient to confer peptide-specific antigen-binding ability. The antigen-binding fragment of the antibody (e.g., the antibody fragment described above) may be obtained from a given antibody (e.g., monoclonal antibody 4D06 or 4D08) using conventional techniques known to those of skill in the art (e.g., recombinant DNA technology or enzymatic or chemical breakage methods) and the antigen-binding fragment of the antibody may be screened for specificity in the same manner as for intact antibodies. The term "Fd fragment" refers to an antibody fragment comprising the VH and CH1 structural domains; the term "Fv fragment" refers to an antibody fragment comprising the VL and VH structural domains of a single arm of the antibody; the term "dAb fragment" refers to an antibody fragment comprising the VH domain (Ward et al., Nature 341 :544-546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 structural domains of the antibody; "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

[0087]

[0086] Techniques described for the production of single chain antibodies (U. S. Patent No. 4,946,778; Bird (1988), Science 242: 423-26; Huston, et al. (1988), Proc. Natl. Acad. Sci. USA, 85: 5879-83; and Ward, et al. (1989), Nature, 334: 544-46) can be adapted to produce gene-single chain antibodies comprising the heavy chain varialbe domain and the light chain variable domain of the invention. Single chain antibodies are typically formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

[0088]

[0087] In various embodiments, the antigen binding fragment may also refer to an antibody fragment that recognize specific epitopes, such as the epitopes of HBV described herein, and may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') 2 fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')2 fragments. The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

[0089]

[0088] In various embodiments, the antigen binding fragment may include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) the Fv fragment consisting of the VL and VH domains of a single antibody, (iii) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (iv) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site, (v) bispecific single chain Fv dimers and (vi) "diabodies" or "triabodies", multivalent or multispecific fragments constructed by gene fusion. In all these antibody variants, the CDR regions as defined herein may be invariable and at least one heavy chain variable region and at least one light chain variable region, as defined herein, are comprised.

[0089] In various embodiments, the antigen-binding fragment may always include the heavy chain and light chain variable domains as disclosed herein or may include the VH and VL CDR regions as disclosed herein.

[0090]

[0090] In various embodiments, the antigen-binding fragment may comprise or consist of the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID NOs. 1-3, respectively, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID NOs. 4-6, respectively.

[0091]

[0091] In various embodiments, the antigen-binding fragment may comprise or consist of the heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:13 or a variant thereof, and / or the light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:14 or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID NOs. 1-6 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0092]

[0092] In various embodiments, the antigen-binding fragment may comprise or consist of the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID NOs. 7-9, respectively, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID NOs. 10-12, respectively.

[0093]

[0093] In various embodiments, the antigen-binding fragment may comprise or consist of the heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:15 or a variant thereof, and / or the light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO:16 or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID NOs. 7-12 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0094]

[0094] In various embodiments, the antigen-binding fragment may be a Fv, a Fab, a Fab’, a F(ab’)2, scFv or a scFv2 fragment. In various embodiments, the antigen-binding fragment may be scFv fragment. In various embodiments, the scFv fragment may comprise VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID NOs. 1-3, respectively, and VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID NOs. 4-6 , respectively; or the scFv fragment may comprise the VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID NOs. 7-9, respectively, and the VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID NOs. 10-12, respectively.

[0095] In various embodiments, the scFv fragment may comprise or consist of the amino acid sequence set forth in SEQ ID NO:55 and 56, or 57 and 58, or variants thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID NOs. 1-12 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0095]

[0096] In various embodiments, the scFv fragment may comprise or consist of the amino acid sequence set forth in SEQ ID NO:33 or 34, or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID NOs. 1-12 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0096]

[0097] The antigen-binding fragment may be modified. For example, the molecules may be stabilized by the incorporation of disulfide bridges linking the VH and VL domains. Examples of antibody formats and architectures are described in Holliger & Hudson, 2006, Nature Biotechnology 23(9) :1 126- 1 136, and Carter 2006, Nature Reviews Immunology 6:343-357.

[0097]

[0098] The exemplified antibodies (mAb-4D06 and mAb-4D08) and antigen-binding fragment disclosed herein (e.g. scFv) were used to generate chimeric antigen receptors (CAR) and constructs thereof, for use in adoptive T cell therapy of hepatitis B virus infection or HBV-induced hepatocellular carcinoma. In particular, these antibodies (mAb-4D06 and mAb-4D08) were cloned as scFvs into a 2ndgeneration CAR format with intracellular CD28 and CD3zeta signalling domains and compared to the existing C8-CAR in terms of expression and functionality on HBsAg and HBV-expressing target cells. More particularly, the antibodies 4D06 and 4D08 variable chain fragments were cloned into a 2ndgeneration CAR format with CD28 and CD3zeta intracellular signalling domains. As known in the art, the term “2ndgeneration CAR format” as used herein refers to the advancement of CAR cell therapy from the 1stgeneration format (i.e. including a single signaling domain), where the 2ndgeneration format includes one costimulatory domain in addition to the signaling domain, which is intended to enhances T cell activation, proliferation, survival, and cytokine production.

[0098]

[0099] Interestingly, the exemplified CAR constructs disclosed herein (derived from mAb-4D06 and mAb-4D08) displayed a higher functional avidity despite lower expression levels on primary human T cells than a previously generated CAR recognizing the HBV envelope proteins (i.e. C8-CAR: SEQ ID NO:41 ). All CAR-T cells proved to be polyfunctional regarding cytokine secretion and killed HBV- positive target cells with similar efficacy. Further, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently the lowest. This data disclosed herein demonstrates an efficient and fast approach to identifying pathogen -specific monoclonal human antibodies from small donor cell numbers for subsequent CAR generation. Moreover, a lower unspecific activation for the 4D08-CAR was observed. This constitutive signal, known as tonic signaling, is considered pivotal to CAR specificity and CAR-T cell function as it predisposes the cells to exhaustion25. Based on the experimental data described herein, in particular the distinct polyfunctionality profile of 4D08-CAR versus 4D06- and C8-CAR, it may be hypothesized that binding of a linear epitope could be one of the contributing factors to lower tonic signaling and may be beneficial for CAR functionality. In addition, antibodies recognizing a linear epitope on the small HBVenv protein may be advantageously potent against escape mutations.

[0099]

[0100] Accordingly, another aspect of the invention therefore relates to a chimeric antigen receptor comprising of an antigen-binding fragment (e.g. scFv) of the antibody disclosed herein.

[0100]

[0101] The term “chimeric antigen receptor” or “CAR”, as used herein has its general meaning in the art and refers to an artificially constructed hybrid protein or polypeptide containing the antigen binding domains of an antibody (e.g., scFv) linked to T- cell signaling domains. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains. Typically, said chimeric antigen receptor comprises at least one VH and / or VL sequence of the antibody disclosed herein.

[0101]

[0102] In various embodiments, the antigen binding fragment comprised in the CAR, may be formed by a single peptide chain, for example in the case of a single chain variable fragment (scFv). It should be noted that even in such case wherein only a single peptide or peptide chain is involved in forming the antigen binding fragment, the terms “VL” and “VH" may stili be used to refer to the regions corresponding to (or derived from) the VL and VH of the antibody disclosed herein.

[0102]

[0103] In various embodiments, the antigen-binding fragment of the CAR may comprise or consist of a single chain variable fragment (scFv) of the antibody disclosed herein

[0103]

[0104] The term “scFv” as used herein refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable domains, or fragments of these VL and VH variable regions comprising the CDRs, in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. In various embodiments, the linker may be a glycine-serine (GS) linker.

[0104]

[0105] In various embodiments, the antigen-binding fragment of the CAR may be a scFv fragment comprising the CDRs of the VH and the VL domains of the antibody disclosed herein.

[0106] In various embodiments, the antigen binding fragment of the CAR is part of an extraceilular domain (ED) for antigen binding, that is the chimeric antigen receptor comprises an extracellular domain comprising the antigen binding fragment, or more particularly the CDRs of the VH and the VL domain of the antibody disclosed herein.

[0105]

[0107] In various embodiments, the chimeric antigen receptor comprises the VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID NOs. 1-3, respectively, and / or the VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID NOs. 4-6, respectively.

[0106]

[0108] In various embodiments, the chimeric antigen receptor may comprise a scFv fragment comprising the amino acid sequence set forth in SEQ ID NO:55 and 56, or variants thereof, wherein the variants are invariable with respect to the CDR regions of SEQ ID NOs. 1-6 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0107]

[0109] In various embodiments, the chimeric antigen receptor may comprise a scFv fragment comprising or consisting of the amino acid sequence set forth in SEQ ID NO:33, or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID NOs. 1-6 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0108]

[0110] In various embodiments, the chimeric antigen receptor comprises the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID NOs. 7-9, respectively, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID NOs. 10-12, respectively.

[0109]

[0111] In various embodiments, the chimeric antigen receptor may comprise a scFv fragment comprising the amino acid sequence set forth in SEQ ID NO:57 and 58, or variants thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID NOs. 7-12 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0110]

[0112] In various embodiments, the chimeric antigen receptor may comprise an scFv fragment comprising or consisting of the amino acid sequence set forth in SEQ ID NO:34, or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID NOs. 7-12 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0113] The chimeric antigen receptor also comprises a transmembrane domain (TD), and one or more intracellular signalling domains(ID), whereby the extracellular domain is joined to the transmembrane domain which is subsequently joined to the intracellular domains, in order (e.g. ED-TD-ID).

[0111]

[0114] The intracellular signaling domain of the CAR may include one or more intracellular T cell signaling domains responsible for activation of at least one of the normal effector functions of a T cell in which the CAR is expressed or placed within. Exemplary T cell signaling domains are provided herein, and are known to the person of ordinary skill in the art. While an entire intracellular T cell signaling domain can be employed in a CAR, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular T cell signaling domain may be used, such truncated portion may be used in place of the intact chain as long as it transduces the relevant T cell effector function signal. Examples of intracellular T cell signaling domains for use in the CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-stimulatory molecules that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. T cell receptor signaling domains regulate primary activation of the T cell receptor complex either in a stimulatory way, or in an inhibitory way. The disclosed CARs can include primary cytoplasmic signaling sequences that act in a stimulatory manner, which may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences that can be included in a disclosed CAR include those from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d proteins.

[0112]

[0115] In various embodiments, the intracellular domain may comprise or consist of two signalling domains. In various embodiments, the intracellular domain may comprise or consist of a CD28 and a CD3zeta signalling domain. In various embodiments, the intracellular domain may comprise or consist of an amino acid sequence set forth in SEQ ID NO:47, or a variant thereof, wherein the variants share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0113]

[0116] With respect to the transmembrane domain, the CAR may be designed to comprise a transmembrane domain that is joined to the extracellular domain and intracellular domain of the CAR. In various embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR may be used. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane -bound or transmembrane protein. Exemplary transmembrane domains for use in the disclosed CARs can include at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.

[0117] In various embodiments, the transmembrane domain may comprise or consist of a CD28 transmembrane domain. In various embodiments, the transmembrane domain may comprise or consist of an amino acid sequence set forth in SEQ ID NO:49, or a variant thereof, wherein the variants share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0114]

[0118] In various embodiments, the extracellular domain may comprise or consist of a hinge region (HR), a linker and a tag, in addition to the antigen binding fragment.

[0115]

[0119] In various embodiments, the linker may be a GS linker comprising or consisting of an amino acid sequence set forth in SEQ ID NO:43, or a variant thereof, wherein the variants share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0116]

[0120] The “hinge region” as used herein is located between the antigen binding fragment of the extracellular domain and the transmembrane domain. A hinge region refers to an amino acid sequence that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular domain relative to the transmembrane domain may be used. The hinge domain may contain about 10-100 amino acids, e.g., about any one of 15-amino acids, 20-50 amino acids, or 30-60 amino acids. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use.

