Manipulated hepatitis B virus neutralizing antibodies and their use

Abstract

The present disclosure relates in part to antibodies, and antigen-binding fragments thereof, capable of binding to the antigenic loop region of hepatitis B surface antigen (HBsAg) and, optionally, neutralizing hepatitis B virus (HBV) infection and, optionally, neutralizing hepatitis delta virus (HDV) infection. The antibodies and antigen-binding fragments disclosed herein have advantageous production characteristics, e.g., reduced aggregate formation and / or improved production titers in transformed host cells, compared to reference antibodies or antigen-binding fragments. The present disclosure also relates to fusion proteins comprising the antigen-binding fragments, as well as nucleic acids encoding such antibodies, antigen-binding fragments, and fusion proteins, and cells that produce such antibodies, antigen-binding fragments, and fusion proteins.

Description

[Technical Field]

[0001] Statement regarding sequence listings The sequence listing relating to this application is provided in text format instead of as a paper copy, and is incorporated herein by reference. The name of the text file containing the sequence listing is 930485.414WO_SEQUENCE_LISTING.txt. The text file is 101KB in size, was created on June 22, 2021, and submitted electronically via EFS-Web. [Background technology]

[0002] background Hepatitis B virus causes potentially life-threatening acute and chronic liver infections. Acute hepatitis B is characterized by viremia with or without symptoms, and with a risk of developing fulminant hepatitis (Liang TJ, Block TM, McMahon BJ, Ghany MG, Urban S, Guo JT, Locarnini S, Zoulim F, Chang KM, Lok AS. Present and future therapies of hepatitis B: From discovery to cure. Hepatology. 2015 Aug 3. doi: 10.1002 / hep.28025. [Epub ahead of print]). Despite the availability of an effective vaccine against hepatitis B since 1982, the WHO reports that 240 million people are chronically infected with hepatitis B, and more than 780,000 people die each year from complications of hepatitis B. Approximately one-third of patients with chronic hepatitis B (CHB) develop cirrhosis, liver failure, and hepatocellular carcinoma, which are major causes of 600,000 deaths per year (Liang TJ, Block TM, McMahon BJ, Ghany MG, Urban S, Guo JT, Locarnini S, Zoulim F, Chang KM, Lok AS. Present and future therapies of hepatitis B: From discovery to cure. Hepatology. 2015 Aug 3. doi: 10.1002 / hep.28025. [Epub ahead of print]).

[0003] In patients infected with HBV, severe complications can develop as a result of co-infection or superinfection with HDV. According to the WHO, hepatitis D infects approximately 15 million people worldwide. HDV is considered a subviral satellite because it can only thrive and replicate in the presence of HBV. HDV is one of the smallest known animal viruses (40 nm), with a genome of only 1.6 kb, encoding S and L HDAg. All other proteins required for HDV genome replication, including RNA polymerase, are provided by the host cell, while the HDV envelope is provided by HBV. Once introduced into a permissive cell, the HDV RNA genome replicates and associates with multiple copies of the HDV-encoded protein to assemble a ribonucleoprotein (RNP) complex. RNPs are expelled from the cell by the HBV envelope protein, which can assemble lipoprotein vesicles that budding into the lumen of the pre-Golgi compartment before being secreted. Furthermore, the HBV envelope protein also provides a mechanism for targeting HDV to uninfected cells, thereby ensuring HDV transmission.

[0004] Complications caused by HDV include a higher likelihood of experiencing liver failure in acute infections, and rapid progression to cirrhosis, along with an increased risk of developing liver cancer in chronic infections. When combined with hepatitis B virus, hepatitis D has the highest mortality rate of all hepatitis infections, at 20% (Fattovich G, Giustina G, Christensen E, Pantalena M, Zagni I, Realdi G, Schalm SW. Influence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B. Gut. 2000 Mar;46(3):420-6). The only approved treatment for chronic HDV infection is interferon-alpha. However, treatment of HDV with interferon-alpha is relatively inefficient and has not shown sufficient tolerance. Interferon-alpha treatment results in a persistent virological response in one-quarter of patients six months after treatment. Furthermore, nucleoside analogs (NAs) have been widely tested in hepatitis delta, but appear to be ineffective. Combination therapy using NAs and interferon has also proven disappointing (Zaigham Abbas, Minaam Abbas Management of hepatitis delta: Need for novel therapeutic Options. World J Gastroenterol 2015 August 28; 21(32): 9461-9465).

[0005] Therefore, new treatment options are needed, such as new therapies with neutralizing activity against hepatitis B and / or D infection. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Liang TJ, Block TM, McMahon BJ, Ghany MG, Urban S, Guo JT, Locarnini S, Zoulim F, Chang KM, Lok AS. Present and future therapies of hepatitis B: From discovery to cure. Hepatology. 2015 Aug 3. doi: 10.1002 / hep.28025. [Epub ahead of print] [Non-Patent Document 2] Fattovich G, Giustina G, Christensen E, Pantalena M, Zagni I, Realdi G, Schalm SW. Influence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B. Gut. 2000 Mar;46(3):420-6 [Non-Patent Document 3] Zaigham Abbas, Minaam Abbas Management of hepatitis delta: Need for novel therapeutic Options. World J Gastroenterol 2015 August 28; 21(32): 9461-9465 [Brief explanation of the drawing]

[0007] The figures provided herein are intended to illustrate in more detail the subject matter covered herein. The figures are not intended to limit the disclosure in any way.

[0008] [Figure 1]Figure 1 shows the binding of the anti-HBV antibody "HBC34-v35-GAALIE-MLNS" (rIgG1m17,1), which includes VH (as described in SEQ ID NO: 38) and VL (as described in SEQ ID NO: 57), and contains mutations G236A, A330L, I332E, M428L, and N434S in Fc, to HBsAg (left) and neutralization of HBV infection (right). The binding of antibodies at various concentrations to HBsAg of 10 genotypes ((A)~(J)) is shown on the left. As shown, neutralization was measured by HBsAg concentration (IU / ml) and HBeAg index. HBC34-v35-GAALIE-MLNS binds to the conserved structural epitope of HBsAg with picomolar affinity and potently neutralizes 10 HBV genotypes.

[0009] [Figure 2] Figure 2 provides size exclusion chromatography (SEC) data showing the high molecular weight species present in purified HBC34-v35-GAALIE-MLNS and HBC34-v35-MLNS (HBC34-v35-GAALIE-MLNS differs from HBC34-v35-GAALIE-MLNS in that it does not contain the G236A, A330L, and I332E Fc mutations) IgG after one week of incubation at room temperature (approximately 75 mg / mL). The largest peak is shown in the inset image in the upper right corner of the graph.

[0010] [Figure 3] Figure 3 provides size exclusion chromatography (SEC) data showing high molecular weight species over time in purified HBC34-v35 Fab. The largest peak is shown in the inset image in the upper right corner of the graph.

[0011] [Figure 4] Figure 4 shows the binding of HBC34-v35 IgG (top row of the panel) and Fab (bottom row of the panel) monomers (center panel) and enriched dimers (right panel) to HBsAg as measured by surface plasmon resonance (SPR). The left side is a schematic diagram showing the binding of IgG and Fab to HBsAg. The HBsAg concentrations used are as shown in the legend.

[0012] [Figure 5A] Figures 5A and 5B show (A) preparative SEC data illustrating the isolated HBC34-v35 recombinant Fab dimer (left peak) and (B) crystallization of the Fab dimer. [Figure 5B] Figures 5A and 5B show (A) preparative SEC data illustrating the isolated HBC34-v35 recombinant Fab dimer (left peak) and (B) crystallization of the Fab dimer.

[0013] [Figure 6A] Figures 6A and 6B show (A) the preparative SEC data of the isolated HBC34-v35 recombinant Fab monomer and (B) the crystallization of the Fab monomer. [Figure 6B] Figures 6A and 6B show (A) the preparative SEC data of the isolated HBC34-v35 recombinant Fab monomer and (B) the crystallization of the Fab monomer.

[0014] [Figure 7] Figure 7 provides (left) a schematic diagram of dimerization involving the antibody CDR and (right) a ribbon model showing the HBC34-v35 Fab dimer.

[0015] [Figure 8] Figure 8 illustrates (right) the involvement of VL-VL interactions in HBC34-v35 dimerization, and (left) a summary of interactions within L-CDR2.

[0016] [Figure 9] Figure 9 provides another diagram of the HBC34-v35 Fab-Fab interaction in L-CDR2 and the light chain framework region.

[0017] [Figure 10] Figure 10 provides diagrams of the conformations of the HBC34-v35 Fab dimer (left) and the Fab monomer (right).

[0018] [Figure 11A] Figures 11A-11C show the reduction in dimerization by a variant Fab manipulated from HBC34-v35 (shown as "L-CDR2 GL Fab" in Figure 11A, and also referred to herein as HBC34-v36), which has three L-CDR2 residues reversed in its germline sequence compared to HBC34-v35 Fab. (A) Percent dimers in purified Fab by absolute size exclusion chromatography (aSEC) at day 0 and days 5-7. (B) SEC analysis of pressurized L-CDR2 GL Fab sample at day 0. (C) SEC analysis of pressurized L-CDR2 GL Fab sample at day 5. [Figure 11B-1] Figures 11A-11C show the reduction in dimerization by a variant Fab manipulated from HBC34-v35 (shown as "L-CDR2 GL Fab" in Figure 11A, and also referred to herein as HBC34-v36), which has three L-CDR2 residues reversed in its germline sequence compared to HBC34-v35 Fab. (A) Percent dimers in purified Fab by absolute size exclusion chromatography (aSEC) at day 0 and days 5-7. (B) SEC analysis of pressurized L-CDR2 GL Fab sample at day 0. (C) SEC analysis of pressurized L-CDR2 GL Fab sample at day 5. [Figure 11B-2]Figures 11A-11C show the reduction in dimerization by a variant Fab manipulated from HBC34-v35 (shown as "L-CDR2 GL Fab" in Figure 11A, and also referred to herein as HBC34-v36), which has three L-CDR2 residues reversed in its germline sequence compared to HBC34-v35 Fab. (A) Percent dimers in purified Fab by absolute size exclusion chromatography (aSEC) at day 0 and days 5-7. (B) SEC analysis of pressurized L-CDR2 GL Fab sample at day 0. (C) SEC analysis of pressurized L-CDR2 GL Fab sample at day 5. [Figure 11C-1] Figures 11A-11C show the reduction in dimerization by a variant Fab manipulated from HBC34-v35 (shown as "L-CDR2 GL Fab" in Figure 11A, and also referred to herein as HBC34-v36), which has three L-CDR2 residues reversed in its germline sequence compared to HBC34-v35 Fab. (A) Percent dimers in purified Fab by absolute size exclusion chromatography (aSEC) at day 0 and days 5-7. (B) SEC analysis of pressurized L-CDR2 GL Fab sample at day 0. (C) SEC analysis of pressurized L-CDR2 GL Fab sample at day 5. [Figure 11C-2] Figures 11A-11C show the reduction in dimerization by a variant Fab manipulated from HBC34-v35 (shown as "L-CDR2 GL Fab" in Figure 11A, and also referred to herein as HBC34-v36), which has three L-CDR2 residues reversed in its germline sequence compared to HBC34-v35 Fab. (A) Percent dimers in purified Fab by absolute size exclusion chromatography (aSEC) at day 0 and days 5-7. (B) SEC analysis of pressurized L-CDR2 GL Fab sample at day 0. (C) SEC analysis of pressurized L-CDR2 GL Fab sample at day 5.