[0117]

[0121] In various embodiments, the hinge region may comprise or consist of an amino acid sequence set forth in SEQ ID NO:45, or a variant thereof, wherein the variants share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0118]

[0122] The “Tag”, as used herein, relates to a group of atoms or a molecule that is attached covalently to an amino acid sequence for the purpose of detection by an appropriate detection system. The term “tag” and “label” may be used interchangeably. In various embodiments, the tag may be a "HA tag" that refers to an epitope tag derived from the influenza hemagglutinin (HA) protein. In the context of chimeric antigen receptors (CARs), an HA tag would be a short peptide sequence from the HA protein that is incorporated into the CAR construct. In various embodiments, the tag may be a HA tag comprising or consisting of an amino acid sequence set forth in SEQ ID NO:53, or a variant thereof, wherein the variants share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0119]

[0123] In various embodiments, the extracellular domain may further comprise a GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor) leader sequence. In various embodiments, the GM-CSF leader sequence may comprise or consist of an amino acid sequence set forth in SEQ ID NO:51 , or a variant thereof, wherein the variants share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0120]

[0124] In various embodiments, the chimeric antigen receptor may comprise or consist of the amino acid sequence set forth in SEQ ID NO:37, or a variant thereof, wherein the variants are invariable with respect to the CDR regions of SEQ ID NOs. 1-6 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence. In various embodiments, the chimeric antigen receptor may comprise or consist of the amino acid sequence set forth in SEQ ID NO:39, or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID NOs. 7-12 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

[0121]

[0125] In various embodiments, the chimeric antigen receptor may be glycosylated, amidated, carboxylated, phosphorylated, esterif ied, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and / or optionally dimerized or polymerized.

[0122]

[0126] In various embodiments, the chimeric antigen receptor may comprise or consist of: (i) an intracellular domain comprising or consisting of two signalling domains; (ii) a transmembrane domain naturally associated with the signaling domain it is joined to; and (iii) an extracellular domain comprising or consisting of a hinge region, an antigen binding fragment comprising a light and heavy chain variable domain of the antibody disclosed herein, a tag and a leader sequence.

[0123]

[0127] In various embodiments, the chimeric antigen receptor may comprise or consist of: (i) an intracellular domain comprising or consisting of a CD28 and a CD3zeta signalling domain; (ii) a CD28 transmembrane domain; and (iii) an extracellular domain comprising or consisting of an lgG4 hinge region, an scFv fragment comprising the light and heavy chain variable domains comprising the CDR regions of the antibody disclosed herein, wherein the light chain variable domain is contiguously linked via a GS linker to the heavy chain variable domain, a HA tag and a GM-CSF leader sequence.

[0124]

[0128] Another aspect of the invention relates to a nucleic acid molecule encoding the antibody, or the antigen-binding fragment, or the chimeric antigen receptor disclosed herein.

[0125]

[0129] All embodiments disclosed above in relation to the antibody, antigen-binding fragment, or chimeric antigen receptor disclosed herein, similarly apply to the nucleic acid molecule disclosed herein, and vice versa.

[0126]

[0130] The term "nucleic acid (molecule)" as used herein, refers to polymeric forms of nucleotides (polynucleotides) and may be refered to or include nucleotide sequences of any length, including naturally occurring and non-naturally occurring nucleic acids. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and / or their analogs. Methods for selection and preparation of nucleic acids are diverse and well described in standard biomolecular protocols. A typical way would be preparative PCR and chromatographic purification starting from existing template DNAs or stepwise synthesis of artificial nucleic acids. Typically, the nucleic acid molecules referred to herein are DNA molecules.

[0127]

[0131] In this regard, the nucleotide sequence can be DNA molecules or RNA molecules. They can exist as an individual strand, as an individual strand complementary to said individual strand, or as a double strand. With DNA molecules in particular, the sequences of both complementary strands in all three possible reading frames are to be considered in each case. Also to be considered is the fact that different codons, i.e. base triplets, can code for the same amino acids, so that a specific amino acid sequence can be coded by multiple different nucleic acids. As a result of this degeneracy of the genetic code, all nucleic acid sequences that can encode one of the above-described amino acid sequence are included in this subject of the invention. The skilled artisan is capable of unequivocally determining these nucleic acid sequences, since despite the degeneracy of the genetic code, defined amino acids are to be associated with individual codons. The skilled artisan can therefore, proceeding from an amino acid sequence, readily ascertain nucleic acids coding for that amino acid sequence. In addition, in the context of nucleic acids sequences, one or more codons can be replaced by synonymous codons. For example, every organism, e.g. a host cell of a production strain, possesses a specific codon usage. "Codon usage" is understood as the translation of the genetic code into amino acids by the respective organism. Bottlenecks in protein biosynthesis can occur if the codons located on the nucleic acid are confronted, in the organism, with a comparatively small number of loaded tRNA molecules. Also, its codes for the same amino acid, the result is that a codon becomes translated in the organism less efficiently than a synonymous codon that codes for the same amino acid. Because of the presence of a larger number of tRNA molecules for the synonymous codon, the latter can be translated more efficiently in the organism.

[0128]

[0132] By way of methods commonly known today such as, for example, chemical synthesis or the polymerase chain reaction (PCR) in combination with standard methods of molecular biology or protein chemistry, a skilled artisan has the ability to manufacture, on the basis of known DNA sequences and / or amino acid sequences, the corresponding nucleic acids all the way to complete genes. Such methods are known, for example, from Sambrook, J., Fritsch, E. F., and Maniatis, T, 2001 , Molecular cloning: a laboratory manual, 3rd edition, Cold Spring Laboratory Press.

[0129]

[0133] In various embodiments, the nucleic acid molecule encodes the heavy and / or light chain variable domain of the antibody disclosed herein.

[0130]

[0134] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the nucleotide sequences set forth in SEQ ID NOs. 17-19, respectively, or degenerate variants thereof, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the nucleotide sequence set forth in SEQ ID NOs. 20-22, respectively, or degenerate variants thereof.

[0131]

[0135] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the VH CDR1 having the nucleotide sequences set forth in SEQ ID NO. 17; the VH CDR2 having the nucleotide sequences set forth in SEQ ID NO. 18; the VH CDR3 having the nucleotide sequences set forth in SEQ ID NO. 19; the VL CDR1 having the nucleotide sequences set forth in SEQ ID NO. 20; the VL CDR2 having the nucleotide sequences set forth in SEQ ID NO. 21 ; and the VL CDR3 having the nucleotide sequences set forth in SEQ ID NO. 22.

[0132]

[0136] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the nucleotide sequences set forth in SEQ ID NOs. 23-25, respectively, or degenerate variants thereof, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the nucleotide sequence set forth in SEQ ID NOs. 26-28, respectively, or degenerate variants thereof.

[0133]

[0137] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the VH CDR1 having the nucleotide sequences set forth in SEQ ID NO. 23; the VH CDR2 having the nucleotide sequences set forth in SEQ ID NO. 24; the VH CDR3 having the nucleotide sequences set forth in SEQ ID NO. 25; the VL CDR1 having the nucleotide sequences set forth in SEQ ID NO. 26; the VL CDR2 having the nucleotide sequences set forth in SEQ ID NO. 27; and the VL CDR3 having the nucleotide sequences set forth in SEQ ID NO. 28.

[0134]

[0138] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable domain as set forth in SEQ ID NO: 29 or 31 , or a variant thereof.

[0135]

[0139] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the light chain variable domain as set forth in SEQ ID NO: 30 or 32 or a variant thereof.

[0136]

[0140] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable domain as set forth in SEQ ID NO: 29 or 31 , or a variant thereof, and a nucleotide sequence encoding the light chain variable domain as set forth in SEQ ID NO: 30 or 32 or a variant thereof.

[0137]

[0141] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a scFv fragment of the antibody disclosed herein, as set forth in SEQ ID NO: 35 or 36, or a variant thereof.

[0142] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a CAR construct disclosed herein, as set forth in SEQ ID NO: 38 or 40, or a variant thereof.

[0138]

[0143] The term "variant" in the context of a nucleic acid (nucleotide) sequence refers has an altered sequence in which one or more of the nucleotides in the reference sequence is deleted, or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Due to the degeneracy of the genetic code, a "variant" of a nucleotide sequence can either result in a change in the respective reference amino acid sequence, i.e. in an amino acid "sequence variant" or not. In various embodiments, a nucleotide sequence variant does not result in an amino acid sequence variant ( e.g ., a silent mutation). In various embodiments, a nucleotide sequence variant that results in one or more "non-silent" mutations is contemplated. In various embodiments, a nucleotide sequence variant shares at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence set forth in SEQ ID Nos. 29-32, 35, 36, 38 or 40. In various embodiments, a nucleotide sequence variant encodes an amino acid sequence that is at least 80%, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the reference amino acid sequence. Nucleotide and amino sequences as disclosed herein refer also to codon-optimized versions of a reference or wild-type nucleotide or amino acid sequence. In any of the embodiments described herein, nucleotide sequence disclosed herein may be codon-optimized for a host cell containing the nucleotide sequence (see, e.g , Scholten et al., Clin. Immunol. 1 19: 135-145 (2006). Codon optimization can be performed using known techniques and tools. Codon-optimized sequences include sequences that are partially codon- optimized (i.e., at least one codon is optimized for expression in the host cell) and those that are fully codon-optimized. In various embodiments, the variants are invariable with respect to the nucleotide sequences set forth in SEQ ID Nos. 17-28 encoding the CDR regions, or their degenerate variants, and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence

[0139]

[0144] The term "degenerate variant" refers to a nucleotide sequence encoding a protein (for example, an antibody or variable region thereof that specifically binds to the HBV envelope, more particularily an HBsAg) that includes a nucleotide sequence that is degenerate as a result of the genetic code. There are twenty natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the resulting antibody, antigenbinding fragment, or chimeric antigen receptor disclosed herein encoded by the nucleotide sequence remains capable of binding to HBV.

[0140]

[0145] In various embodiments, the nucleic acid molecule may be a vector. "Vectors" are understood for purposes herein as elements - made up of nucleic acids - that contain a nucleic acid sequence contemplated herein as a characterizing nucleic acid region. They enable said nucleic acid to be established as a stable genetic element in a species or a cell line over multiple generations or cell divisions. In particular, when used in bacteria, vectors are special plasmids, i.e. circular genetic elements. Included among the vectors are, for example, those whose origins are bacterial plasmids, viruses, or bacteriophages, or predominantly synthetic vectors or plasmids having elements of widely differing derivations. Using the further genetic elements present in each case, vectors are capable of establishing themselves as stable units in the relevant host cells over multiple generations. They can be present extrachromosomally as separate units, or can be integrated into a chromosome resp. into chromosomal DNA. The term “bacterial plasmid” as used herein refers to a circular DNA molecule capable of replication in a bacterial host cell. A bacterial plasmid may contain an appropriate origin of replication (ori), which is a sequence of DNA sufficient to enable the replication of the plasmid in a host bacterial cell and a bacterial backbone sequence. A bacterial plasmid may also contain a selectable marker sequence within said bacterial backbone sequence, which encodes a selectable marker conferring cellular resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol, and tetracycline.

[0141]

[0146] In various embodiments, the vector may be an expression vector. Expression vectors encompass nucleic acid sequences which are capable of replicating in the host cells. In various embodiments, the vectors described herein thus also contain regulatory elements that control expression of the nucleic acids. Expression is influenced in particular by the promoter or promoters that regulate transcription. Expression can occur in principle by means of the natural promoter originally located in front of the nucleic acid to be expressed, but also by means of a host-cell promoter furnished on the expression vector or also by means of a modified, or entirely different, promoter of another organism or of another host cell. In the present case at least one promoter for expression of a nucleic acid as contemplated herein is made available and used for expression thereof. Expression vectors can furthermore be regulated, for example by way of a change in culture conditions or when the host cells containing them reach a specific cell density, or by the addition of specific substances, in particular activators of gene expression. The expression vector may be based on plasmids well known to person skilled in the art such as pBR322, puC16, pBluescript (RTM) and the like. Thus, the expression vector may be termed as an expression plasmid.

[0142]

[0147] In various embodiments, the vector may be selected from the group of plasmids, binary vectors, DNA vectors, mRNA vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, transposon-based vectors, and artificial chromosomes. In various embodiments, the vector may be a viral vector. The vector essentially is used for delivery of the nucleic acid molecule or construct either in vitro, ex vivo or in vivo.

[0143]

[0148] Accordingly, there is also provided an expression system, or more particularly an expression vector, comprising: (i) a first gene encoding for a light chain variable domain or the complete light chain; and (ii) a second gene encoding for a heavy chain variable domain or the complete heavy chain; wherein the expression system is optionally inducible to express an antibody, antigen-binding fragment, or chimeric antigen receptor disclosed herein. In various embodiments, the expression system may be a cell-based expression system in a host cell.

[0149] In various embodiments, the nucleic acid molecule disclosed herein may be comprised in an “expression construct” which refers to a functional unit built in the vector for the purpose of recombinantly expressing the antibody, antigen-binding fragment, or chimeric antigen receptor disclosed herein, when introduced into an appropriate host cell. In various embodiments, the nucleic acid molecule may comprise or consist of a chimeric antigen receptor expression construct encoding a SvFc fragment disclosed herein. The term "recombinant'1, as used herein (e.g. a recombinant antibody, a recombinant protein, a recombinant nucleic acid, or the like, refers to any molecule (antibody, protein, nucleic acid, or the like) which is prepared, expressed, created or isolated by recombinant means, and which is not naturally occurring. "Recombinant" can be used synonymously with "engineered" or "nonnatural" and can refer to to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene or operon.

[0144]

[0150] Accordingly, another aspect of the invention relates to a host cell comprising the nucleic acid molecule disclosed herein.

[0145]

[0151] All embodiments disclosed above in relation to the antibody, antigenic binding fragment, CAR, and nucleic acid molecule disclosed herein, similarly apply to the host cell, and vice versa.

[0146]

[0152] All cells are in principle suitable as host cells, i.e. prokaryotic or eukaryotic cells. Those host cells that can be manipulated in a genetically advantageous fashion. The term "host cell" as used herein refers to a living ceil into ’which a ONA sequence of interest (i.e. nucleic acid molecule, vector or expression construct) is to be or has been introduced. The living ceil includes both a cultured cell and a cell within a living organism. In various embodiments, host cells can be engineered to incorporate a desired gene or expression construct on its chromosome or in its genome. The host cell may be any cell that is commonly used for expression, i.e. transcription and translation of the nucleic acid molecule for the production of the antibody, antigen-binding fragment, or chimeric antigen receptor disclosed herein. In particular, the term “host cell” relates to prokaryotes, eukaryotes, plants, insect cells or mammalian cells, cell lines and cell culture systems. Host cells include, without limitation, bacterial, microbial, plant or animal cells.

[0147]

[0153] In various embodiments, the host cell is represented by those host cells whose activity can be regulated on the basis of genetic regulation elements that are made available, for example, on the vector, but can also be present a priori in those cells. They can be stimulated to expression, for example, by controlled addition of chemical compounds that serve as activators, by modifying the culture conditions, or when a specific cell density is reached. This makes possible economical production of the proteins contemplated herein.

[0148]

[0154] A nucleic acid molecule disclosed herein or a vector containing said nucleic acid molecule may be transfected, tansduced or transformed into the host cell, which all generally refers to the incorporation of the nucleic acid molecule into the host cell, wherein the nucleic acid molecule may be incorporated into the genome of the host cell ( e.g., chromosome, plasmid, plastid, or mitochondrial DNA), and converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0149]

[0155] In various embodiments, the nucleic acid molecule disclosed herein or a vector containing said nucleic acid molecule may be transformed into the host cell. Methods for the transformation of cells are established in the existing art and are sufficiently known to the skilled artisan. Those host cells that can be manipulated in genetically advantageous fashion, e.g. as regards transformation using the nucleic acid or vector and stable establishment thereof, are preferred, for example single-celled fungi or bacteria. In addition, preferred host cells are notable for being readily manipulated in microbiological and biotechnological terms. This refers, for example, to easy culturability, high growth rates, low demands in terms of fermentation media, and good production and secretion rates for polypeptides. The polypeptides can furthermore be modified, after their manufacture, by the cells producing them, for example by the addition of sugar molecules, formylation, amination, etc. Post-translation modifications of this kind can functionally influence the polypeptide.

[0150]

[0156] In various embodiments, the nucleic acid molecule disclosed herein or a vector containing said nucleic acid molecule may be transduced into the host cell. The term "transduction" in this context is well known in the art and generally refers to the use of a retroviral vector to introduce genetic material into a cell, and optionally the subsequent integration into the genome of the cell via the retroviral vector. In various embodiments, the nucleic acid molecule disclosed herein or a vector containing said nucleic acid molecule may be introduced into the host cell by retroviral transduction.

[0151]

[0157] In various embodiments, the nucleic acid molecule disclosed herein or a vector containing said nucleic acid molecule may be transfected into the host cell, whereby the host cell may be an eukaryotic host cell. The term "transfection" as used herein means the uptake of exogenous or heterologous RNA or DNA by a cell. A cell has been "transfected" by exogenous or heterologous RNA or DNA when such RNA or DNA has been introduced inside the cell. Methods for the transfection of cells are established in the existing art and are sufficiently known to the skilled artisan. In contrast to prokaryotic cells, eukaryotic cells are capable of post-translational ly modifying the protein that is formed. In various embodiments, the eukaryotic host cell is a higher eukaryotic host cell. The term “higher eukaryotic cell’’ as used herein refers to eukaryotic cells that are not cells from unicellular organisms. In other words, a higher eukaryotic cell is a cell from (or derived from, in case of cell cultures) a multicellular eukaryote such as a human cell line or another mammalian cell line. Particularly, the term generally refers to mammalian cells, human cell lines and insect cell lines. More particularly, the term refers to vertebrate cells, even more particularly to mammalian cells or human cells. This may be particularly advantageous, for example, when the polypeptides, in connection with their synthesis, are intended to experience specific modifications made possible by such systems. Among the modifications that eukaryotic systems carry out in particular in conjunction with protein synthesis are, for example, the bonding of low-molecular-weight compounds such as membrane anchors or oligosaccharides.

[0152]

[0158] In various embodiments, the eukaryotic host cell may be a mammalian cell. The mammalian cell can include, but are not limited to a human, simian, murine, mice, rat, monkey, rabbit, rodent, hamster, goat, bovine, sheep or pig cell. In various embodiments, the eukaryotic host cell may be a human cell. In various embodiments, the eukaryotic host cell is a cell from a cell line including, but are not limited to Chinese hamster ovary (CHO) cells, murine myeloma cells such as NSO and Sp2 / 0 cells, COS cells, Hela cells and human embryonic kidney (HEK-293) cells. In various embodiments, the eukaryotic host cell is a human embryonic kidney (HEK-293) cell, more preferably a human Expi293F cell. In particular, Expi293F cells may be used as transgenic master cell lines with modular features as a basis for biopharmaceutical testing / production and innovative therapeutic applications such as transplantable cell-encapsulated mini bioreactors.

[0153]

[0159] In this regard, a host cell may include “transfected cells”, “transduced cells”, or “transformed cells,” which include the primary transformed / transfected / transduced host 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.

[0154]

[0160] In various embodiments, the host cell contemplated herein may be used to manufacture the antibody, antigen-binding fragment, or chimeric antigen receptor disclosed herein.

[0155]

[0161] A further aspect of the invention is therefore a method for manufacturing the antibody, antigen-binding fragment disclosed herein, comprising culturing a host cell disclosed herein; and isolating the antibody, or antigen-binding fragment, from the culture medium or from the host cell. Culture conditions and mediums can be selected by those skilled in the art based on the host organism used by resorting to general knowledge and techniques known in the art. The method may comprise the step of transforming or transfecting the nucleic acid molecule or vector into the host cell at suitable conditions to induce said transformation or transfection.

[0156]

[0162] Once isolated, the antibody, or antigen-binding fragment, can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like. The antibody, or antigen-binding fragment, need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.

[0163] In various embodiments, the host cell may be engineered to express the chimeric antigen receptor disclosed herein. The chimeric antigen receptor may be expressed on a cell surface of the host cell, and the host cell may be used for cellular, and more preferably adoptive, immunotherapy. For this purpose, the host cell may be a mammalian cell, more preferably a human cell. This host cell may be termed herein as a “CAR cell”.

[0157]

[0164] In various embodiments, preparation of trie engineered CAR ceils may include one or more culture and / or preparation steps. The host ceil for introduction of the CAR construct may be isolated from a sample obtained from or derived from a subject. In various embodiments, the subject from which the cell is isolated is one having a disease or condition or in need of cell immunotherapy or to which cell immunotherapy will be administered. The subject in various embodiments is a human in need of a particular therapeutic intervention, such as the adoptive immunotherapy for which ceils are being isolated, processed, and / or engineered.

[0158]

[0165] The “sample” as used herein, generally includes biological samples such as tissues and bodily fluids. “Bodily fluids” may include, but are not limited to, blood, serum, plasma, saliva, cerebral spinal fluid, pleural fluid, tears, lactal duct fluid, lymph, sputum, urine, amniotic fluid, and semen. A sample may include a bodily fluid that is “acellular”. The “biological sample” includes any suitable sample isolated from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and / or incubation. Suitable samples include, but are not limited to, a sample containing tissues, cells, and / or biological fluids isolated from a subject. Examples of biological samples include, but are not limited to, tissues, organ samples, cells, biopsies, autopsies, pathology specimens, blood, lymph, serum, sweat, plasma, urine, saliva, mucus and tears, including processed samples derived therefrom.

[0159]

[0166] In various embodiments, the sample from which the host cell is derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell immunotherapy, e.g., adoptive cell immunotherapy, samples from autologous and allogeneic sources.

[0160]

[0167] In various embodiments, the isolation of the host cell includes one or more preparation and / or non-affinity based cell separation steps. In various embodiments, host cell may be washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In various embodiments, the host cell may be separated based on one or more properties, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0161]

[0168] In various embodiments, the host cell from the circulating blood of a subject may be obtained, e.g., by apheresis or leukapheresis. The samples may contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and may contain cells other than red blood cells and platelets.

[0162]

[0169] In various embodiments, the host cell may be a peripheral blood lymphocyte (PBL), peripheral blood mononuclear ceil (PBMC), T ceil, a NK ceil, or a NK-T cell. In various embodiments, the host cell may be a T cell. The T cel! may be isolated from the peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC) of a subject or a sample obtained therefrom. The T cell may be any T cell, such as a cultured T ceil, e.g., a primary T cell, or a I cell from a cultured T cell line, e.g., Jurkat, SupTI, or a T cell obtained from a mammal. II obtained from a mammal, the I cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. The T cell can be any type of T ceil and can be of any developmental stage, including but not limited to, CD4+ / CD84- double positive T cells, CD4+ helper T cells, e.g., Th2 cells, CD8+ T ceils (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like. The T cell may be a CD8+ T ceil or a CD4+ T ceil.