[0019] [Figure 12] Figure 12 shows the binding of HBC34-v35 and HBC34-v36 to HBsAg as determined by ELISA. The antibody was expressed as IgG1 with wild-type Fc (allotype G1m17,1).

[0020] [Figure 13] Figure 13 shows the in vitro neutralization of HBV genotype D infection by HBC34-v35 and HBC34-v36. Antibodies were expressed as IgG1 with wild-type Fc (allotype G1m17,1). Neutralization was measured by the percentage of target cells expressing HBsAg (left) or HBeAg (right). N=1 experiment.

[0021] [Figure 14A] Figures 14A-14E show the binding of additional antibodies HBC34-v37-HBC34-v50 (unpurified supernatant from CHO cells) to HBsAg as determined by ELISA. Purified HBC34-v35 was included as a control. The antibodies were expressed as IgG1 with wild-type Fc (allotype G1m17,1). The calculated EC50 values ​​are shown below each graph. [Figure 14B] Figures 14A-14E show the binding of additional antibodies HBC34-v37-HBC34-v50 (unpurified supernatant from CHO cells) to HBsAg as determined by ELISA. Purified HBC34-v35 was included as a control. The antibodies were expressed as IgG1 with wild-type Fc (allotype G1m17,1). The calculated EC50 values ​​are shown below each graph. [Figure 14C] Figures 14A-14E show the binding of additional antibodies HBC34-v37-HBC34-v50 (unpurified supernatant from CHO cells) to HBsAg as determined by ELISA. Purified HBC34-v35 was included as a control. The antibodies were expressed as IgG1 with wild-type Fc (allotype G1m17,1). The calculated EC50 values ​​are shown below each graph. [Figure 14D] Figures 14A-14E show the binding of additional antibodies HBC34-v37-HBC34-v50 (unpurified supernatant from CHO cells) to HBsAg as determined by ELISA. Purified HBC34-v35 was included as a control. The antibodies were expressed as IgG1 with wild-type Fc (allotype G1m17,1). The calculated EC50 values ​​are shown below each graph. [Figure 14E] Figures 14A-14E show the binding of additional antibodies HBC34-v37-HBC34-v50 (unpurified supernatant from CHO cells) to HBsAg as determined by ELISA. Purified HBC34-v35 was included as a control. The antibodies were expressed as IgG1 with wild-type Fc (allotype G1m17,1). The calculated EC50 values ​​are shown below each graph.

[0022] [Figure 15] Figure 15 shows the neutralization of HBV genotype D with HBC34-v35 and certain antibodies of the present disclosure, using HBeAg as the viral readout information. The calculated EC50 values ​​for each mAb are shown on the right. HBC34-v35 (purified IgG and supernatant) and HBC34-v36 (purified IgG) were used as controls.

[0023] [Figure 16A] Figures 16A–16D provide size exclusion chromatography (SEC) data showing high molecular weight species (HMWS) present in HBC34-v35 and nine purified variant antibodies of the present disclosure over a 32-day period. HBC34-v35 and variant antibodies were concentrated to approximately 25 mg / mL and incubated at different temperatures. HMWS were evaluated by SEC on days -1, 0, 5, 15, and 32. The sample on day -1 was evaluated before concentration. The antibody compositions were incubated at 4°C (Figure 16A), 25°C (Figure 16B), or 40°C (Figure 16C) over the course of the experiment. The frequency of HMWS after 32 days of incubation at 40°C is summarized in Figure 16D. [Figure 16B]Figures 16A–16D provide size exclusion chromatography (SEC) data showing high molecular weight species (HMWS) present in HBC34-v35 and nine purified variant antibodies of the present disclosure over a 32-day period. HBC34-v35 and variant antibodies were concentrated to approximately 25 mg / mL and incubated at different temperatures. HMWS were evaluated by SEC on days -1, 0, 5, 15, and 32. The sample on day -1 was evaluated before concentration. The antibody compositions were incubated at 4°C (Figure 16A), 25°C (Figure 16B), or 40°C (Figure 16C) over the course of the experiment. The frequency of HMWS after 32 days of incubation at 40°C is summarized in Figure 16D. [Figure 16C] Figures 16A–16D provide size exclusion chromatography (SEC) data showing high molecular weight species (HMWS) present in HBC34-v35 and nine purified variant antibodies of the present disclosure over a 32-day period. HBC34-v35 and variant antibodies were concentrated to approximately 25 mg / mL and incubated at different temperatures. HMWS were evaluated by SEC on days -1, 0, 5, 15, and 32. The sample on day -1 was evaluated before concentration. The antibody compositions were incubated at 4°C (Figure 16A), 25°C (Figure 16B), or 40°C (Figure 16C) over the course of the experiment. The frequency of HMWS after 32 days of incubation at 40°C is summarized in Figure 16D. [Figure 16D] Figures 16A–16D provide size exclusion chromatography (SEC) data showing high molecular weight species (HMWS) present in HBC34-v35 and nine purified variant antibodies of the present disclosure over a 32-day period. HBC34-v35 and variant antibodies were concentrated to approximately 25 mg / mL and incubated at different temperatures. HMWS were evaluated by SEC on days -1, 0, 5, 15, and 32. The sample on day -1 was evaluated before concentration. The antibody compositions were incubated at 4°C (Figure 16A), 25°C (Figure 16B), or 40°C (Figure 16C) over the course of the experiment. The frequency of HMWS after 32 days of incubation at 40°C is summarized in Figure 16D.

[0024] [Figure 17A] Figures 17A–17J show the binding of 10 genotypes ((A)–(J)) to HBsAg by HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 17B] Figures 17A–17J show the binding of 10 genotypes ((A)–(J)) to HBsAg by HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 17C] Figures 17A–17J show the binding of 10 genotypes ((A)–(J)) to HBsAg by HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 17D] Figures 17A–17J show the binding of 10 genotypes ((A)–(J)) to HBsAg by HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 17E]Figures 17A–17J show the binding of 10 genotypes ((A)–(J)) to HBsAg by HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 17F] Figures 17A–17J show the binding of 10 genotypes ((A)–(J)) to HBsAg by HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 17G] Figures 17A–17J show the binding of 10 genotypes ((A)–(J)) to HBsAg by HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 17H] Figures 17A–17J show the binding of 10 genotypes ((A)–(J)) to HBsAg by HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 17I] Figures 17A–17J show the binding of 10 genotypes ((A)–(J)) to HBsAg by HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 17J]Figures 17A–17J show the binding of 10 genotypes ((A)–(J)) to HBsAg by HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control.

[0025] [Figure 18A] Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) relative to antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 18B] Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) relative to antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 18C] Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) relative to antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 18D]Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 18E] Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 18F] Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 18G] Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 18H] Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) against antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 18I] Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) relative to antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 18J] Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) relative to antibody concentration (ng / ml). A false stain is included as a negative control. [Figure 18K] Figures 18A–18K show the binding of HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50 to HBsAg genotype D and 10 HBsAg genotype D variants as determined by FACS. Data are reported as mean fluorescence intensity (MFI) relative to antibody concentration (ng / ml). A false stain is included as a negative control.

[0026] [Figure 19] Figure 19 shows the antibody titers produced by transfection with HBC34-v35, HBC34-v40, HBC34-v44, HBC34-v45, and HBC34-v50. Both 5 ml and 100 ml transfection systems were evaluated, and the 100 ml systems were tested in 2- or 3-series configurations. Antibody titers from individual 5 ml and 100 ml tests, as well as the average titer from the 100 ml tests, are shown (reported as mg / L).

[0027] [Figure 20]Figure 20 shows SEC data reflecting the thermostability of HBC34-v35 ("HBC35" in the legend), HBC34-v40 ("HBC40"), HBC34-v44 ("HBC44"), HBC34-v45 ("HBC45"), and HBC34-v50 ("HBC50"). The antibodies were concentrated to 25 mg / ml and incubated at 40°C for 4 days, after which HMWS was quantified.

[0028] [Figure 21A] Figures 21A–21C, 22A–22C, and 23A–23C show the light chain amino acid residues selected to manipulate the HBC34-v35 to reduce aggregation. Figures 21A, 22A, and 23A show the HBC34-v35 light chain CDR2 residues selected for manipulation. Figures 21B, 22B, and 23B show the framework residues selected for manipulation. Figures 21C, 22C, and 23C show the sequence alignment of the partial VL sequence of the variant antibody. [Figure 21B] Figures 21A–21C, 22A–22C, and 23A–23C show the light chain amino acid residues selected to manipulate the HBC34-v35 to reduce aggregation. Figures 21A, 22A, and 23A show the HBC34-v35 light chain CDR2 residues selected for manipulation. Figures 21B, 22B, and 23B show the framework residues selected for manipulation. Figures 21C, 22C, and 23C show the sequence alignment of the partial VL sequence of the variant antibody. [Figure 21C] Figures 21A–21C, 22A–22C, and 23A–23C show the light chain amino acid residues selected to manipulate the HBC34-v35 to reduce aggregation. Figures 21A, 22A, and 23A show the HBC34-v35 light chain CDR2 residues selected for manipulation. Figures 21B, 22B, and 23B show the framework residues selected for manipulation. Figures 21C, 22C, and 23C show the sequence alignment of the partial VL sequence of the variant antibody. [Figure 22A]Figures 21A–21C, 22A–22C, and 23A–23C show the light chain amino acid residues selected to manipulate the HBC34-v35 to reduce aggregation. Figures 21A, 22A, and 23A show the HBC34-v35 light chain CDR2 residues selected for manipulation. Figures 21B, 22B, and 23B show the framework residues selected for manipulation. Figures 21C, 22C, and 23C show the sequence alignment of the partial VL sequence of the variant antibody. [Figure 22B] Figures 21A–21C, 22A–22C, and 23A–23C show the light chain amino acid residues selected to manipulate the HBC34-v35 to reduce aggregation. Figures 21A, 22A, and 23A show the HBC34-v35 light chain CDR2 residues selected for manipulation. Figures 21B, 22B, and 23B show the framework residues selected for manipulation. Figures 21C, 22C, and 23C show the sequence alignment of the partial VL sequence of the variant antibody. [Figure 22C] Figures 21A–21C, 22A–22C, and 23A–23C show the light chain amino acid residues selected to manipulate the HBC34-v35 to reduce aggregation. Figures 21A, 22A, and 23A show the HBC34-v35 light chain CDR2 residues selected for manipulation. Figures 21B, 22B, and 23B show the framework residues selected for manipulation. Figures 21C, 22C, and 23C show the sequence alignment of the partial VL sequence of the variant antibody. [Figure 23A] Figures 21A–21C, 22A–22C, and 23A–23C show the light chain amino acid residues selected to manipulate the HBC34-v35 to reduce aggregation. Figures 21A, 22A, and 23A show the HBC34-v35 light chain CDR2 residues selected for manipulation. Figures 21B, 22B, and 23B show the framework residues selected for manipulation. Figures 21C, 22C, and 23C show the sequence alignment of the partial VL sequence of the variant antibody. [Figure 23B]Figures 21A–21C, 22A–22C, and 23A–23C show the light chain amino acid residues selected to manipulate the HBC34-v35 to reduce aggregation. Figures 21A, 22A, and 23A show the HBC34-v35 light chain CDR2 residues selected for manipulation. Figures 21B, 22B, and 23B show the framework residues selected for manipulation. Figures 21C, 22C, and 23C show the sequence alignment of the partial VL sequence of the variant antibody. [Figure 23C] Figures 21A–21C, 22A–22C, and 23A–23C show the light chain amino acid residues selected to manipulate the HBC34-v35 to reduce aggregation. Figures 21A, 22A, and 23A show the HBC34-v35 light chain CDR2 residues selected for manipulation. Figures 21B, 22B, and 23B show the framework residues selected for manipulation. Figures 21C, 22C, and 23C show the sequence alignment of the partial VL sequence of the variant antibody. [Modes for carrying out the invention]

[0029] Detailed explanation This disclosure relates to the field of immunotherapy for hepatitis B virus (HBV) and hepatitis delta virus (HDV). The disclosed binding proteins, such as antibodies, antigen-binding fragments, and fusion proteins, are capable of binding to epitopes located in the antigenic loop region of the S domain of the HBV envelope protein (HBsAg) and are capable of neutralizing HBV infection and, in some embodiments, HDV infection.