[0163]

[0170] In various embodiments, the host cell may be a T cell engineered to express the chimeric antigen receptor disclosed herein. The chimeric antigen receptor may be expressed on the surface of the T cell, and used for cellular adoptive immunotherapy.

[0164]

[0171] in various embodiments, the host cell may be a Treg. As used herein, the term“Treg” or “T regulatory cell” denotes a T lymphocyte endowed with a given antigen specificity imprinted by the TCR it expresses and with regulatory properties defined by the ability to suppress the response of conventional T lymphocytes or other immune cells. Such responses are known in the art and include, but are not limited to, cytotoxic activity against antigen-presenting target cells and secretion of different cytokines.

[0165]

[0172] Also provided is a population of cells comprising at least one host cell disclosed herein. The population of cells may be a heterogeneous population comprising the host cell comprising any of the nucleic acid molecule disclosed herein, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the nucleic acid molecule disclosed herein, or a cell other than a T cell. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the nucleic acid molecule disclosed herein. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising the nucleic acid molecule or vector disclosed herein, such that all cells of the population comprise the expression vector. In various embodiments, the population of cells is a clonal population comprising host cells comprising the nucleic acid molecule or vector disclosed herein.

[0166]

[0173] The antibody, antigen-binding fragment, chimeric antigen receptor, nucleic acid molecule, and host cell disclosed herein may be suitable for use in methods of preventing, treating or ameliorating an HBV infection or a disease associated with HBV infection, or for neutralizing the virulence of HBV, in a subject, or detecting a HBV infection in a subject.

[0167]

[0174] All embodiments disclosed above in relation to the antibody, antigenic binding fragment, CAR, nucleic acid molecule, host cell disclosed herein, similarly apply to the methods and uses disclosed herein, and vice versa.

[0168]

[0175] In this regard, for the purpose of brevity, the antibody, antigen-binding fragment, chimeric antigen receptor, nucleic acid molecule, and host cell disclosed herein may be collectively referred to as the “active agent” responsible for eliciting the desired effects once administered to a subject, such that recitation of “active agent” herein may refer to any one of the antibody, antigen-binding fragment, chimeric antigen receptor, nucleic acid molecule, and host cell disclosed herein. In addition, the antibody, antigen-binding fragment, chimeric antigen receptor or host cell expressing the CAR on its surface, as disclosed herein may be collectively referred to as the “binding agent" each being capable of binding to the HBV, such that recitation of “binding agent" herein may refer to any one of the antibody, antigen-binding fragment, chimeric antigen receptor, or host cell expressing the CAR on its surface.

[0169]

[0176] In various embodiments, the HBV infection may be a chronic HBV infection. In various embodiments, the disease associated with HBV infection may include hepatitis, liver fibrosis, liver cirrhosis, and liver cancer, preferably HBV-induced hepatocellular carcinoma. In various embodiments, the hepatitis is hepatitis B, and examples include acute hepatitis, chronic hepatitis, and fulminant hepatitis.

[0170]

[0177] Accordingly, another aspect of the invention therefore relates to a method of preventing, treating or ameliorating an HBV infection or a disease associated with HBV infection, or for neutralizing the virulence of HBV, in a subject. Said method may comprise administering a therapeutically effective amount of the active agent to said subject. Also encompassed is the active agent for use as a medicament, for example, for use in a method of preventing, treating or ameliorating an HBV infection or a disease associated with HBV infection.

[0171]

[0178] As used herein, the terms "treating" and "treatment" refer to reduction in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, prevention of the occurrence of symptoms and / or their underlying cause, and improvement or remediation of damage. As used herein, the term “preventing" refers to the prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented and those in whom reoccurrence of the disorder needs to be prevented. In various embodiments, the terms “treating”, “ameliorating”, “delaying” or “preventing”, as used herein refer to achieving one or more of the following in the subject: (a) reducing the severity of a given condition; (b) limiting or preventing the development of a condition; (c) removing a given condition; (d) limiting or preventing the recurrence of a given condition; (e) alleviation of the condition and / or its symptoms; and (f) delay the onset of a condition. Any one or more of these effects may be achieved in a subject who previously had or currently has or is suspected to develop a glucose metabolism disease or condition. In particular, the therapeutic terms “treating, ameliorating or preventing”, may refer to reducing the likelihood of a particular condition or disease state from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms do not necessarily indicate complete or absolute prevention or treatment. Those in need of such treatment include an individual or patient that has been diagnosed with HBV infection or a disease associated with HBV infection.

[0172]

[0179] As used herein, the term "subject" refer to all mammals, including humans. Examples of subjects include humans, cows, dogs, cats, horses, goats, sheep, pigs, and rabbits, and may be used interchangeably with “patient” or “individual”. In various embodiments, the subject may refer to a warmblooded animal, preferably a mammal, more preferably a human.

[0173]

[0180] Preferably the active agent may be administered to the subject is in a therapeutically effective amount sufficient to inhibit, prevent or reduce HBV infection in one or more anatomical sites, or organs within the subject. As used herein a "therapeutically effective amount" refers to an amount of the active agent that produces the effects for which it is administered, and essentially is capable of inhibiting, reducing, or reducing HBV infection. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques in the art of clinical pharmacology or pharmacokinetics. An effective amount of the active agent to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the subject. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 10 ng to up to 100 mg more per day, preferably about 1 pg to 10 mg per day.

[0174]

[0181] Another aspect of the invention relates to a composition for preventing, treating or ameliorating an HBV infection or a disease associated with HBV infection, or for neutralizing the virulence of HBV, in a subject, comprising the active agent, and a carrier and / or excipient. In various embodiments the composition may be a pharmaceutical composition for use as a medicament.

[0175]

[0182] Formulations of the composition disclosed herein may be prepared for storage by mixing at the active agent, for example the antibody or antigen-binding fragment, at a desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980), for example in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are pharmaceutically acceptable, i.e. nontoxic to recipients, at the dosages and concentrations employed, and include buffers such as 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 orbenzyl 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 other 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; sweeteners and other flavoring agents; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; additives; coloring agents; salt- forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0176]

[0183] In various embodiments, the composition comprising the active agent may be in a water- soluble form, such as comprising certain ingredients in form of pharmaceutically acceptable salts, such as acid and / or base addition salts. The formulations to be used for in vivo administration are typically sterile. This is readily accomplished by known methods.

[0177]

[0184] In various embodiments, where the active agent is an antibody or antigenic fragment thereof, the composition may be formulated as immunoliposomes or in microcapsules. Techniques for preparing such formulations are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.

[0178]

[0185] Administration of the composition in the form of a sterile aqueous solution, may be done in a variety of ways, including, but not limited to orally, subcutaneously, intravenously, intranasally, intraotically, transdermally, topically (e.g., gels, salves, lotions, creams, etc.), intraperitoneally, intramuscularly, intrapulmonary, vaginally, parenterally, rectally, or intraocularly. As is known in the art, the composition may be formulated accordingly depending upon the manner of administration. Protein therapeutics are often delivered by IV infusion or bolus.

[0179]

[0186] The amounts and frequencies of administration are, in various embodiments, selected to be therapeutically or prophylactically effective. As is known in the art, adjustments for protein degradation, systemic versus localized delivery, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0187] In various embodiments, only a single dose of the active agent is used. In other embodiments, multiple doses of the active agent may be administered. The elapsed time between administrations may be less than 1 hour, about 1 hour, about 1 -2 hours, about 2-3 hours, about 3-4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 2-4 days, about 4-6 days, about 1 week, about 2 weeks, or more than 2 weeks. In other embodiments the active agent may be administered in metronomic dosing regimes, either by continuous infusion or frequent administration without extended rest periods. Such metronomic administration may involve dosing at constant intervals without rest periods. Typically such regimens encompass chronic low-dose or continuous infusion for an extended period of time, for example 1 -2 days, 1 -2 weeks, 1 -2 months, or up to 6 months or more. The use of lower doses may minimize side effects and the need for rest periods.

[0180]

[0188] In various embodiments, the active agent and one or more other prophylactic or therapeutic agents are cyclically administered to the patient. Cycling therapy involves administration of a first agent at one time, a second agent at a second time, optionally additional agents at additional times, optionally a rest period, and then repeating this sequence of administration one or more times. The number of cycles is typically from 2 - 10. Cycling therapy may reduce the development of resistance to one or more agents, may minimize side effects, or may improve treatment efficacy.

[0181]

[0189] The active agent of the present invention may be administered concomitantly with one or more other therapeutic regimens or agents. The additional therapeutic regimes or agents may be used to improve the efficacy or safety of the active agent. Also, the additional therapeutic regimes or agents may be used to treat the same disease or a comorbidity rather than to alter the action of the active agent. The terms "in combination with" and "co-administration" are not limited to the administration of the prophylactic or therapeutic agents at exactly the same time. Instead, it is meant that the active agent of the present invention and the other agent or agents are administered in a sequence and within a time interval such that they may act together to provide a benefit that is increased versus treatment with only either the active agent of the present invention or the other agent or agents. In one embodiment, that the active agent and the other agent or agents act additively, in another embodiment they act synergistically. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The skilled medical practitioner can determine empirically, or by considering the pharmacokinetics and modes of action of the agents, the appropriate dose or doses of each therapeutic agent, as well as the appropriate timings and methods of administration.

[0182]

[0190] In various embodiments, such co-administration also requires co-formulation of the active agent in one pharamceutical composition, as defined herein.

[0183]

[0191] In various embodiments, the active agent of the present invention may be administered in combination with one or more additional molecules, such as other antibodies or Fc molecules. The active agent may be co-administered with one or more other antibodies or antigen binding fragments that have efficacy in treating the same disease or an additional comorbidity, for example two antibodies may be administered that recognize two surface antigens of HBV.

[0184]

[0192] In various other embodiments, the active agent may be administered with one or more immunomodulatory agents. Such agents may increase or decrease production of one or more cytokines, up- or down-regulate self-antigen presentation, mask MHC antigens, or promote the proliferation, differentiation, migration, or activation state of one or more types of immune cells. Immunomodulatory agents include but are not limited to non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoralac, oxaprozin, nabumentone, sulindac, tolmentin, rofecoxib, naproxen, ketoprofen, and nabumetone, steroids (e.g. glucocorticoids, dexamethasone, cortisone, hydroxycortisone, methylprednisolone, prednisone, prednisolone, trimcinolone, azulfidineicosanoids such as prostaglandins, thromboxanes, and leukotrienes, as well as topical steroids such as anthrahn, calcipotriene, clobetasol, and tazarotene), cytokines such as TGFz, IFND , IFN D , IFN D , IL-2, IL-4, IL- 10, cytokine, chemokine, or receptor antagonists. In various embodiments, the subject may be on nucleoside analogue therapy if indicated by guidelines. In various embodiments, to improve T cell engraftment, lymphodepletion using e.g. fludarabine I cyclophosphamide or any other drug with similar mode of action may be administered to the subject.

[0185]

[0193] In various embodiments, the active agent of the present invention may be co-administered with a cytokine. The term "cytokine" as used herein is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines. In various embodiments, the cytokines or other agents co-administered serve to stimulate cells of the immune system. Such a mode of treatment may enhance desired effector function. For example, agents that stimulate NK cells, including but not limited to IL-2 may be co-administered. In another embodiment, agents that stimulate macrophages, including but not limited to C5a, formyl peptides such as N-formyl-methionyl-leucyl-phenylalanine (Beigier-Bompadre et al. (2003) Scand J. Immunol. 57. 221 -8), may be co-administered. Also, agents that stimulate neutrophils, including but not limited to G-CSF, GM-CSF, and the like may be administered Furthermore, agents that promote migration of such immunostimulatory cytokines may be used. Also additional agents including but not limited to interferon gamma, IL-3 and IL-7 may promote one or more effector functions. Alternatively, cytokines or other agents that inhibit effector cell function may be coadministered with the active agent of the present invention Such a mode of treatment may limit unwanted effector function.

[0186]

[0194] It is of course contemplated that the active agent of the invention may employ in combination with still other therapeutic techniques such as surgery.

[0195] It is also contemplated, that the binding molecule (i.e. antibody, antigen-binding fragment, chimeric antigen receptor, or host cell expressing the CAR) disclosed herein may be used to inhibit or neutralize HBV in an in vitro method or model of HBV infection.

[0187]

[0196] Accordingly, another aspect of the invention therefore also relates to an in vitro method of inhibiting HBV in a sample, which comprises the step of contacting the sample with a binding molecule disclosed herein.

[0188]

[0197] The term “contacting” as used herein, generally refers to any suitable means for delivering, exposing or bringing the sample and the binding molecule together in a controlled experimental setting under suitable conditions. As will be appreciated by the skilled person, the "contacting" step can vary depending on the nature of the sample and binding molecule involved, whereby the contacting may involve physically mixing the sample with the binding molecule in a test tube, petri dish or other container, or it could involve applying the binding molecules to the sample through techniques such as pipetting, incubation, or surface immobilization, or it could involve introducing the binding molecules into the sample (i.e. cells), such as through transfection.The purpose of the "contacting" step is to allow the binding molecules to interact with the sample and exert its inhibitory effect on the target molecule (i.e. HBV). In various embodiments, contacting occurs -n a solution in which the sample and binding molecules are mixed in a common solution and are allowed to freely associate, or the contacting can occur at or otherwise within a cell or in a cell-free environment. The contacting step may be carried out under conditions suitable to form an immune complex between the binding molecule and the HBV.

[0189]

[0198] In various embodiments, the sample may comprises a serum sample, a blood sample, or a plasma sample. A sample may be obtained directly from a subject or a control (e.g., by blood or tissue sampling) or from a third party (e.g., received from an intermediary, such as a healthcare provider or lab technician). The sample may be obtained from a subject that is suffering from, or suspected of suffering from, HBV infection or a disease associated with HBV infection. If the method is used for pathogen detection, any sample type useful and known for such purpose may be used. A biological sample, as contemplated herein, may also include cultured biological materials, including a sample derived from cultured cells, such as culture medium collected from cultured cells or a cell pellet. Accordingly, a biological sample may refer to a lysate, homogenate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. A biological sample may also be modified prior to use, for example, by purification of one or more components, dilution, and / or centrifugation. In various embodiments the biological sample may be obtained from an HBV infected subject or an animal model of HBV infection.

[0190]

[0199] Also provided are methods involving the use of the binding molecule disclosed herein, for detection, prognosis, diagnosis, and / or informing treatment decisions in a subject, such as by the detection of HBV in the subject. In various embodiments, the methods are diagnostic and / or prognostic methods in association with HBV or a disease associated with HBV.

[0200] Accordingly, another aspect of the invention therefore also relates to a method of detecting a HBV infection in a subject, comprising: (a) contacting a biological sample that has been obtained from the subject with the binding molecule disclosed herein, under conditions sufficient to form an immune complex; and (b) detecting the presence of the immune complex on the biological sample, wherein the presence of the immune complex on the biological sample indicates that the subject has an HBV infection or a disease associated with HBV.

[0191]

[0201] In various embodiments, the method includes incubating and / or probing the biological sample with the binding molecules and / or administering the binding molecules to a subject. In various embodiments, the biological sample includes a cell or tissue or portion thereof. In various embodiments, the contacting is under conditions permissive for binding of the binding molecules to HBV present in the sample. In various embodiments, the methods include detecting whether an immune complex is formed between the binding molecules and HBV in the sample, such as detecting the presence or absence or level of such binding. Such a method may be an in vitro or in vivo method. In various embodiments, a binding molecule may be used in methods for selecting subjects eligible for therapy with an active agent disclosed herein, or determining the susceptibility of a subject to treatment with the active agent disclosed herein, where HBV is a biomarker for the selection of the subject or determinaiton of susceptibility the subject.

[0192]

[0202] Conditions sufficient to form an immune complex, include conditions which allow the binding molecules to bind to its epitope to a detectably greater degree than, and / or to the substantial exclusion of, binding to substantially all other epitopes. Conditions sufficient to form an immune complex are dependent upon the format of the binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Harlow & Lane, Antibodies, A Laboratory Manual, 2nded. Cold Spring Harbor Publications, New York (2013), for a description of immunoassay formats and conditions. The conditions employed in the methods are "physiological conditions" which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.

[0193]

[0203] The formation of an immune complex may be detected through conventional methods known to the skilled artisan, for instance immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging, CT scans, X-ray and affinity chromatography. Immunological binding properties of selected antibodies may be quantified using methods well known in the art.

[0204] In various embodiments, the binding molecule may be directly labeled with a detectable marker. Suitable labels for the binding molecule may include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non -limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, betagalactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non- limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.

[0194]

[0205] For purposes of diagnosis, the binding molecule may be labeled with a detectable moiety including but riot limited to radioisotopes, fluoroscent labels, and various enzyme-substrate labels know in the art. Methods of conjugating labels to such binding molecules are known in the art. In various embodiments, binding molecules need not be labeled, and the presence thereof can be detected using a labeled antibody which binds to any of the binding molecules. The binding molecules disclosed herein may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). In various embodiments, the binding molecules disclosed herein may also be used for in vivo diagnostic assays, such as in vivo imaging. Generally, the binding agent is labeled with a radionuclide (such as111in, "To,!4C,131i,125i, or3H) so that the cells or tissue of interest can be localized in vivo following administration to a subject. The binding agent may also be used as staining reagent in pathology, e.g., using known techniques.

[0195]

[0206] The present invention is further illustrated by the following examples. However, it should be understood, that the invention is not limited to the exemplified embodiments.

[0196]

[0207] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0197]

[0208] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. EXAMPLES

[0198] Materials & Methods

[0199]

[0209] Antibody production and purification: HEK293 cells were transiently transfected as described above in T150 tissue culture flasks. Cell culture supernatant was collected at day 3, 5 and 7 post-transfection and filtered with a 0.45 pm sterile filter. Purification was performed with a protein G column (GE Healthcare, Uppsala, Sweden) and low pH elution. Elution fractions were pooled, concentrated via filtration and submitted to a buffer exchange to PBS. Antibody concentrations after purification were determined via Bradford Assay with a bovine gamma globulin protein standard (Pierce, Thermo Fisher).

[0200]

[0210] SDS-PAGE and Western blot: Antibody purity was assessed with a 12% polyacrylamide gel electrophoresis (PAGE) under reducing and non-reducing conditions followed either by Coomassie brilliant blue G250 (Carl Roth GmbH) staining or western blotting using a HRP-coupled goat anti-human IgG antibody (Sigma Aldrich) for detection. For Coomassie staining, 5 pg of each antibody was loaded to the gel, whereas for Western blot 100 ng were used. Under reducing conditions, the antibodies were heated for 10’ at 95°C in Laemmli sample buffer. Under non-reducing conditions antibodies were loaded to the gel diluted in non-reducing LDS sample buffer (Thermo Fisher).

[0201]

[0211] Testing anti-HBs antibodies in HDV background: Virus production: HDV enveloped with different genotypes of HBV envelope protein (genotype A-H) was produced by co-transfection of Huh7 cells with pJC126 (HDV genotype 1 , kindly provided by John Taylor) and the corresponding plasmids pLX304-HB2.7 coding for genotypes A-H, respectively. The cell supernatant containing HDV virus was harvested from day 10-13 post transfection. Infection and neutralization assay: Huh7-NTCP cells were seeded in 96-well plates 1 day prior infection (25,000 cells / well). On the day of infection, antibodies were serially diluted in medium and then mixed with HDV-containing medium. The virus / antibody mixtures were incubated for 60’ at 37°C, then diluted 1 :2 with medium containing 8% PEG and used as the inoculum for Huh7-NTCP cells. The final concentration of antibodies in the inoculum ranged from 0.01 -1000nM. One day after infection, the cells were washed with PBS and further cultivated with a medium changed every 3 days. Cells were fixed and analyzed by an immunofluorescent assay at day 6 or 7 post infection: To this end, cells were fixed in 4% PFA for 30’, followed by 30’ incubation in permeabilization buffer (PBS, 0.25% Triton X-100). After 30’ incubation in blocking buffer (PBS, 0.05% Tween-20, 3% BSA), cells were incubated for 1 h with anti-HDAg antibody diluted in blocking buffer. After washing, secondary antibodies goat anti-rabbit IgG Alexa Fluor555 (Invitrogen) were added for 1 h and the cells were imaged and analyzed by automated immunofluorescence microscopy.

[0202]

[0212] Identification of HBsAg-specific B cell clones and antibodies: PBMC were isolated via a standard Ficoll gradient. Informed consent in writing was obtained from each donor. B cells were enriched from PBMC using Dynabeads Untouched Human B cells Kit (Thermo Fisher). For further analysis, B cells were incubated with hFc block (BD Biosciences) for 10’ at RT, followed by biotinylated HBsAg for 30’ on ice (HBsAg by Roche Diagnostics; EZ-Link Sulfo-NHS-Biotinylation kit, Thermo Fisher). Memory B cell staining: 30’ on ice in PBS+0.5% BSA with live-dead stain (Thermo Fisher), anti- CD19-eF450 (eBioscience), anti-lgG-Pe-Cy7 (BD) and Streptavidin-APC (eBioscience). Cells were analyzed via flow cytometry on Cytoflex S (Beckman Coulter) or sorted with MoFlo II (Beckman Coulter) into lysis buffer, i.e. PBS+12U RNasin (Promega), 100mM DTT (Thermo Fisher). PCR plates were sealed and stored at -80°C. IgG gene identification via PCR and sequencing from single B cells was performed as described elsewhere15.

[0203]

[0213] B-cell ELISpot: PBMC were stimulated with 1 pg / mL R848 and 10ng / mL IL2 (MabTech) at 2x106cells / mL for 5d at 37°C. ELISpot plates (Merck) were activated and coated ON at 4°C with 2pg / mL HBsAg (Roche Diagnostics), washed 1 x with PBS and blocked with RPMI for 2h at RT. Stimulated PBMC were added to ELISpot plates at 1 -2x105 / well in RPMI and incubated for 18h at 37°C. B cells were subsequently removed, plates were washed 5x with PBS. 1 pig / ml detection antibody MT78-145 (MabTech) was added in PBS+0.5%BSA for 2h at RT. After washing, Streptavidin-HRP (MabTech) in PBS+0.5%BSA was added for 1 h at RT, followed by TMB (MabTech). Spot development was stopped by rinsing with H?O and spot numbers were quantified with ImmunoSpot Reader (CTL Europe).

[0204]

[0214] Antibody expression and ELISA: HEK293 cells were transiently transfected with heavy and light chains using Fugene HD transfection reagent (Promega). 3 days post-transfection, the supernatant was analysed via sandwich ELISA. The following reagents were used for detecting hlgG: goat antihuman IgG for coating (Thermo Fisher), PBS+5%BSA for blocking and PBS+0.05% Tween-20 for washing as well as polyclonal goat anti-human IgG-HRP (Sigma Aldrich) and TMB (Thermo Fisher) for detection. For the detection of HBsAg-specific antibodies, pre-coated Protein G ELISA plates (Pierce, Thermo Fisher) were used. After adding samples, HBsAg-biotin was added 1 :200 in PBS and Avidin- HRP (eBioscience) and TMB were used for development, as described above. Absorbance at 450nm was measured with Tecan Reader (Tecan Group AG).