[0030] The binding proteins disclosed herein have advantageous production characteristics (e.g., reduced antibody dimerization and / or increased production in host cells) compared to a reference anti-HBV antibody, for example, the CDR of "HBC34-v35" disclosed in PCT Publication No. WO2020 / 132091, and optionally VH and VL. Briefly, while the HBC34-v35 antibody has favorable binding and neutralizing properties, it may form antibody dimers via light chain interactions during antibody production / purification, as disclosed herein. The HBC34-v35 dimer has a reduced ability to bind to HBsAg compared to the HBC34-v35 antibody monomer. Reducing dimerization can improve, for example, the efficiency of antibody (or antigen-binding fragment) production and the efficacy of the antibody (or antigen-binding fragment) dose.

[0031] In certain embodiments, the binding proteins disclosed herein can bind to any or all known HBsAg genotypes, as well as HBsAg variants, and can neutralize HBV infection, as well as HDV infection. In certain embodiments, the binding proteins disclosed herein can bind to HBV and / or HDV, and / or neutralize them, with similar or even increased efficacy compared to HBC34-v35.

[0032] Nucleic acids encoding such binding proteins, and host cells expressing such binding proteins, are also provided herein. Furthermore, this disclosure provides methods for using the binding proteins described herein in the diagnosis, prevention, and treatment of diseases, as well as in screening methods.

[0033] For example, embodiments of antibodies, antigen-binding fragments, and fusion proteins described herein may be used in methods for preventing, treating, attenuating, or diagnosing HBV and HDV. In certain embodiments, the antibodies, antigen-binding fragments, and fusion proteins described herein bind to two or more different genotypes and two or more different infectious variants of the hepatitis B virus surface antigen. In specific embodiments, the antibodies, antigen-binding fragments, and fusion proteins described herein bind to all known genotypes and all known infectious variants of the hepatitis B virus surface antigen.

[0034] This disclosure also provides a method for treating chronic HBV infection in a subject requiring such treatment, comprising administering an anti-HBV antibody or antigen-binding fragment to the subject in combination with an agent that reduces the HBV antigen load. This disclosure also provides a method for treating chronic HBV infection in a subject requiring such treatment, comprising administering an anti-HBV antibody or antigen-binding fragment to the subject in combination with an inhibitor of HBV gene expression.

[0035] In some of the methods, compositions for use, or uses described herein, the agents that reduce the HBV antigen load or inhibitors of HBV gene expression are RNAi agents (e.g., siRNA, e.g., HBV001, HBV002, or HBV003).

[0036] Before describing this disclosure in more detail, providing definitions of certain terms used herein may aid in its understanding. Additional definitions are provided throughout this disclosure.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art.

[0038] Throughout this disclosure, unless the context requires otherwise, the term “comprise,” and its variations, e.g., “comprises,” and “comprising,” are used synonymously with, for example, “having,” “has,” “including,” and “includes,” and are understood to imply that they include, but do not exclude, any other unmentioned members, ratios, integers, concentrations, or steps, as they are specified, including fractions thereof, where appropriate, such as one-tenth and one-hundredth of an integer. Any range of concentration, percentage, ratio, or integer should be understood to include, unless otherwise indicated, any integer values ​​within the enumerated range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer). Similarly, any range of numbers enumerated herein with respect to any physical characteristics such as polymer subunits, size, or thickness should be understood to include, unless otherwise indicated, any integers within the enumerated range.

[0039] The term "consisting essentially of" is not equivalent to "comprising" and refers to a material or step specified in the claims, or a step that does not substantially affect the fundamental characteristics of the subject matter described in the claims. For example, a protein domain, region, or module (e.g., a binding domain), or protein, is "consistently of" a particular amino acid sequence if its amino acid sequence, in combination, contributes at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein, and does not substantially affect the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of the binding protein) (i.e., does not reduce the activity by more than 50%, such as 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%), and includes elongation, deletion, mutation, or a combination thereof (e.g., amino acids at the amino or carboxy terminus, or between domains).

[0040] Furthermore, it should be understood that individual compounds or groups of compounds resulting from various combinations of structures and substituents described herein are disclosed to the same extent as each compound or group of compounds would be described individually. Therefore, the selection of a particular structure or particular substituent is within the scope of this disclosure.

[0041] The terms “a,” “an,” and “the,” as well as similar references, used in connection with describing this disclosure (including in relation to the claims), shall be construed to cover both singular and plural forms unless otherwise indicated herein or unless clearly inconsistent with the context. The use of alternatives (e.g., “or”) should be understood to mean one of those alternatives, both, or any combination thereof. The enumeration of value ranges herein is intended to serve as a simplified way of referring individually to each separate value that falls within that range. Unless otherwise indicated herein, each individual value is incorporated into this disclosure as it is individually enumerated herein. The language herein should not be construed to indicate any non-claimed element as essential to the practice of the subject matter disclosed herein.

[0042] The word “substantially” does not exclude “completely”; for example, a composition “substantially” containing Y may not contain Y completely. In certain embodiments, “substantially” means a given amount, effect, or activity of a composition, method, or use of the Disclosure compared to a reference composition, method, or use, and describes a reduction in amount, effect, or activity of 50% or less of the amount, effect, or activity of the reference composition, method, or use, e.g., 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%, or less.

[0043] As used herein, the term “about” means ±20% of the indicated range, value, or structure unless otherwise indicated. In certain embodiments, “about” includes ±15%, ±10%, or ±5%.

[0044] "As needed" or "as required" means that the elements, components, events, or circumstances described thereafter may or may not be present, and that the description includes examples of the presence and absence of such elements, components, events, or circumstances.

[0045] As used herein, “amino acids” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as later modified amino acids, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a different structure from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0046] As used herein, the terms “peptide,” “polypeptide,” and “protein,” as well as variations thereof, refer to molecules comprising at least two amino acids linked to one another by (ordinary or modified) peptide bonds. Thus, a protein or polypeptide comprises a polymer of amino acid residues. For example, a peptide, polypeptide, or protein may comprise, or be composed of, a plurality of amino acids selected from the 20 amino acids defined by the genetic code, or from amino acid analogs or mimics, each linked to at least one another by peptide bonds. A peptide, polypeptide, or protein may comprise, or be composed of, L-amino acids and / or D-amino acids (or their analogs or mimics). The terms “peptide,” “polypeptide,” and “protein” also include “peptide mimics,” defined as peptide analogs containing non-peptidic structural elements, which are capable of mimicking or antagonizing the biological action(s) of a native parent peptide. In certain embodiments, peptide mimics lack features such as enzymatically cleavable peptide bonds.

[0047] Peptides, polypeptides, or proteins may contain, or be composed of, amino acids other than the 20 amino acids defined by the genetic code, in addition to these amino acids. In certain embodiments, with respect to this disclosure, peptides, polypeptides, or proteins may contain amino acids modified by natural processes, e.g., post-translational maturation processes, or by chemical processes (e.g., synthetic processes), which are known in the art and are described herein. Such modifications may appear anywhere in the polypeptide; e.g., in the peptide backbone; in the amino acid chain; or at the carboxyl or amino terminus. Peptides or polypeptides may be branched, e.g., after ubiquitination, or may be cyclic with or without branching. The terms “peptide,” “polypeptide,” and “protein” also include modified peptides, polypeptides, and proteins. For example, peptide, polypeptide, or protein modifications may include acetylation, acylation, ADP-ribosylation, amidation, covalent fixation of nucleotides or nucleotide derivatives, covalent fixation of lipids or lipid derivatives, covalent fixation of phosphatidylinositol, covalent or non-covalent crosslinking, cyclization, disulfide bond formation, demethylation, glycosylation including pegylation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfation, amino acid addition, such as arginylation or ubiquitination.Such modifications are described in the literature (see Proteins Structure and Molecular Properties (1993) 2nd Ed., TE Creighton, New York; Post-translational Covalent Modifications of Proteins (1983) BC Johnson, Ed., Academic Press, New York; Seifter et al. (1990) Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182: 626-646 and Rattan et al., (1992) Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663: 48-62). Therefore, the terms “peptide,” “polypeptide,” and “protein” may include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, etc. Variants of the proteins, peptides, and polypeptides of this disclosure are also intended. In certain embodiments, the variant proteins, peptides, and polypeptides include or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of a defined amino acid sequence or reference amino acid sequence described herein.

[0048] As used herein, "(poly)peptide" and "protein" may be used interchangeably with respect to amino acid residues linked by peptide bonds, for example, polymers of multiple amino acid monomers.

[0049] A "nucleic acid molecule," "polynucleotide," or "polynucleic acid" refers to a polymer compound containing covalently linked nucleotides, which may consist of natural subunits (e.g., purine or pyrimidine bases) or unnatural subunits (e.g., morpholine rings). Examples of purine bases include adenine, guanine, hypoxanthine, and xanthine, while examples of pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid monomers may be linked by phosphodiester bonds or analogs of such links. Analogs of phosphodiester links include phosphorothioates, phosphorodioates, phosphoroselenoates, phosphorodiodiselenoates, phosphoranilothioates, phosphoranilidates, and phosphoramidates.

[0050] Nucleic acid molecules include polydeoxyribonucleic acid (DNA), including polyribonucleic acid (RNA), cDNA, genomic DNA, and synthetic DNA, all of which can be single-stranded or double-stranded. In the case of single-stranded molecules, the nucleic acid molecule can be a coding strand or a non-coding (antisense) strand. MicroRNA, siRNA, viral genomic RNA, and synthetic RNA are also considered. Polynucleotides (including oligonucleotides) and their fragments can be generated, for example, by polymerase chain reaction (PCR) or in vitro translation, or by ligation, cleavage, endonuclease activity, or exonuclease activity.

[0051] Nucleic acid molecules that encode an amino acid sequence contain all nucleotide sequences that encode the same amino acid sequence. Some versions of a nucleotide sequence may also contain introns to the extent that introns may be removed by cotranscription or posttranscriptional mechanisms. Different nucleotide sequences may encode the same amino acid sequence as a result of genetic coding duplication or degeneracy, by splicing, or both.