[0205]

[0215] Epitope determination with dot blot and Western blot: An Immuno-Blot PVDF membrane (Biorad) was activated and calibrated with pure methanol and PBS for the dot blot. Drops of 2 pL were placed on the membranes (3 drops per piece) containing HBsAg (1 pg / mL) either diluted in PBS (nonreducing condition) or cooked for 10’ at 95°C in dot blot buffer (0.125 M Tris-HCI, 4% SDS, 20% Glycerol, 10% |3-mercaptoethanol) (reducing condition). After complete drying, the membranes were blocked with 5% milk in TBS-T buffer for 1 h at RT. Membranes were then transferred into 5% milk TBS-T solutions containing the primary antibodies 4D06, 4D08 or HB-1 with a concentration of 1 pg / ml. After incubation for 1.5 h at RT, the membranes were washed 3x for 10’ in TBS-T buffer and placed into 5% milk in TBS-T buffer containing secondary antibody goat anti-human IgG HRP (Sigma Aldrich) diluted 1 :10,000 for 1 h at RT. After final washing, an ECL detection solution (GE Healthcare) was added to develop and analyze the blots. The HBsAg Western blot was performed in a similar manner. HBsAg was heated for20’ at 95°C in dot blot buffer and loaded onto a 12% SDS-PAGE gel. Separated proteins were wet-blotted onto an Immuno-Blot PVDF membrane (Biorad) and blocked in the same way as described above for the dot blot. The antibodies were used at 1 pg / mL diluted in 5% milk in TBS-T buffer. All further steps were performed as in the dot blot.

[0206]

[0216] HBsAg-ELISA: ELISA plates were coated ON at 4°C with 1 pg / mL HBsAg. Plates were washed 4x times with PBS+0.05% Tween20 (Sigma Aldrich) and blocked with 5% BSA in PBS for 1 h at RT. Purified antibodies 4D06 and 4D08 were serially diluted in PBS from 1000 nM to 0.03 nM and incubated for 2 h at RT. After washing, the HRP-conjugated detection antibodies goat anti-human IgG (1 :1000 in PBS; Sigma Aldrich) was added and incubated for 1 h at RT. Finally, the assay was developed with TMB, and the reaction was stopped after 5’ upon adding 2N H2SO4. Absorbance was measured at 450 nm in a Tecan reader (Tecan Group AG). EC50 values were calculated by non-linear regression with GraphPad Prism.

[0207]

[0217] For the analysis of different genotypes, pre-coated Murex HBsAg Version 3 ELISA stripes (DiaSorin) were activated as described by the manufacturer. HBsAg-positive human plasma containing 15 different HBV sub- / genotypes was added at 1 lU / well (Paul-Ehrlich-lnstitute, 1s’ WHO International Reference Panel for HBV Genotypes for HBsAg Assays). Human serum from a patient tested negative for HBsAg was used as a negative control. After incubation for 1 h at 37°C, biotinylated antibodies 4D06 and 4D08 were added at 2 pg / mL in PBS and further incubated for 30’ at 37°C. Biotinylation was performed in-house using an EZ-Link Sulfo-NHS-Biotinylation kit (Thermo Fisher). 1 :500 HRP-coupled Avidin in PBS was added for detection and incubated for 30’ at 37°C. The development and measurement was done as described above.

[0208]

[0218] HBV uptake inhibition: The capability of mAbs 4D06 and 4D08 to prevent HBV uptake was tested in vitro using HepG2 cells stably expressing human NTCP (HepG2-NTCP). For this, cells were seeded in collagen-coated 24-well plates with a density of 3x105cells / well in DMEM differentiation medium (DMEM diff, i.e. 10% FCS, 1 % penicillin-streptomycin, 1% glutamine, 1 % NEAA, supplemented with 2.5% DMSO). Cells were differentiated for 3 days before infection. On the day of infection, 10OnM of 4D06 and 4D08 mAbs were pre-incubated with purified HBV with an MOI 100 for 30’ at 37°C in DMEM diff medium. Heparin (Ratiopharm) and HBIg (0.3 ILJ / well; Nabi-HB, Biotest Pharmaceuticals Corporation) were handled similarly and served as controls. Cells were pre-chilled on ice for 15’ before inoculation. The inoculum with HBV and antibodies was incubated for 1 h, enabling the virus to bind to the cell surface. After incubation, cells were washed 2x with PBS, trypsinized for 3’, and shifted back to 37 °C for 3-24h. The total cellular DNA was extracted from cell lysate using a NucleoSpin tissue kit (Macherey-Nagel) to determine intracellular HBV cccDNA as markers of HBV uptake. DNA was frozen at -20°C until cccDNA was quantified by qPCR as previously described28.

[0209]

[0219] Analysis of antibody neutralization capacity: HepG2 NTCP cells were seeded with 3x105cells / well in collagen-coated 24-well plates in DMEM diff medium and differentiated for 3 days prior to infection. Purified HBV (MO1 100) was pre-incubated with serial dilutions of mAbs 4D06 and 4D08 from 0.01 nM to 1000 nM for 3 h at 37°C. PEG6000 was added to the inoculum at a final concentration of 4% (v / v). The inoculum was incubated for 18 h before. Cells were then washed 3x with PBS, and a fresh DMEM diff medium was added. The supernatant was collected for HBeAg measurement at days 4 and 8 post infection to monitor the infection. The experiment was terminated at day 8 post infection when cells were lysed, and total DNA was extracted with the NucleoSpin tissue kit supplied by Macherey-Nagel. Intracellular cccDNA was quantified by qPCR as previously described28. HBeAg was determined by commercial immunoassay (Siemens Molecular Diagnostics).

[0210]

[0220] Retroviral transduction of T cells: T cells were enriched and stimulated using human T activator CD3 / CD28 Dynabeads (Thermo Fisher) for 2 days in T cell medium with FBS: RPMI, 10% FBS, 1 % pen / strep, 1% glutamine, 1 % sodium pyruvate, 1% NEAA, 10 mM HEPES, 16.6 pg / ml Gentamycin (all Thermo Fisher), supplemented with 300 U / ml IL2. 0.45 pm-filtered retrovirus cell culture supernatant from stable producer cell lines was centrifuged at 2000xg, 32°C for 2h on non-tissue culture-treated plates (Corning) coated with 20 pg / ml RetroNectin for 2h (Takara). Retrovirus cell culture supernatant was removed and T cells were spinoculated onto the virus-coated plate at 1000xg for 10’. A 2ndtransduction was performed after 24h. CAR expression was determined by flow cytometry with anti-human CD4-APC (eBioscience), anti-human CD8-PB (BioLegend) and anti-HA-PE (BioLegend), diluted in PBS with 0.1% BSA (Sigma-Aldrich). Cells were analyzed using a GytoFLEX S (Beckman Coulter) and data were analyzed with FlowJo 10.4 software.

[0211]

[0221] Co-cultures on plate-bound antigen: HBsAg was coated on cell culture 96-well plates ON at 37°C in concentrations from 0.1 -10pg / ml. Plates were washed 2x with ELISA and CAR-transduced T cells (CD4+to CD8+ratio ~1 :1 ) were added with 1 x105transduced cells / well. IFNy in the cell culture supernatant was detected after 48h using an ELISA kit (Thermo Fisher) according to the manufacturer’s instructions. ECso values were calculated by non-linear regression with GraphPad Prism.

[0212]

[0222] Co-cultures of CAR-T cells with HepG2-derived target cells: HepG2-derived cell lines were seeded in DMEM diff medium on collagen-coated plates, medium was changed every 2-3 days for 10- 12 days. For coculture, T cells (CD4+to CD8+ratio ~ 1 :1 ) were added in equal amounts of T cell medium (final concentration of 1% DMSO in coculture) at different effector-to-target ratios. IFNy in the cell culture supernatant was detected using an ELISA kit (Thermo Fisher) according to the manufacturer’s instructions. For intracellular cytokine staining, 2 pg / ml Brefeldin A (Sigma-Aldrich) was added 16h after co-culture start and staining was performed after 48h, using the Fixation / Permeabilization Solution kit (BD), live / dead Fixable Aqua stain (Thermo Fisher), anti-human CD4-PerCP (BioLegend), anti-human CD8-FITC (Thermo Fisher), anti-HA-PE (BioLegend), anti-human IFNy -AF700 (BD Biosciences), antihuman TNFa-APC (BioLegend), anti-human IL2-PE-Cy7 (Thermo Fisher), anti-human GrzB-PB (Thermo Fisher). For proliferation analysis, CellTrace Violet Stain (Thermo Fisher) was used according to manufacturer’s instructions.

[0213]

[0223] Real-time cytotoxicity measurement: HepG2-derived cell lines were differentiated in cell culture flasks with a change of DMEM diff every 2-3 days for 7-9 days. Cells were then seeded onto 96- well electronic microtiter plates (ACEA Biosciences) with 5x104 / well and differentiated for an additional 3 days. CAR-transduced T cells (CD4+ to CD8+ ratio ~1 :1 ) were added at different effector to target ratios. Electrical impedance was measured every 30’ with an xCELLigence SP real-time cell analyzer (ACEA Biosciences).

[0214] Results

[0215] Example 1 : Isolation of HBsAg-specific memory B cells with ELISpot and flow cytometry

[0216]

[0224] For the generation of monoclonal antibodies from peripheral blood of an immune donor, single B-cell sorting technology15was used. This approach relies on the frequency of circulating antigenspecific memory B cells, which is generally extremely low and highly dependent on the immune status of a person16. Thus, identifying and isolating those rare antigen-specific B cells is challenging and depends on a suitable donor. In addition, an optimized sorting strategy is also required to separate the signal from the background.

[0217]

[0225] To identify low-frequency HBV-specific human memory B cells from peripheral blood, HBsAg was purified from human serum and labeled with biotin. The resulting HBsAg-biotin was used as a decoy to selectively bind to anti-HBs B-cell receptors on the surface of lgG+CD19+ human memory B cells. B cells were enriched from PBMC by negative selection to decrease unspecific binding of HBsAg-biotin to non-B cells. HBsAg-specific memory B cells were identified through incubation with HBsAg-biotin and fluorophore-coupled streptavidin via a four-step gating strategy (FIG. 1A) from a donor with a high serum anti-HBs titer who had previously resolved an acute HBV infection and observed a frequency of 0.42% HBsAg+CD19+ lgG+B cells out of the total lgG+cells (FIG. 1 B). Incubation with streptavidin only served as a control for non-specific binding to B-cell receptors and indicated background binding of 0.03% of lgG+B cells. Staining of an HBV-naTve donor sample showed background frequencies of 0.15% and 0.04%, respectively (FIG. 2). Considering that lgG+B cells comprise approximately 15% of the B-cell population17, the frequency of HBsAg-specific B cells of CD19+B cells was calculated with 0.06% for the donor with resolved acute HBV infection.

[0218]

[0226] To further verify the frequency of antigen-specific B cells obtained by flow cytometry, an HBsAg- specific B cell, ELISpot was performed in parallel. PBMC were stimulated in vitro for five days with IL2 and the immune adjuvant R848 to induce the differentiation of memory B cells into antibody-secreting cells. After transferring the cells to IgG- and HBsAg-coated ELISpot plates, antibody secretion was determined by spot counting. While total IgG secretion was high both for the donor with resolved HBV infection as well as a naive donor sample which served as negative control, anti-HBs antibodies were secreted only by cells from the donor with resolved infection with frequencies ranging at approximately 0.01 % (20 spots per 2.0x105cells) of HBsAg+ cells in PBMC (FIG.1C).

[0219]

[0227] After successfully evaluating HBsAg-biotin as a specific decoy to capture HBsAg-reactive memory B cells, single-cell sorting of those cells was performed. B cells were enriched from the PMBC of the donor with resolved infection and incubated with HBsAg-biotin. CD19+lgG+and HBsAg+memory B cells were single-cell-sorted based on the gating strategy described above (Error! Reference source not found. A). A total number of 238 antigen-positive single cells were sorted directly into 96-well PCR plates containing lysis buffer and frozen immediately on dry ice.

[0220]

[0228] To date, different approaches have been used for the generation of HBV -specific mAbs, mostly using classical hybridoma technology, phage display technologies or immortalization of human peripheral blood B cells with Epstein-Barr virus. However, these methods have drawbacks, such as the high probability of inducing harmful immune responses in humans against murine protein sequences or the rather inefficient process of B-cell immortalization. Single B cell antibody technology represents a major improvement to the existing processes, whereby single antigen-specific human B cells can be isolated from small quantities of donor cells with an unbiased approach, and recombinant antibodies can be generated based on amplified immunoglobulin variable chain genes.

[0221] Example 2: Identification of human immunoglobulin variable chain genes and cloning of full- length, recombinant anti-HBs antibodies

[0222]

[0229] To generate recombinant human monoclonal antibodies, mRNA was isolated from each singlesorted HBsAg+memory B cell and reverse transcribed into cDNA. Human immunoglobulin variable heavy, kappa light, and lambda light chain genes were recovered using nested PCR according to the protocol published by Tiller etal5. While the overall amplification efficacy was low, corresponding PCR products of heavy and light chain genes were obtained for 27 single-sorted B cells, which were then purified and sequenced. Most PCR products represented productive rearranged Ig entities, and germline V(D)J-gene segments with the highest identity were identified using the international immunogenetics information system (IMGT)18. Based on their specific sequence, corresponding variable heavy and light chain couples were cloned as recombinant, full-length IgG 1 antibodies, and 20 clones were expressed in HEK293 cells. Whereas most constructs showed a high secretion of total IgG, only two antibodies, namely 4D06 and 4D08, gave a strong anti-HBs signal (FIG.1D) and were selected for further analysis. 4D06 and 4D08 were then re-expressed in HEK293 cells and purified by protein G column affinity purification to a purity of >95% (FIG. 3).

[0223] Example 3: 4D06 and 4D08 mAbs recognize a conformational versus a linear epitope of the HBV S-protein

[0224]

[0230] A dot blot was performed to evaluate whether 4D06 and 4D08 recognize a linear or a conformational epitope within the HBV env protein. Recombinant HBsAg was dotted onto a membrane either under non-reducing conditions, keeping the native three-dimensional structure of HBsAg, or under reducing conditions, leading to protein linearization. Both mAbs 4D06 and 4D08 recognized the dotted antigen under native conditions; however, the reactivity was lost for 4D06 under reducing conditions, while it remained intact for 4D08, indicating the recognition of a conformational epitope for the former and a linear epitope for the latter (FIG. 4A). To verify this observation, a Western blot of HBV-positive cell lysates separated by SDS-PAGE was performed. Two bands at a size of 27 kDa and 24 kDa representing glycosylated and non-glycosylated HBsAg were detected with 4D08 and HB-1 antibodies. This control antibody recognizes a known linear epitope in the small HBV env protein at position S119-125 in the antigenic loop19. Additionally, one band with a molecular weight of approximately 50 kDa was detected, most likely representing a dimer of the S protein20(FIG. 4B). In conclusion, 4D06 recognizes a conformational epitope on the HBVenv proteins, whereas 4D08 binds a linear epitope.

[0225] Example 4: 4D06 and 4D08 are high-affinity antibodies with broad neutralization capacity

[0226]

[0231] Antibody affinities were determined by ELISA using serial dilutions of mAbs 4D06 and 4D08 on plate-bound HBsAg. The calculation of half-maximal effective concentrations (ECso) via non-linear regression revealed high affinities of both antibodies in the nanomolar range, i.e. , 18.5 nM for 4D06 and 158 nM for 4D08 (FIG.4C).

[0227]

[0232] Next, the reactivity of 4D06 and 4D08 against the eight major HBV genotypes (A-H) was investigated, five of which (genotypes A, B, C, D and E) are responsible for causing 96% of chronic HBV infections worldwide21. An indirect ELISA using a WHO genotype reference panel for HBsAg assays from the Paul-Ehrlich Institute (Langen, Germany) showed a broad reactivity of 4D06 and 4D08 towards all tested genotypes and serotypes adw, ayw. and adr (FIG.4D).

[0228]

[0233] The capacity of 4D06 and 4D08 to prevent virus uptake in vitro was analysed. Differentiated HepG2-NTCP cells, susceptible to HBV infection, were used as target cells and pre-incubated with mAbs 4D06 and 4D08 or polyclonal anti-HBs antiserum (HBIg) or heparin, both of which are known to inhibit HBV uptake22and served as positive controls. Target cells were then inoculated with HBV at 4“C, allowing virus attachment, before shifting to 37°C to induce synchronized HBV uptake. 24 hours after inoculation, no nuclear transcription template of HBV, the so-called cccDNA, was detected via qPCR, indicating successful infection prevention by pre-incubation with 4D06 and 4D08 (FIG. 4E). Using the hepatitis Delta virus enveloped with HBVenv of a broad range of different genotypes, it was determined that both mAb, 4D06, and 4D08 are broadly neutralizing (FIG. 5).

[0229]

[0234] Last, the neutralization capacity of both antibodies was quantified using the mAbs at increasing concentrations. The establishment of an HBV infection was quantified through cccDNA qPCR in cell lysates at day 8, where a superior neutralization capacity was observed for 4D06 compared to 4D08 (FIG. 4F). The measurement of secreted HBeAg at days 4 and 8 post-infection showed a concentration- and time-dependent increase of HBeAg, with 4D06 achieving complete neutralization at a concentration of 100 nM, whereas 4D08 only reached a comparable result at 1000 nM (FIG. 4G). In summary, 4D06 and 4D08 are high-affinity antibodies that can broadly neutralize different HBV geno- and serotypes.

[0230] Example 5: CAR constructs derived from 4D06 and 4D08 mAbs are functional in primary human T cells

[0235] To explore the potential of our novel mAbs 4D06 and 4D08 for adoptive T-cell therapy, 2ndgeneration CARs composed of an antibody-binding domain with an HA-tag, an lgG4 hinge region, a CD28 transmembrane domain, and intracellular signaling moieties of CD28 and CD3zeta were constructed (FIG. 6A). The respective binding domain of each CAR construct consisted of a scFv using a glycine-serine linker connecting codon-optimized variable heavy and light chains of either the 4D06- or the 4D08-antibody. The C8-binder from an HBV-specific S-CAR was used as previously generated13as a control. After retroviral transduction, all CAR constructs expressed well in primary human CD4+or CD8+T cells from healthy donors, with the C8-CAR consistently generating higher transduction rates than the 4D06- and 4D08-CARs (FIG. 6B). A higher expression level of the C8-CAR than the 4D06- and 4D08-CARs was confirmed by considering the mean fluorescence intensity of flow cytometry staining (FIG. 6C).

[0231]

[0236] CAR-transduced T cells were cultured on titrated plate-bound HBsAg to compare the novel CAR constructs to the existing C8-CAR regarding their binding affinity. Unlike their mAb counterparts, 4D06- and 4D08-CARs showed very comparable ECso values with 0.15 and 0.19 pg / ml, whereas the C8-CAR ranged approximately three times lower with an ECso of 0.53 pg / ml (FIG. 6D). Taken together, all three CAR constructs were functional in primary human T cells and showed affinities in a nanomolar range.

[0232] Example 6: 4D06- and 4D08-CARs are polyfunctional and able to kill HBV-expressing target cells

[0233]

[0237] To further characterize the functionality of our new constructs, we performed co-culture experiments of CAR-transduced T cells with a set of hepatoma cell lines, where HepG2 cells represent the non-HBV-expressing control, HepG2-SML cells express the HBVenv proteins S, M and L under the endogenous HBV promotors, and HepG2-2.15 cells carry several copies of a dimer HBV genome as integrates. When co-culturing CAR-T cells at decreasing effector-to-target (E:T) ratios, we observed that CAR constructs C8 and 4D06 induced IFNg-secretion not only on HBV-expressing target cells but also on HepG2 control cells at 1 :1 and 1 :3 E:T ratios, whereas 4D08-CAR showed no unspecific activity. On HBV-expressing target cells, however, cytokine secretion from T cells grafted with all three CARs was comparable and strongly depended on the E:T ratio (FIG. 7A). Cell trace violet staining in flow cytometry showed a marked proliferation of T cells grafted with all three CAR constructs when exposed to their cognate antigen on HepG2-SML and on HepG2.215 cells. On HepG2.2.15 cells, T cells grafted with the 4D06-CAR slightly outperformed those grafted with the C8-CAR, followed by the 4D08-CAR (FIG. 7B). However, C8- and 4D06-CAR T cells also exhibited a pronounced, antigen-independent proliferation on HepG2 control cells. Interestingly, this was lower for 4D08-CAR T cells. All three constructs induced the secretion of varying amounts of cytokines as observed through intracellular staining in flow cytometry, with CD4+T cells generally secreting higher amounts of TNFa and IL2 and CD8+ T cells secreting more IFNy and GrzB (FIG. 7C).

[0234]

[0238] Once again, C8- and 4D06-CARs displayed a distinct background activation on HepG2 cells compared to the 4D08-CAR, which was not activated in the absence of antigen but also outperformed the 4D08-CAR in terms of specific cytokine secretion on HepG2-SML and HepG2-2.15 cells. Interestingly, when looking at polyfunctionality of T cells on HepG2-SML cells, it appeared that C8- and 4D06-transduced CAR T cells shared a rather similar profile with a substantial subset of triple or quadruple positive cells among both CD4+and CD8+T cells, whereas 4D08-transduced T cells seemed to have a much higher proportion of single positive cells or cells that didn’t secrete any cytokines at all in particular in the CD4+subset (FIG. 7D). Last but not least, CAR-transduced T cells were able to effectively kill HBsAg-expressing target cells, as shown by an xCELLigence cytotoxicity assay (FIG. 7E). C8-CAR T cells once again showed substantial background activation and cytotoxicity on HepG2 control cells, which was less pronounced for the 4D06-CAR and absent for the 4D08-CAR. Surprisingly, on HBV-expressing cells HepG2-SML and HepG2-2.15, all three CAR constructs performed similarly well.

[0235]

[0239] In conclusion, T cells transduced with all three CAR constructs show a polyfunctional cytokine secretion profile. However, it seems that the 4D08-CAR shows an overall diminished background activation compared to C8- and 4D06-CARs, which also concurs with a different cytokine secretion profile, while the killing efficacy remains the same for all three.

[0236] List of Sequence ID NOs described herein

[0237]

[0240] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an xml file, which is incorporated by reference herein. In the accompanying sequence listing:

[0238] Amino Acid Sequences:

[0239] Nucleotide Sequences:

[0240] References:

[0241] 1. World Health Organization. Hepatitis B 2019. https: / / www.who.int / news-room / fact- sheets / detail / hepatitis-b

[0242] 2. Wiktor S. Viral Hepatitis. In: Holmes KK, Bertozzi S, Bloom BR, Jha P, eds. Major Infectious Diseases. Washington (DC): The International Bank for Reconstruction and Development / The World Bank

[0243] © 2017 International Bank for Reconstruction and Development / The World Bank.; 2017.