[0052] Variants of the nucleic acid molecules of this disclosure are also contemplated. The variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the defined or reference polynucleotide nucleic acid molecules described herein, or they hybridize to polynucleotides under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate, about 65–68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide, about 42°C. The nucleic acid molecule variants retain the ability to encode a fusion protein or its binding domain having functionalities described herein, such as specific binding of a target molecule.

[0053] As used herein, the term “sequence variant” means any sequence having one or more changes compared to a reference sequence, thereby the reference sequence being any publicly available sequence and / or any sequence listed in the “Sequence and Sequence Number List” (Sequence Listing) herein. Thus, the term “sequence variant” includes nucleotide sequence variants and amino acid sequence variants. In certain embodiments of a sequence variant relating to a nucleotide sequence, the reference sequence is also a nucleotide sequence, while in certain embodiments of a sequence variant relating to an amino acid sequence, the reference sequence is also an amino acid sequence. As used herein, a “sequence variant” may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference sequence.

[0054] "Sequence identity percentage" refers to the relationship between two or more sequences determined by comparing them. Methods for determining sequence identity may be designed to provide the best possible match between the sequences being compared. For example, sequences may be aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). Furthermore, non-homologous sequences may be ignored for comparison purposes. Sequence identity percentages referred to herein are calculated over the length of the reference sequence unless otherwise specified. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignment and calculation of identity percentages may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithms used in the BLAST program can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. In the context of this disclosure, when sequence analysis software is used for analysis, it is understood that the results of the analysis are based on the “default values” of the program referred to. “Default values” means any set of values ​​or parameters that are originally loaded into the software when it is first initialized.

[0055] With respect to nucleic acid (nucleotide) sequences, a “sequence variant” has an altered sequence in which one or more nucleotides in the reference sequence are deleted or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred herein by standard single-letter designations (A, C, G, or T). Due to the degeneracy of the genetic code, a “sequence variant” of a nucleotide sequence may or may not result in changes in the respective reference amino acid sequence, i.e., changes in the amino acid “sequence variant.” In certain embodiments, a nucleotide sequence variant may not result in an amino acid sequence variant (e.g., a silent mutation). In some embodiments, a nucleotide sequence variant that results in one or more “non-silent” mutations is intended. In some embodiments, the nucleotide sequence variants of the Disclosure encode amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference amino acid sequence. The nucleotides and amino sequences disclosed herein also refer to codon-optimized versions of reference or wild-type nucleotide or amino acid sequences. In any of the embodiments described herein, the polynucleotides of the Disclosure may be codon-optimized for host cells containing the polynucleotide. Codon optimization may be performed using known techniques and tools, for example, using the GenScript® OptimumGene® tool or the GeneArt Gene Synthesis Tool (Thermo Fisher Scientific). Codon-optimized sequences include partially codon-optimized sequences (i.e., at least one codon is optimized for expression in host cells) and fully codon-optimized sequences.

[0056] With respect to amino acid sequences, a "sequence variant" has a modified sequence in which one or more amino acids are deleted, substituted, or inserted compared to a reference amino acid sequence. As a result of the modification, such a sequence variant has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference amino acid sequence. For example, a variant sequence having 10 or fewer changes per 100 amino acids of the reference sequence, i.e., any combination of deletions, insertions, or substitutions, is "at least 90% identical" to the reference sequence.

[0057] A "conservative substitution" refers to an amino acid substitution that does not significantly affect or alter the binding characteristics of a particular protein. Generally, a conservative substitution is one in which the substituted amino acid residue is replaced by an amino acid residue with a similar side chain. Conservative substitutions include substitutions found in one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). In addition, or alternatively, amino acids can be classified into groups of conserved substitutions based on similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, the aliphatic classification may include Gly, Ala, Val, Leu, and Ile, depending on the purpose of the substitution. Other groups of conserved substitutions include: sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. For further information, see Creighton (1984) This can be found in Proteins, WH Freeman and Company.

[0058] Amino acid sequence insertions can include amino-terminus and / or carboxyl-terminus fusions, as well as intrasequence insertions of single or multiple amino acid residues, ranging in length from one residue to polypeptides containing 100 or more residues. Examples of terminal insertions include the fusion of an amino acid sequence to the N-terminus or C-terminus of a reporter molecule or enzyme.

[0059] In general, changes in sequence variants do not cause loss of or significant reduction of the desired functionality of the respective reference sequence. For example, it is preferable that the variant sequences of the present disclosure do not significantly reduce or negate the functionality of the antibody or antigen-binding fragment to bind to the same epitope, the functionality to adequately neutralize HBV and HDV infection, and / or cause or increase antibody dimerization, and / or are not produced at lower titers in host cells, compared to antibodies or antigen-binding fragments having (or encoded by) the reference sequence.

[0060] As used herein, a nucleic acid sequence or amino acid sequence "derived from" a specified nucleic acid, peptide, polypeptide, or protein refers to the origin of that nucleic acid, peptide, polypeptide, or protein. A nucleic acid sequence or amino acid sequence derived from a particular sequence may have an amino acid sequence that is essentially identical to the sequence or portion from which it originates, and thereby "essentially identical" includes the sequence variants as defined above. A nucleic acid sequence or amino acid sequence derived from a particular peptide or protein may originate from a corresponding domain in a particular peptide or protein. In this regard, "corresponding" refers to the possession of the same functionality or feature of the interest. For example, "extracellular domain" may correspond to another "extracellular domain" (of another protein), or "transmembrane domain" may correspond to another "transmembrane domain" (of another protein). Thus, the "corresponding" portions of peptides, proteins, and nucleic acids are readily identifiable to those skilled in the art. Similarly, a sequence "derived from" another (e.g., "source") sequence can be identified by those skilled in the art as having its origin in the source sequence.

[0061] A nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be identical to the starting nucleic acid, peptide, polypeptide, or protein from which it originates. However, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may also have one or more mutations compared to the starting nucleic acid, peptide, polypeptide, or protein from which it originates, and in particular, a nucleic acid sequence or amino acid sequence derived from another nucleic acid, peptide, polypeptide, or protein may be a functional sequence variant of the starting nucleic acid, peptide, polypeptide, or protein from which it originates. For example, in a peptide / protein, one or more amino acid residues may be substituted with other amino acid residues, or one or more amino acid residue insertions or deletions may exist.

[0062] As used herein, the term “mutation” refers to changes in a nucleic acid sequence and / or amino acid sequence compared to a reference sequence, e.g., the corresponding genome, wild type, or reference sequence. For example, a mutation compared to a reference genome sequence may be, for example, a somatic mutation (naturally occurring), a spontaneous mutation, an induced mutation, e.g., a mutation induced by an enzyme, chemical or radiation, or a mutation obtained by site-directed mutagenesis (a molecular biological method for causing specific and intentional changes in a nucleic acid sequence and / or amino acid sequence). Thus, the term “mutation” or “mutate” should be understood to also include, for example, physically causing or inducing a mutation in a nucleic acid sequence or amino acid sequence. Mutations include substitutions, deletions and / or insertions of one or more nucleotides or amino acids, and inversions of several consecutive nucleotides or amino acids. To achieve a mutation in an amino acid sequence, the mutation may be introduced into the nucleotide sequence encoding the amino acid sequence in order to express a (recombinant) mutated polypeptide. Mutations can be achieved, for example, by altering the codon of a nucleic acid molecule encoding a single amino acid (e.g., by site-directed mutagenesis) to provide a codon encoding a different amino acid or a codon encoding the same amino acid (e.g., by altering one, two, or three nucleotide bases within it), or by synthesizing sequence variants.

[0063] "Functional variant" means a polypeptide or polynucleotide that is structurally similar to or substantially structurally similar to the parent compound or reference compound of this disclosure, but has a slightly different composition (e.g., one base, atom or functional group is different, added, or removed), and as a result, the polypeptide or encoded polypeptide can perform at least one function of the parent polypeptide with at least 50% efficiency, preferably at at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the activity of the parent polypeptide. In other words, a functional variant of the polypeptide of this disclosure or the polypeptide encoded herein has “similar binding,” “similar affinity,” or “similar activity” if, in a selected assay such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining for measuring association constant (Ka) or dissociation constant (KD)), the functional variant exhibits a reduction in performance of 50% or less compared to the parent compound or reference polypeptide.

[0064] As used herein, “functional portion” or “functional fragment” means a polypeptide or polynucleotide comprising only the domain, portion, or fragment of a parent compound or reference compound, wherein the polypeptide or encoded polypeptide retains at least 50% of the activity associated with the domain, portion, or fragment of the parent compound or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the activity of the parent polypeptide, or provides a biological benefit (e.g., effector function). A “functional portion” or “functional fragment” of a polypeptide or encoded polypeptide in this disclosure has “similar binding” or “similar activity” if, in a selected assay, the functional portion or fragment exhibits a performance reduction of 50% or less (preferably 20% or less or 10% or less, or, in terms of affinity, no more than a logarithmic difference compared to the parent or reference) compared to the parent polypeptide or reference polypeptide.

[0065] The term “isolated” means that a material has been removed from its original environment (e.g., its natural environment, if it exists naturally). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide is isolated if it has been separated from some or all of the material coexisting in the natural system. Such a nucleic acid may be part of a vector and / or such a nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate), and such a vector or composition is still isolated in that it is not part of the natural environment for the nucleic acid or polypeptide. In some embodiments, “isolated” may describe antibodies, antigen-binding fragments, fusion proteins, polynucleotides, vectors, host cells, or compositions located outside the human body.

[0066] The term "gene" refers to a segment of DNA or RNA involved in the production of polypeptide chains; in certain contexts, the term "gene" includes the regions preceding and following the coding region (e.g., the 5' untranslated region (UTR) and 3'UTR) as well as the intervening sequences (introns) between individual coding segments (exons).

[0067] In relation to the insertion of nucleic acid molecules into cells, the term “introduced” means “transfection,” or “transformation,” and includes reference to the uptake of nucleic acid molecules into eukaryotic or prokaryotic cells, which may be taken up into the cell’s genome (e.g., chromosomes, plasmids, plastids, or mitochondrial DNA), converted into autonomous replicons, or transiently expressed (e.g., transfected mRNA).

[0068] The term “recombinant,” as used herein (e.g., recombinant antibody, recombinant protein, recombinant nucleic acid), refers to any molecule (such as an antibody, protein, nucleic acid) that is prepared, expressed, created, or isolated by recombinant means and does not exist in nature. “Recombinant” may be used synonymously with “manipulated” or “unnatural” and may refer to an organism, microorganism, cell, nucleic acid molecule, or vector that contains at least one genetic alteration or is modified by the introduction of an exogenous nucleic acid molecule, such alteration or modification being introduced by genetic engineering (i.e., human intervention). Examples of genetic alterations include modifications that introduce an expressible nucleic acid molecule encoding a protein, fusion protein, or enzyme, or the addition, deletion, substitution, or other functional disruption of the cellular genetic material of other nucleic acid molecules. Additional modifications include, for example, non-coding regulatory regions in which the modification alters the expression of a polynucleotide, gene, or operon.