[0244] 3. Fanning GC, Zoulim F, Hou J, Bertoletti A. Therapeutic strategies for hepatitis B virus infection: towards a cure. Nat Rev Drug Discov 2019; 18(11 ): 827-44.

[0245] 4. Block TM, Gish R, Guo H, et al. Chronic hepatitis B: what should be the goal for new therapies? Antiviral research 2013; 98(1 ): 27-34.

[0246] 5. Bertoletti A, Le Bert N. Immunotherapy for Chronic Hepatitis B Virus Infection. Gut Liver 2018; 12(5): 497-507.

[0247] 6. Rehermann B. Pathogenesis of chronic viral hepatitis: differential roles of T cells and NK cells. Nat Med 2013; 19(7): 859-68.

[0248] 7. Bohne F, Protzer U. Adoptive T-cell therapy as a therapeutic option for chronic hepatitis B. J Viral Hepat 2007; 14 Suppl 1 : 45-50.

[0249] 8. Lau GK, Lok AS, Liang RH, et al. Clearance of hepatitis B surface antigen after bone marrow transplantation: role of adoptive immunity transfer. Hepatology 1997; 25(6): 1497-501 .

[0250] 9. Tan AT, Schreiber S. Adoptive T-cell therapy for HBV-associated HCC and HBV infection. Antiviral research 2020; 176: 104748.

[0251] 10.Wisskirchen K, Kah J, Malo A, et al. T cell receptor grafting allows virological control of Hepatitis B virus infection. J Clin Invest 2019; 129(7): 2932-45.

[0252] 11 . Krebs K, Bottinger N, Huang LR, et al. T cells expressing a chimeric antigen receptor that binds hepatitis B virus envelope proteins control virus replication in mice. Gastroenterology 2013; 145(2): 456- 65.

[0253] 12.Festag MM, Festag J, Frassle SP, et al. Evaluation of a Fully Human, Hepatitis B Virus-Specific Chimeric Antigen Receptor in an Immunocompetent Mouse Model. Mol Ther2019; 27(5): 947-59.

[0254] 13. Bohne F, Chmielewski M, Ebert G, et al. T cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes. Gastroenterology 2008; 134(1 ): 239-47. 14. Zhao L, Chen F, Quitt O, et al. Hepatitis B virus envelope proteins can serve as therapeutic targets embedded in the host cell plasma membrane. Cell Microbiol 2021 ; 23(12): e13399.

[0255] 15. Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann H. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. Journal of immunological methods 2008; 329(1 -2): 1 12-24.

[0256] 16. Cox KS, Tang A, Chen Z, et al. Rapid isolation of dengue-neutralizing antibodies from single cell- sorted human antigen-specific memory B-cell cultures. MAbs 2016; 8(1 ): 129-40.

[0257] 17. Seifert M, Kuppers R. Human memory B cells. Leukemia 2016; 30(12): 2283-92.

[0258] 18.Lefranc MP, Giudicelli V, Duroux P, et al. IMGT(R), the international ImMunoGeneTics information system(R) 25 years on. Nucleic acids research 2015; 43(Database issue): D413-22.

[0259] 19.Kucinskaite-Kodze I, Pleckaityte M, Bremer CM, et al. New broadly reactive neutralizing antibodies against hepatitis B virus surface antigen. Virus Res 2016; 21 1 : 209-21 .

[0260] 2O.Suffner S, Gerstenberg N, Patra M, et al. Domains of the Hepatitis B Virus Small Surface Protein S Mediating Oligomerization. Journal of Virology 2018; 92(1 1 ).

[0261] 21 .Velkov S, Ott JJ, Protzer U, Michler T. The Global Hepatitis B Virus Genotype Distribution Approximated from Available Genotyping Data. Genes (Basel) 2018; 9(10).

[0262] 22.Lempp FA, Urban S. Inhibitors of hepatitis B virus attachment and entry. Intervirology 2014; 57(3- 4): 151 -7.

[0263] 23. Tan W, Meng Y, Li H, et al. A bispecific antibody against two different epitopes on hepatitis B surface antigen has potent hepatitis B virus neutralizing activity. / WAbs 2013; 5(6): 946-55.

[0264] 24.Tajiri K, Ozawa T, Jin A, et al. Analysis of the epitope and neutralizing capacity of human monoclonal antibodies induced by hepatitis B vaccine. Antiviral research 2010; 87(1 ): 40-9.

[0265] 25. Long AH, Haso WM, Shern JF, et al. 4-1 BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 2015; 21 (6): 581 -90.

Claims

CLAIMSWhat is claimed is:

1. An antibody comprising a heavy chain variable domain and a light chain variable domain, wherein:(i) the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 1 -3, respectively, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 4-6 , respectively; or(ii) the heavy chain variable domain comprises VH GDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 7-9, respectively, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 10-12, respectively, wherein the antibody binds to Hepatitis B virus (HBV), more preferably binds to an HBV envelope protein.

2. The antibody of claim 1 , wherein the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 1 -3, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 4-6, and wherein the heavy chain variable domain (VH) comprises or consists of an amino acid sequence set forth in SEQ ID NO: 13 or a variant thereof, and a light chain variable domain (VL) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 14 or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID Nos. 1 - 6 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

3. The antibody of claim 1 , wherein the heavy chain variable domain comprises VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 7-9, respectively, and the light chain variable domain comprises VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 10-12, respectively, and wherein the heavy chain variable domain (VH) comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof, and a light chain variable domain (VL) comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 16 or a variant thereof, wherein the variants are invariable with respect to the CDR regions in SEQ ID Nos. 7- 12 and share at least 80 %, preferably at least 90 %, more preferably at least 95% sequence identity with the respective reference sequence.

4. The antibody of any one of claims 1 -3, wherein the antibody is an IgG immunoglobulin, preferably an lgG1 immunoglobulin.

5. The antibody of claim 4, wherein the antibody comprises a IgG constant region within a heavy chain of the immunoglobulin and a IgG constant region within a light chain of the immunoglobulin.

6. The antibody of any one of claims 1 -5, wherein the antibody is a human antibody, a monoclonal antibody, a purified antibody, or a single chain antibody, preferably a human monoclonal antibody.

7. An antigen-binding fragment of the antibody according to any one of claims 1 -6.

8. The antigen-binding fragment of claim 7, wherein the antigen-binding fragment is a Fv, a Fab, aFab’, a F(ab’)2, scFv or a scFv2 fragment.

9. The antigen-binding fragment of claim 7 or 8, wherein the antigen-binding fragment comprises:(i) the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 1 -3, respectively, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 4-6, respectively; or(ii) the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the amino acid sequences set forth in SEQ ID Nos. 7-9, respectively, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the amino acid sequence set forth in SEQ ID Nos. 10-12, respectively.

10. A chimeric antigen receptor (CAR) comprising the antigen-binding fragment, preferably a scFv fragment, of any one of claims 7-9.11 . The chimeric antigen receptor of claim 10, further comprising an intracellular domain and a transmembrane domain, wherein the antigen-binding fragment is comprised in an extracellular domain.

12. The chimeric antigen receptor of claim 11 , wherein the intracellular domain comprises a CD28 and a CDSzeta signalling domain, the transmembrane domain comprises a CD28 transmembrane domain, and the extracellular domain further comprises a hinge region, a linker and a tag.

13. The chimeric antigen receptor of any one of claims 10-12 for use in adoptive immunotherapy.

14. A nucleic acid molecule encoding the antibody of any one of claims 1 -6, or the antigen-binding fragment of any one of claims 7-9, or the chimeric antigen receptor of any one of claims 10-13.

15. The nucleic acid molecule of claim 14, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the heavy and / or light chain variable domain of the antibody of any one of claims 1 -6.

16. The nucleic acid molecule of claim 15, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable domain as set forth in SEQ ID Nos: 29 or 31 , or a variant thereof.

17. The nucleic acid molecule of claim 15 or 16, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the light chain variable domain as set forth in SEQ ID Nos: 30 or 32 or a variant thereof.

18. The nucleic acid molecule of any one of claims 14-17, comprising:(i) a nucleotide sequence encoding the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the nucleotide sequences set forth in SEQ ID Nos. 17-19, respectively, or degenerate variants thereof, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the nucleotide sequence set forth in SEQ ID Nos. 20-22, respectively, or degenerate variants thereof; or(ii) a nucleotide sequence encoding the heavy chain variable domain comprising VH CDR1 , VH CDR2, and VH CDR3 regions having the nucleotide sequences set forth in SEQ ID Nos. 23-25, respectively, or degenerate variants thereof, and / or the light chain variable domain comprising VL CDR1 , VL CDR2, and VL CDR3 regions having the nucleotide sequence set forth in SEQ ID Nos. 26-28, respectively, or degenerate variants thereof.

19. The nucleic acid molecule of any one of claims 14-18, wherein the nucleic acid molecule is a vector.

20. A host cell comprising the nucleic acid molecule of any one of claims 14-19.21 . The host cell of claim 20, wherein the host cell has been transfected, transduced or transformed by the nucleic acid molecule.

22. The host cell of claim 20 or 21 , wherein the nucleic acid molecule encodes the chimeric antigen receptor of any one of claims 10-13, wherein the host cell is engineered to express the chimeric antigen receptor.

23. The host cell of claim 22, wherein the host cell is a T cell for use in adoptive immunotherapy.

24. A composition for preventing, treating or ameliorating an HBV infection or a disease associated with HBV infection, in a subject, comprising the antibody of any one of claims 1 -6, or the antigen-binding fragment of any one of claims 7-9, the chimeric antigen receptor of any one of claims 10-13, or the nucleic acid molecule of any one of claims 14-19, or the host cell of any one of claims 20-23; and a pharmaceutically acceptable carrier and / or excipient.

25. A method of preventing, treating or ameliorating an HBV infection or a disease associated with HBV infection in a subject, comprising administering to the subject a therapeutically effective amount of the antibody of any one of claims 1 -6, the antigen-binding fragment of any one of claims 7-9, the chimeric antigen receptor of any one of claims 10-13, the nucleic acid molecule of any one of claims 14-19, the host cell of any one of claims 20-23, or the composition of claim 24.

26. Use of the antibody of any one of claims 1 -6, the antigen-binding fragment of any one of claims 7-9, the chimeric antigen receptor of any one of claims 10-13, the nucleic acid molecule of any one of claims 14-19, the host cell of any one of claims 20-23, or the composition of claim 24, in the manufacture of a medicament for preventing, treating or ameliorating an HBV infection or a disease associated with HBV infection in a subject.

27. The composition of claim 24, or the method of claim 25, or the use of claim 26, wherein the disease associated with HBV infection comprises hepatitis, liver fibrosis, liver cirrhosis, and liver cancer, preferably HBV-induced hepatocellular carcinoma.

28. An in vitro method of inhibiting HBV in a sample, comprising contacting the sample with the antibody according to any one of claims 1 to 6, the antigen-binding fragment of any one of claims 7-9, the chimeric antigen receptor of any one of claims 10-13 or the host cell of claim 22, under conditions suitable to form an immune complex.

29. A method of detecting a HBV infection or a disease associated with HBV, in a subject, comprising:(a) contacting a biological sample that has been obtained from the subject with the antibody of any of claims 1 -6, or antigen binding fragment of any of claims 7-9, or the chimeric antigen receptor of any one of claims 10-13 or the host cell of claim 22, under conditions sufficient to form an immune complex; and(b) detecting the presence of the immune complex on the biological sample, wherein the presence of the immune complex on the biological sample indicates that the subject has an HBV infection or a disease associated with HBV.

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