[0069] As used herein, “heterogeneous,” “non-endogenous,” or “exogenous” means any gene, protein, compound, nucleic acid molecule, or activity that is not native to the host cell or subject, or any gene, protein, compound, nucleic acid molecule, or activity that is native to the host cell or subject but has been modified. Examples of heterogeneous, non-endogenous, or exogenous genes, proteins, compounds, or nucleic acid molecules that are mutated or otherwise modified so that they differ in structure, activity, or both between the native and modified genes, proteins, compounds, or nucleic acid molecules. In certain embodiments, a heterogeneous, non-endogenous, or exogenous gene, protein, or nucleic acid molecule may not be endogenous to the host cell or subject, but instead, a nucleic acid encoding such a gene, protein, or nucleic acid molecule may be added to the host cell by conjugation, transformation, transfection, electroporation, etc., where the added nucleic acid molecule may be integrated into the host cell’s genome or may exist as extrachromosomal gene material (e.g., as a plasmid or other self-replicating vector). The terms “homologous” or “homogenetic” refer to genes, proteins, compounds, nucleic acid molecules, or activities found in or derived from a host cell, species, or strain. For example, a heterogeneous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene, encoding a homologous polypeptide or activity, but the polynucleotide or polypeptide may have a modified structure, sequence, expression level, or any combination thereof. Non-endogenous polynucleotides or genes, and the polypeptides or activities they encode, may originate from the same species, different species, or a combination thereof.

[0070] As used herein, the terms “endogenous” or “native” refer to polynucleotides, genes, proteins, compounds, molecules, or activities that are normally present in a host cell or subject.

[0071] As used herein, the term "expression" refers to the process by which polypeptides are produced based on the coding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional regulation, post-transcriptional modification, translation, post-translational regulation, post-translational modification, or any combination thereof. The nucleic acid molecule being expressed is typically operably ligated to an expression control sequence (e.g., a promoter).

[0072] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is influenced by the other. For example, a promoter is operably linked to a coding sequence if it can influence the expression of that sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Not linked" means that the related genetic elements are not closely related to each other, and the function of one does not affect the other.

[0073] As described herein, one or more heterogeneous nucleic acid molecules can be introduced into a host cell as separate nucleic acid molecules, as multiple individually controlled genes, as polycistronic nucleic acid molecules, as a single nucleic acid molecule encoding a protein (e.g., the heavy chain of an antibody), or in any combination thereof. When two or more heterogeneous nucleic acid molecules are introduced into a host cell, it is understood that two or more heterogeneous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., in a single vector), in separate vectors, incorporated into a single or multiple sites on a host chromosome, or in any combination thereof. The number of heterogeneous nucleic acid molecules or protein activities referred to means the number of encoding nucleic acid molecules or protein activities, not the number of separate nucleic acid molecules introduced into the host cell.

[0074] Where used herein, the terms “cell,” “cell line,” and “cell culture” are interchangeable, and all such names include offspring. Therefore, the terms “transformer,” “transformed cell,” and “host cell” include primary target cells and cultures derived therefrom, regardless of the number of transfers. It is also understood that, due to intentional or accidental mutations, not all offspring may be exactly identical in DNA content. This includes variant offspring that have the same or substantially the same function, phenotype, or biological activity as those screened in the originally transformed cells. Where a distinctly different name is intended, it will be evident from the context.

[0075] The term “construction” refers to any polynucleotide (or, where the context clearly indicates, a fusion protein) containing a recombinant nucleic acid molecule.

[0076] In certain embodiments, the polynucleotides of this disclosure may be functionally ligated to certain elements of a vector. For example, polynucleotide sequences necessary to bring about the expression and processing of a coding sequence to be ligated may be functionally ligated. The regulatory sequences may include appropriate transcription start, terminate, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and optionally, sequences that enhance protein secretion. The regulatory sequences may be functionally ligated if they are in close proximity to the gene of interest and the regulatory sequences that act in trans or at a distance that controls the gene of interest.

[0077] Antibody and antigen-binding fragments Embodiments disclosed herein include antibodies and antigen-binding fragments thereof that are capable of binding to the antigenic loop region of HBsAg (HBsAg and the antigenic loop region are described in further detail herein) and, if necessary, capable of neutralizing infection by genotypes D, A, B, C, E, F, G, H, I, or J, or any combination thereof; i.e., any one, two, three, four, five, six, seven, eight, nine, or all ten of these genotypes of hepatitis B virus (HBV). If discussed further herein, the antibodies and antigen-binding fragments disclosed herein have other advantages, including, but are not limited to, features that are advantageous for production in host cells, and a reduced tendency to form undesirable aggregates, such as dimers.

[0078] As used herein, unless the context explicitly indicates otherwise, “antibody” means an intact antibody comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds (however, heavy-chain antibodies lacking light chains are generally still encompassed by the term “antibody,” although preferred embodiments of this disclosure are understood to include both VH and VL, and in some embodiments, both heavy and light chains), as well as any antigen-binding portion or fragment of an intact antibody having or retaining the ability to bind to an antigen target molecule recognized by the intact antibody, such as scFv, Fab, or F(ab')2 fragment. Therefore, the term “antibody” as used herein is used in its broadest sense and includes polyclonal and monoclonal antibodies, complete antibodies, and their functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain variable fragments (scFv), and single-domain antibody fragments (e.g., sdAb, sdFv, nanobody). The term also encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, e.g., intrabody, peptibody, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, polyspecificity, e.g., bispecificity antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv, and other antibody forms known in the art. Unless otherwise specified, the term “antibody” should be understood to encompass its functional antibody fragments. The term "antibody" includes complete antibodies or full-length antibodies, and any class or subclass of IgG or its subclasses, including IgG and its subclasses (IgG1, IgG2, IgG4), IgM, IgE, IgA, and IgD.

[0079] As used herein, the terms “antigen-binding fragment,” “fragment,” and “antibody fragment” are used interchangeably to refer to any fragment of the antibody of this disclosure that retains the antigen-binding activity of the antibody. Examples of antibody fragments include, but are not limited to, single-chain antibodies, Fab, Fab', F(ab')2, Fv, or scFv.

[0080] Human antibodies are well known (e.g., van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can be produced in transgenic animals (e.g., mice) that are capable of producing a complete repertoire or selection of human antibodies without the production of endogenous immunoglobulins during immunization. Transplantation of a human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies during antigen challenge (see, for example, Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).Human monoclonal antibodies can be prepared by using improved EBV-B cell immortalization, as described in Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. (2004): An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 10(8):871-5. The term “human antibody,” as used herein, also includes such antibodies whose variable or constant regions have been modified, for example, to produce the properties of the antibody and antibody fragments described herein.

[0081] The antibodies according to this disclosure may be of any isotype (e.g., IgA, IgG, IgM, IgE, IgD; i.e., including α, γ, μ, ε, or δ heavy chains). Of the IgG isotypes, for example, the antibody may be of the IgG1, IgG2, IgG3, or IgG4 subclass. In a specific embodiment, the antibody according to this disclosure is an IgG1 antibody. The antibodies or antigen-binding fragments provided herein may contain a κ or λ light chain. Preferably, the antibodies or antigen-binding fragments may contain a λ light chain. In a particular embodiment, the HBsAg-specific antibody described herein is of an IgG isotype (e.g., IgG1M,171 allotype) and can block the release of HBV and HBsAg from infected cells. Thus, in a particular embodiment, the antibodies described herein can bind intracellularly, thereby blocking the release of HBV virions and HBsAg.

[0082] The term “V L " or "VL" and "V H"VL" or "VH" refers to the variable regions (also called variable domains) derived from the antibody light chain and antibody heavy chain, respectively; typically, these regions are directly involved in the binding of the antibody or antigen-binding fragment to the antigen. The VL (and CL or light chain) may be of the kappa (κ) class (also "VK" herein) or lambda (λ) class. The variable binding region includes distinct subregions known as "complementarity-determining regions" (CDRs) and "framework regions" (FRs). The terms "complementarity-determining regions" and "CDRs" are synonymous with "hypervariable regions" or "HVRs" and refer to sequences of amino acids within the antibody variable region that generally collectively confer the antigen specificity and / or binding affinity of the antibody, where consecutive CDRs (i.e., CDR1 and CDR2, CDR2 and CDR3) are separated from each other by framework regions in the primary amino acid sequence. Within each variable region, there are three CDRs (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also called CDRH and CDRL, respectively). In certain embodiments, antibody VH contains four FRs and three CDRs as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; antibody VL contains four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. Generally, VH and VL together form an antigen-binding site by their respective CDRs, but in some cases, the binding site may be formed by or contain one, two, three, four, or five CDRs, and these CDRs may be located in VH, in VL, or both.

[0083] In certain embodiments, the amino acid numbering of the antibody CDR and variable region follows a system developed by the Chemical Computing Group (“CCG”); for example, using Molecular Operating Environment (MOE) software (www.chemcomp.com).

[0084] In certain embodiments, the amino acid numbering of the antibody CDR and variable region follows the IMGT numbering scheme (see, for example, Lefranc et al., Dev. Comp. Immunol. 27:55, 2003).

[0085] Equivalent residue positions can be annotated for different molecules being compared using the Antigen Receptor Numbering and Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300).

[0086] As used herein, a “variant” of a CDR refers to a functional variant of a CDR sequence (provided herein) having up to one to three amino acid substitutions, deletions, or combinations thereof.

[0087] In certain embodiments, the Disclosure relates to an antibody or antigen-binding fragment thereof comprising (i) a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 34, the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36, and the amino acid sequence of SEQ ID NO: 37; and (ii) a light chain variable region (VL) containing any one of the amino acid sequences of SEQ ID NOs: 41, 40, 42, and 43, the amino acid sequence described in any one of SEQ ID NOs: 49, 44-48, and 50-53, and the amino acid sequence described in SEQ ID NO: 55 or 56, If necessary, VL may contain R60N substitution mutations, R60A substitution mutations, R60K substitution mutations, S64A substitution mutations, I74A substitution mutations, or any combination thereof, compared to SEQ ID NO: 58, with the amino acid numbering of the substitution mutations following SEQ ID NO: 58, and further if necessary, VL may not contain any further mutations compared to SEQ ID NO: 58. The antibody or its antigen-binding fragment can bind to the antigenic loop region of HBsAg and, if necessary, neutralize infection by hepatitis B virus (HBV) genotypes D, A, B, C, E, F, G, H, I, or J, or any combination thereof. The present invention provides an antibody or an antigen-binding fragment thereof.

[0088] In some embodiments, the antibody or its antigen-binding fragment contains (i) the amino acid sequence described in SEQ ID NOs. 34, 35, and 37 in VH, and the amino acid sequence described in SEQ ID NOs. 41, 45, and 55 in VL, respectively; (ii) the amino acid sequence described in SEQ ID NOs. 34, 35, and 37 in VH, and the amino acid sequence described in SEQ ID NOs. 41, 46, and 55 in VL, respectively; (iii) the amino acid sequence described in SEQ ID NOs. 34, 35, and 37 in VH, and the amino acid sequence described in SEQ ID NOs. 41, 47, and 55 in VL, respectively; (iv) the amino acid sequence described in SEQ ID NOs. 34, 35, and 37 in VH, and the amino acid sequence described in SEQ ID NOs. 41, 48, and 55 in VL, respectively; (v) the amino acid sequence described in SEQ ID NOs. 34, 35, and 37 in VH, respectively. (vi) The amino acid sequence and the VL contain the amino acid sequences described in SEQ ID NOs: 41, 49, and 55, respectively; (vi) The VH contains the amino acid sequences described in SEQ ID NOs: 34, 35, and 37, respectively, and the VL contains the amino acid sequences described in SEQ ID NOs: 41, 50, and 55, respectively; (vii) The VH contains the amino acid sequences described in SEQ ID NOs: 34, 35, and 37, respectively, and the VL contains the amino acid sequences described in SEQ ID NOs: 41, 51, and 55, respectively; (viii) The VH contains the amino acid sequences described in SEQ ID NOs: 34, 35, and 37, respectively, and the VL contains the amino acid sequences described in SEQ ID NOs: 41, 52, and 55, respectively; or (ix) The VH contains the amino acid sequences described in SEQ ID NOs: 34, 35, and 37, respectively, and the VL contains the amino acid sequences described in SEQ ID NOs: 41, 53, and 55, respectively.

[0089] In certain embodiments, the Disclosure provides an antibody or antigen-binding fragment thereof comprising (i) a heavy chain variable region (VH) comprising the CDRH1 amino acid sequence described in SEQ ID NO: 34, the CDRH2 amino acid sequence described in SEQ ID NO: 35 or 36, and the CDRH3 amino acid sequence described in SEQ ID NO: 37; and (ii) a light chain variable region (VL) comprising the CDRL1 amino acid sequence described in any one of SEQ ID NOs: 40-43, the CDRL2 amino acid sequence described in any one of SEQ ID NOs: 45-53, and the CDRL3 amino acid sequence described in SEQ ID NO: 55 or 56, wherein the CDR is, according to CCG, the antibody or antigen-binding fragment is capable of binding to the antigenic loop region of HBsAg and is capable of neutralizing infection by hepatitis B virus (HBV) of genotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof.

[0090] In a particular embodiment, the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are (i) SEQ ID NOs. 34, 35, 37, 41, 45, and 55, respectively; (ii) SEQ ID NOs. 34, 35, 37, 41, 46, and 55, respectively; (iii) SEQ ID NOs. 34, 35, 37, 41, 47, and 55, respectively; (iv) SEQ ID NOs. 34, 35, 37, 41, 48, and 55, respectively. 5;(v) Sequence IDs 34, 35, 37, 41, 49, and 55 respectively;(vi) Sequence IDs 34, 35, 37, 41, 50, and 55 respectively;(vii) Sequence IDs 34, 35, 37, 41, 51, and 55 respectively;(viii) Sequence IDs 34, 35, 37, 41, 52, and 55 respectively;or(ix) As described in Sequence IDs 34, 35, 37, 41, 53, and 55 respectively, the CDR follows the CCG.

[0091] Table 1 provides the CDR amino acid sequence numbers for a particular antibody, where the CDR is defined according to the CCG.

[0092] [Table 1]

[0093] In certain embodiments, the antibody or antigen-binding fragment is CDRH1, CDRH2, CDRH1 of HBC34-v36;HBC34-v37;HBC34-v38;HBC34-v39;HBC34-v40;HBC34-v41;HBC34-v42;HBC34-v43;HBC34-v44;HBC34-v45;HBC34-v46;HBC34-v47;HBC34-v48;HBC34-v49;or HBC34-v50. The CDR includes RH3, CDRL1, CDRL2, and CDRL3, and the VL further includes R60N substitution mutations, R60A substitution mutations, R60K substitution mutations, S64A substitution mutations, I74A substitution mutations, or any combination thereof, as specified, with the amino acid numbering of the substitution mutations following the SEQ ID NO: 58, and further, as specified, the VL does not include any other mutations as specified by SEQ ID NO: 58.

[0094] Table 2 provides the CDR amino acid sequence numbers for a particular antibody, where the CDRs are defined according to IMGT (short and long versions of CDRH2 and CDRL2 are disclosed).

[0095] [Table 2]

[0096] In certain embodiments, the antibody or antigen-binding fragment is CDRH1, CDRH2, CDR of HBC34-v36;HBC34-v37;HBC34-v38;HBC34-v39;HBC34-v40;HBC34-v41;HBC34-v42;HBC34-v43;HBC34-v44;HBC34-v45;HBC34-v46;HBC34-v47;HBC34-v48;HBC34-v49;or HBC34-v50. The CDR includes H3, CDRL1, CDRL2, and CDRL3, and the CDR, as specified, further includes R60N substitution mutations, R60A substitution mutations, R60K substitution mutations, S64A substitution mutations, I74A substitution mutations, or any combination thereof, compared to SEQ ID NO: 58, and the amino acid numbering of the substitution mutations is as specified in SEQ ID NO: 58, and further, as specified, the VL does not include any other mutations compared to SEQ ID NO: 58.

[0097] Table 3 provides the VH and VL amino acid sequence numbers for a particular antibody.

[0098] [Table 3]

[0099] In certain embodiments, the antibody or antigen-binding fragment comprises the VH and VL amino acid sequences of HBC34-v36;HBC34-v37;HBC34-v38;HBC34-v39;HBC34-v40;HBC34-v41;HBC34-v42;HBC34-v43;HBC34-v44;HBC34-v45;HBC34-v46;HBC34-v47;HBC34-v48;HBC34-v49; or HBC34-v50.

[0100] In certain embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein (i) the VH contains or consists of an amino acid sequence having at least 90% identity (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value in between) with the amino acid sequence described in SEQ ID NOs. 38 or 39; and / or (ii) the VL contains or consists of an amino acid sequence having at least 90% identity (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any non-integer value in between) with the amino acid sequence described in any one of SEQ ID NOs. 58-66, 69, 71, or 72. In certain embodiments, VH and VL include or consist of amino acid sequences having at least 90% identity (i.e., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any non-integer value in between) with respect to the amino acid sequences described in (i) SEQ ID NOs. 38 and 58, respectively; (ii) SEQ ID NOs. 38 and 59, respectively; (iii) SEQ ID NOs. 38 and 60, respectively; (iv) SEQ ID NOs. 38 and 61, respectively; (v) SEQ ID NOs. 38 and 63, respectively; (vii) SEQ ID NOs. 38 and 64, respectively; (viii) SEQ ID NOs. 38 and 65, respectively; (ix) SEQ ID NOs. 38 and 66, respectively; (x) SEQ ID NOs. 38 and 71, respectively; or (xi) SEQ ID NOs. 38 and 72, respectively. As a non-limiting example, in a particular embodiment, VH includes an amino acid sequence having at least 90% identity with SEQ ID NO: 38, and VL includes an amino acid sequence having at least 90% identity with SEQ ID NO: 62.

[0101] In some embodiments, VH includes or consists of the amino acid sequences described in SEQ ID NO: 38 or 39; and / or VL includes or consists of the amino acid sequences described in any one of SEQ ID NOs: 58-66, 69, 71, or 72. In certain embodiments, VH and VL include or consist of the amino acid sequences described in (i) SEQ ID NOs: 38 and 58, respectively; (ii) SEQ ID NOs: 38 and 59, respectively; (iii) SEQ ID NOs: 38 and 60, respectively; (iv) SEQ ID NOs: 38 and 61, respectively; (v) SEQ ID NOs: 38 and 62, respectively; (vi) SEQ ID NOs: 38 and 63, respectively; (vii) SEQ ID NOs: 38 and 64, respectively; (viii) SEQ ID NOs: 38 and 65, respectively; (ix) SEQ ID NOs: 38 and 66, respectively; (x) SEQ ID NOs: 38 and 71, respectively; or (xi) SEQ ID NOs: 38 and 72, respectively.

[0102] In a particular embodiment, the antibody or antigen-binding fragment comprises a VH containing or derived from the amino acid sequence described in SEQ ID NO: 38, and a VL containing or derived from the amino acid sequence described in any one of SEQ ID NOs. 58 to 72.

[0103] In a particular embodiment, the antibody or antigen-binding fragment comprises a VH containing or consisting of the amino acid sequence described in SEQ ID NO: 38, and a VL containing or consisting of the amino acid sequence described in any one of SEQ ID NOs. 59 to 72.

[0104] In another aspect, the Disclosure provides an antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH and VL comprise or consist of (i) the amino acid sequences described in SEQ ID NOs. 38 and 67, respectively; or (ii) the amino acid sequences described in SEQ ID NOs. 38 and 68, respectively, and the antibody or antigen-binding fragment is capable of binding to the antigenic loop region of HBsAg and neutralizing infection by hepatitis B virus (HBV) of genotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof.

[0105] Also provided are antibody or antigen-binding fragments comprising the VH described in SEQ ID NO: 38 or 39 and one VL variant from SEQ ID NOs: 57-72, which includes one or more of the following mutations (determined by CCG numbering in framework region 3): R60A, R60N, R60K, S64A, and I74A. In certain further embodiments, the VL variant does not include any further mutations compared to SEQ ID NOs: 57-72 (each).

[0106] Also provided are an antibody or antigen-binding fragment comprising the VH described in SEQ ID NO: 38 and one of the VL variants from SEQ ID NOs: 57-72, which contains a substitution mutation (e.g., a conservative amino acid substitution or a mutation to an amino acid encoded in the germline) in Q78, D81, or both (CCG numbering).

[0107] Also provided are an antibody or antigen-binding fragment comprising the VH described in SEQ ID NO: 39 and one of the VL variants from SEQ ID NOs: 57-72, which contains a substitution mutation (e.g., a conservative amino acid substitution or a mutation to an amino acid encoded in the germline) in Q78, D81, or both (CCG numbering).

[0108] As will be further discussed herein, the antibodies and antigen-binding fragments disclosed herein have a reduced tendency to form aggregates (e.g., dimers) and / or improved productivity in host cells (e.g., higher titers) and / or similar, substantially identical, or even improved: binding to HbsAg; HBV neutralization; and / or thermal stability compared to the reference antibodies disclosed herein.

[0109] It is understood that the “reference” antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment that is identical to the target antibody or antigen-binding fragment, except for differences in identified or enumerated features (e.g., differences in the CDR and / or variable region framework sequence(s)). In some embodiments, the reference antibody includes the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs. 34, 35, 37, 41, 44, and 55, respectively, and optionally includes the VH amino acid sequence described in SEQ ID NOs. 38 and the VL amino acid sequence described in SEQ ID NOs. 57.

[0110] As a non-limiting example, the antibody or antigen-binding fragment of this disclosure may be an IgG1 isotype and may include a wild-type IgG1 Fc moiety, and the reference antibody or antigen-binding fragment may include the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs. 34, 35, 37, 41, 44, and 55, respectively, and optionally include the VH amino acid sequence described in SEQ ID NOs. 38 and the VL amino acid sequence described in SEQ ID NOs. 57, and may be an IgG1 isotype and include a wild-type IgG1 Fc moiety. When comparing an antibody or antigen-binding fragment disclosed herein with a reference antibody or antigen-binding fragment under certain conditions, it is further understood that, unless otherwise expressly stated, these conditions (e.g., amount of starting material, temperature, buffer, host cell line, culture conditions, duration of appropriate period, codon optimization of the encoding polynucleotide, etc.) are identical, or as close to identical as the conditions allow (e.g., two antibodies may differ in their amino acid sequences by one or more amino acids, but are otherwise identical), and encoded by comparable polynucleotides (e.g., each antibody may be encoded by its own codon-optimized polynucleotide).

[0111] As a non-limiting example, the antibodies and antigen-binding fragments disclosed herein produce fewer aggregates (e.g., in the form of antibody:antibody dimers, antibody:antigen-binding fragment dimers, or antigen-binding fragment:antigen-binding fragment dimers) and / or have a higher titer in host cells compared to a reference antibody or antigen-binding fragment, respectively.

[0112] In this context, a dimer is a complex or aggregate containing two antibody or antigen-binding fragment molecules (e.g., antibody:antibody dimer, Fab:Fab dimer, or antibody:Fab dimer). Where further discussion herein lies, the dimerization relating to this may differ distinctly from the typical associations between antibody heavy chain components and antibody light chain components, or between two antibody heavy chain polypeptides, that occur in the formation of intact tetrameric antibodies, Fv, or Fab, and may involve association between two monomers. Therefore, in this context, “dimer” is understood to mean or not mean the association of an antibody heavy chain and an antibody light chain that provides a semi-antibody containing a functional Fab, and does not include the association of two heavy chains of an antibody (e.g., hinge-hinge and Fc-Fc) or, for example, the VH-VL association in Fv or Fab (e.g., occurring via a disulfide bond).

[0113] In certain embodiments, dimers are formed by the association of VLs of two distinct antibody or antigen-binding fragment molecules. A diagram of a dimer formed by the association of two VLs of distinct antibody molecules is shown in Figure 7 of this specification. Such dimerization may reduce, for example, the binding titer and / or binding affinity and / or avidity and / or neutralizing effectiveness of one or both of the antibody or antigen-binding fragment molecules contained therein. In general, an increase in the presence of such dimers containing multiple antibody or antigen-binding fragments in a composition reduces the overall binding and / or neutralizing effectiveness of the composition.

[0114] Antibody or antigen-binding fragment dimers can be identified using known techniques, such as size exclusion chromatography. A dimer has a molecular weight higher than the molecular weight of each of its individual (monomer) subunits, typically equal to or close to the sum of the molecular weights of its individual subunits. For example, a homodimer (i.e., containing two antibody molecules with identical or substantially identical amino acid sequences) generally has a molecular weight about twice that of each of its monomer subunits. For instance, a typical human IgG1 immunoglobulin molecule has a molecular weight of approximately 150 kilodaltons (e.g., each of the two heavy chains weighs approximately 50 kilodaltons, and each of the two light chains weighs approximately 25 kilodaltons), and a dimer containing two such immunoglobulin molecules has a molecular weight of approximately 300 kilodaltons. Of course, it is understood that, for example, due to any differences in their respective amino acid sequences, at least in part, one antibody may have a molecular weight slightly or somewhat different from other antibodies of the same general structure and isotype.

[0115] As another non-limiting example, an antibody molecule may have a molecular weight between 140 and 160 kilodaltons, and an antibody dimer containing two antibody molecules may have a molecular weight between 280 and 320 kilodaltons. The dimer may be called a "high molecular weight species" or "HMWS".

[0116] The presence of dimers in a composition or sample containing multiple antibody (and / or antigen-binding fragment) molecules can be assessed, for example, using absolute size exclusion chromatography (aSEC). The amount of dimers in a composition or sample can be expressed as a percentage of the total antibody or antigen-binding fragment molecules present as dimers in the composition or sample. For example, in an antibody composition containing 12% dimers, 88% of the total antibody molecules in the sample exist as monomers.

[0117] In any of the embodiments disclosed herein, when a sample containing multiple antibodies or antigen-binding fragments (i.e., multiple antibody or antigen-binding fragment molecules) is incubated at approximately 40°C for approximately 120 to approximately 168 hours, the sample contains less than 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the multiple as dimers, and the presence of dimers is determined, if necessary, by absolute size exclusion chromatography.

[0118] In any of the embodiments disclosed herein, incubation of multiple antibody or antigen-binding fragment molecules disclosed herein results in a reduction in dimer formation compared to incubation of multiple reference antibody or antigen-binding fragment molecules, wherein the reference antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs. 34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence described in SEQ ID NOs. 38 and the VL amino acid sequence described in SEQ ID NOs. 57, and optionally the presence of antibody dimers is determined by absolute size exclusion chromatography. Such reference antibody or antigen-binding fragment (e.g., Fv, Fab) may, in some embodiments, form dimers in the sample (e.g., when incubated at about 40°C for about 120 to about 168 hours) that collectively contain more than 2%, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, or up to 12% of the antibody or antigen-binding fragment molecule. In other words, in some embodiments, up to 12% or more of the reference antibody or antigen-binding fragment molecule is included as a dimer, while a lower percentage, preferably less than 2%, of the antibody or antigen-binding fragment molecule disclosed herein is included as a dimer.

[0119] In some embodiments, the antibody or antigen-binding fragments disclosed herein form dimers in lower amounts compared to the reference antibody, and / or at a reduced frequency and / or as a lower percentage of the total antibody or antigen-binding fragment molecules in the sample or composition (determined, for example, by size exclusion chromatography), in (i) incubation at 4°C for 5 days, 15 days, and / or 32 days; in (ii) incubation at 25°C for 5 days, 15 days, and / or 32 days; and / or (iii) incubation at 40°C for 5 days, 15 days, and / or 32 days. The reference antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs. 34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence described in SEQ ID NOs. 38 and the VL amino acid sequence described in SEQ ID NOs. 57.

[0120] In some embodiments, the percentage of the antibody or antigen-binding fragment molecules disclosed herein that are included as dimers in the composition is less than 4 / 5, less than 3 / 4, less than 1 / 2, less than 1 / 3, less than 1 / 4, less than 1 / 5, less than 1 / 6, less than 1 / 7, less than 1 / 8, less than 1 / 9, or less than 1 / 10 of the percentage of the reference antibody molecules present as dimers in the composition. As a non-limiting example, after incubation at 40°C for 32 days (768 hours), 22% or more of the reference antibody molecules in the composition may be present as dimers, while less than 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the antibody or antigen-binding fragment molecules disclosed herein in the composition may be present as dimers.

[0121] In some embodiments, a host cell (e.g., CHO cell, e.g., ExpiCHO cell) transfected with a polynucleotide encoding the antibody or antigen-binding fragment disclosed herein provides 1.5 times or more, 2 times or more, 3 times or more, or 4 times or more of the antibody or antigen-binding fragment (e.g., measured as a concentration in mg / mL) than a reference host cell transfected with the polynucleotide encoding the reference antibody or antigen-binding fragment, the reference antibody or antigen-binding fragment comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs. 34, 35, 37, 41, 44, and 55, respectively, and optionally comprising the VH amino acid sequence described in SEQ ID NOs. 38 and the VL amino acid sequence described in SEQ ID NOs. 57.

[0122] In some embodiments, the antibodies or antigen-binding fragments disclosed herein are produced in transfected cells with a higher titer compared to the reference antibody or antigen-binding fragment produced in the transfected reference cells, wherein the reference antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs. 34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence described in SEQ ID NOs. 38 and the VL amino acid sequence described in SEQ ID NOs. 57.

[0123] In some embodiments, the antibodies or antigen-binding fragments disclosed herein are produced in transfected cells at a titer at least 1.5 times, at least 2 times, at least 3 times, or at least 4 times higher than the titer at which the reference antibody or antigen-binding fragment is produced, wherein the reference antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs. 34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence described in SEQ ID NOs. 38 and the VL amino acid sequence described in SEQ ID NOs. 57.

[0124] In any of the embodiments disclosed herein, the antibody or antigen-binding fragment can bind to HBsAg (e.g., of subtype adw) at an EC50 (ng / ml) of 3.5 or less, about 3.2 or less, less than 3.0, less than 2.5, less than 2.0, less than 1.5, or less than 1.0. In some embodiments, the antibody or antigen-binding fragment can bind to HBsAg (e.g., of subtype adw) at an EC50 (ng / ml) of less than 3.5, less than 3.4, less than 3.3, less than 3.2, less than 3.1, less than 3.0, less than 2.9, less than 2.8, less than 2.7, less than 2.6, less than 2.5, less than 2.4, less than 2.3, less than 2.1, less than 2.0, less than 1.9, less than 1.8, less than 1.7, less than 1.6, less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1.1, or less than 1.0. In some embodiments, the antibody or antigen-binding fragment can bind to HBsAg (e.g., of subtype adw) at an EC50 (ng / ml) between 0.9 and 2.0, or between 0.9 and 1.9, or between 0.9 and 1.8, or between 0.9 and 1.7, or between 0.9 and 1.6, or between 0.9 and 1.5, or between 0.9 and 1.4, or between 0.9 and 1.3, or between 0.9 and 1.2, or between 0.9 and 1.1, or between 0.9 and 1.0, or between 1.0 and 2.0. In certain embodiments, the antibody or antigen-binding fragment can bind to HBsAg (e.g., of subtype adw) at an EC50 (ng / ml) of 2.0 or less. In some embodiments, the binding EC50 is determined by ELISA (e.g., a direct antigen-binding ELISA assay, where the binding curve is determined by fitting the curve using Graphpad prism).

[0125] In any of the embodiments disclosed herein, the antibody or its antigen-binding fragment can neutralize hepatitis B virus infection with an infection neutralizing EC50 of less than 20 ng / ml, preferably less than 15 ng / ml, and more preferably less than 10 ng / mL. In some embodiments, the antibody or its antigen-binding fragment can neutralize hepatitis B virus infection with an infection neutralizing EC50 of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, or 7 ng / mL. In some embodiments, the antibody or its antigen-binding fragment may contain the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs. 34, 35, 37, 41, 44, and 55, respectively, and may optionally contain the VH amino acid sequence described in SEQ ID NOs. 38 and the VL amino acid sequence described in SEQ ID NOs. 57. It is possible to neutralize hepatitis B virus infection with a lower infection neutralization EC50 than that of a reference antibody or antigen-binding fragment (using the same assay). In some embodiments, the infection neutralization EC50 is determined after incubation of cultured cells, e.g., differentiated HepaRG cells, with a fixed amount of HBV in or without the antibody under test. In such embodiments, incubation may be carried out, for example, over 16 hours at 37°C. This incubation may be carried out in a culture medium (e.g., supplemented with 4% PEG8000). After incubation, the cells may be washed and further cultivated. To measure viral infectivity, for example, the levels of hepatitis B surface antigen (HBsAg) and / or hepatitis B e antigen (HBeAg) secreted into the culture supernatant from 7 to 11 days after infection can be determined by enzyme-linked immunosorbent assay (ELISA). The levels of HBsAg and / or HBeAg from treated cells can be compared to those from untreated cells to determine the presence and extent of neutralization.

[0126] An "Fv" is a small antibody fragment containing a complete antigen recognition and binding site. This fragment typically consists of a dimer of one heavy chain variable domain and one light chain variable domain that are closely and non-covalently associated. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can have the ability to recognize and bind to an antigen, although typically with lower affinity than the entire binding site.

[0127] A "single-stranded Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. In some embodiments, the scFv polypeptide includes a polypeptide linker positioned between the VH and VL domains, linking them, which allows the scFv to maintain or form a desired structure for antigen binding. Such peptide linkers can be incorporated into the fusion polypeptide using standard techniques well known in the art. Furthermore, or alternatively, the Fv may have a disulfide bond formed between VH and VL, stabilizing VH and VL. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see Borrebaeck 1995 below. In certain embodiments, the antibody or antigen-binding fragment comprises an scFv including a VH domain, a VL domain, and a peptide linker that ligates the VH domain to the VL domain. In certain embodiments, the scFv comprises a VH domain ligated to the VL domain by a peptide linker, which may be in the VH-linker-VL direction or the VL-linker-VH direction. Any scFv of the present disclosure may be manipulated such that the C-terminus of the VL domain is ligated to the N-terminus of the VH domain by a short peptide sequence, or vice versa (i.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C)). Alternatively, in some embodiments, the linker may be ligated to the N-terminal portion of the VH domain, the VL domain, or both, or to the terminal of the VH domain, the VL domain, or both.

[0128] Peptide linker sequences may be selected based, for example, on (1) their ability to adopt flexible elongation conformations, (2) their inability to adopt or ability to adopt secondary structures that can interact with functional epitopes on the first and second polypeptides and / or the target molecule, and / or (3) the absence or relative absence of hydrophobic or charged residues that may react with the polypeptide and / or the target molecule. Other considerations for linker design (e.g., length) may include conformations or ranges of conformations in which VH and VL can form functional antigen-binding sites. In certain embodiments, the peptide linker sequence contains, for example, Gly, Asn, and Ser residues. Other nearly neutral amino acids such as Thr and Ala may also be included in the linker sequence. Other amino acid sequences that may be useful as linkers include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Patent No. 4,935,233, and U.S. Patent No. 4,751,180. Other exemplary, non-limiting examples of linkers include, for example, those disclosed by Chaudhary et al., Proc. Natl. Acad. Sci. USA 87:1066-1070 (1990) and Bird et al., Science 242:423-426 (1988), as well as pentamers of four consecutively linked glycine residues, where the C-terminal glycine of the sequence is linked to a single serine, whether existing as a single repeat or 1 to 5 or more times.Any suitable linker may be used, generally of about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 15, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid lengths, or less than about 200 amino acid lengths, preferably comprising a flexible structure (which can provide flexibility and space for conformational movement between two regions, domains, motifs, fragments, or modules connected by the linker), preferably being biologically inactive and / or having a low risk of immunogenicity in humans.

[0129] scFv can be constructed using any combination of VH and VL sequences disclosed herein, or any combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences. In some embodiments, for example, if the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric interference, a linker sequence is not required.

[0130] In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region (or a portion or fragment thereof), a heavy chain constant region (or a portion or fragment thereof), or both. The term "CL" refers to the "immunoglobulin light chain constant region" or "light chain constant region," i.e., the constant region derived from the antibody light chain. The term "CH" refers to the "immunoglobulin heavy chain constant region" or "heavy chain constant region," which can be further divided into CH1, CH2, and CH3 (IgA, IgD, IgG) or CH1, CH2, CH3, and CH4 domains (IgE, IgM), depending on the antibody isotype. The Fc region of the antibody heavy chain is further described herein. In any of the embodiments disclosed herein, the antibody or antigen-binding fragment of this disclosure comprises one or more of CL, CH1, CH2, and CH3. In certain embodiments, CL includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 79. In certain embodiments, CH1-CH2-CH3 includes an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 73, or a variant thereof containing one or more of the following amino acid substitutions (EU numbering): G236A; A330L; I332E; M428L; N434S. The Fc portion is described elsewhere in this specification.

[0131] For example, it is understood that production in mammalian cell lines may remove one or more C-terminal lysines from the antibody heavy chain (see, for example, Liu et al. mAbs 6(5):1145-1154 (2014)). Thus, the antibody or antigen-binding fragments of the Disclosure may comprise a heavy chain, CH1-CH3, CH3, or Fc polypeptide, with or without a C-terminal residue; in other words, embodiments in which the C-terminal residue of the heavy chain, CH1-CH3, or Fc portion is not lysine, and embodiments in which lysine is the C-terminal residue, are included. In certain embodiments, the composition comprises multiple antibodies and / or antigen-binding fragments of the Disclosure, one or more antibody or antigen-binding fragments may not contain a lysine residue at the C-terminus of the heavy chain, CH1-CH3, or Fc portion, and one or more antibody or antigen-binding fragments may contain a lysine residue at the C-terminus of the heavy chain, CH1-CH3, or Fc portion.

[0132] A "Fab" (antigen-binding fragment) is the portion of the antibody that binds to the antigen and contains a variable region of the heavy chain and CH1 linked to the light chain via interchain disulfide bonds. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of the antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of crosslinking the antigen. Both Fab and F(ab')2 are examples of "antigen-binding fragments". Fab' fragments differ from Fab fragments in that they have few further residues at the carboxyl terminus of the CH1 domain, including one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein designation for Fab' fragments in which the cysteine ​​residue(s) of the constant domain have a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. Fab fragments may be linked, for example, by peptide linkers to form a single-chain Fab, also referred to herein as “scFab”. In these embodiments, interchain disulfide bonds present in the native Fab may be absent, and the linker functions, in whole or in part, to link or connect the Fab fragments with a single polypeptide chain. Heavy-chain derived Fab fragments (e.g., containing, consisting of, or essentially consisting of VH+CH1, or “Fd”) and light-chain derived Fab fragments (e.g., containing, consisting of, or essentially consisting of VL+CL) may be linked in any configuration to form an scFab. For example, the scFab may be configured in an N-terminal to C-terminal direction according to (heavy-chain Fab fragment-linker-light-chain Fab fragment) or (light-chain Fab fragment-linker-heavy-chain Fab fragment). Peptide linkers and exemplary linker sequences for use in scFab are discussed in further detail herein.

[0133] In any of the embodiments disclosed herein, the antibody, or its antigen-binding fragment, includes a human antibody, a monoclonal antibody, a purified antibody, a single-chain antibody, Fab, Fab', F(ab')2, Fv, or scFv.

[0134] The antibody fragments described herein can be obtained from antibodies by methods including digestion with enzymes, such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, antibody fragments can be obtained by cloning and expression of a portion of the heavy chain or light chain sequence. This disclosure includes, for example, single-chain Fv fragments (scFv) derived from the heavy and light chains of antibodies described herein, including scFv containing a CDR (and optionally a variable region) derived from the antibody described herein, monomers and dimers of the heavy or light chain (i.e., VH-VL dimer, HC-LC dimer, HC-HC dimer), single-domain heavy chain antibodies, single-domain light chain antibodies, and single-chain antibodies in which the variable domains or regions of the heavy and light chains are linked by a peptide linker.

[0135] In certain embodiments, the antibody or antigen-binding fragment thereof according to the Disclosure includes a purified antibody, a monoclonal antibody, a single-chain antibody, Fab, Fab', F(ab')2, Fv, or scFv.

[0136] The antibodies and antigen-binding fragments of this disclosure may be polyspecific (e.g., dispecific, trispecific, tetraspecific, etc.) in embodiments and may be provided in any polyspecific form disclosed herein. In certain embodiments, the antibody or antigen-binding fragment of this disclosure is a polyspecific antibody, for example, a dispecific or trispecific antibody. Forms of bispecific antibodies are disclosed, for example, in Spiess et al., Mol. Immunol. 67(2):95 (2015) and Brinkmann and Kontermann, mAbs 9(2):182-212 (2017). These bispecific forms and methods for producing them are incorporated herein by reference, including, for example, Bispecific T cell Engager (BiTE), DART, Knobs-Into-Holes (KIH) assembly, scFv-CH3-KIH assembly, KIH Common Light-Chain antibody, TandAb, Triple Body, TriBi Minibody, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCab, Intrabody, CrossMab, Dual Action Fab (DAF) (two-in-one or four-in-one), DutaMab, DT-IgG, Charge Pair, and Fab-arm. Examples include Exchange, SEEDbody, Triomab, LUZ-Y assembly, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG (Four-in-One).A bispecific or polyspecific antibody may include the HBV-specific and / or HDV-specific binding domain of the Disclosure in combination with another HBV-specific and / or HDV-specific binding domain of the Disclosure, or in combination with a different binding domain that specifically binds to HBV and / or HDV (e.g., in the same or different epitopes), or a binding domain that specifically binds to a different antigen.

[0137] The antibody fragments of this disclosure may confer monovalent or polyvalent interactions and may be contained in various structures as described above. For example, the scFv molecule may be synthesized to create a trivalent "triabody" or a tetravalent "tetrabody." The scFv molecule may contain a domain in the Fc region to give rise to a divalent minibody. Furthermore, the sequences of this disclosure may be components of a polyspecific molecule, where the sequence of this disclosure targets the epitope of this disclosure, and other regions of the molecule bind to other targets. Exemplary molecules include, but are not limited to, dispecific Fab2, trispecific Fab3, dispecific scFv, and diabody (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

[0138] Antibodies or their antigen-binding fragments, such as scFv, described herein, may, in certain embodiments, be contained within a fusion protein capable of specifically binding to the antigens described herein.

[0139] As used herein, “fusion protein” means a protein having at least two distinctly different domains or motifs in a single chain, where these domains or motifs are not found together in nature or in a given arrangement within the protein. The polynucleotide encoding the fusion protein may be constructed using PCR, recombined, etc., or such fusion protein may be synthesized.

[0140] In some embodiments, the fusion protein can be expressed on the surface of host cells, such as T cells, NK cells, or NK-T cells. In certain embodiments, the fusion protein includes (i) an extracellular component comprising an antibody or its antigen-binding fragment (e.g., scFv); (ii) a transmembrane component (e.g., a transmembrane domain derived from CD4, CD8, CD27, CD28, or a functional variant or moiety thereof, or any combination thereof); and (iii) a signaling domain derived from a costimulatory protein, or a functional variant or moiety thereof (e.g., CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD2, CD5, ICAM-1 (CD54), LFA-1 (CD11a / CD18), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, MKG2C, SL The intracellular components include ligands that specifically bind to AMF7, NKp80, CD160, B7-H3, CD83, or functional variants thereof, or signaling domains derived from any combination thereof, and / or effector domains (e.g., derived from CD3ε, CD3δ, CD3ζ, CD25, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof).

[0141] In certain embodiments, the fusion protein comprising an antibody or antigen-binding fragment comprises a chimeric antigen receptor molecule (CAR) that can be expressed on the cell surface of host cells, for example, T cells, NK cells, or NK-T cells for use in cellular immunotherapy. CAR molecules and design principles are described, for example, in Sadelain et al., Cancer Discov., 3(4):388 (2013); Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016); Stone et al., Cancer Immunol. Immunother., 63(11):1163 (2014); Xu et al., 2018 Oncotarget 9:13991; Androulla et al., 2018 Curr. Pharm. Biotechnol. Volume 19 (April 2018); Wu et al., 2016 Expert Opin. Biol. Ther. 16:1469; and Ren et al., 2017 Protein Cell 8:634, and these CAR molecules, CAR designs, and CAR design principles are incorporated herein by reference in their entirety.

[0142] Throughout this disclosure, antibodies, their antigen-binding fragments, and fusion proteins may be referred to individually or collectively (for example, in any combination) as “binding proteins.”

[0143] The binding proteins described herein may be provided in a purified form. For example, the antibody may be present in a composition substantially free of other polypeptides, where, for example, less than 90% (by weight), usually less than 60%, and more usually less than 50%, of the composition consists of other polypeptides.

[0144] The binding proteins of this disclosure may be immunogenic in humans and / or non-human (or heterologous) hosts; for example, in mice. For example, an antibody may have an idiotope that is immunogenic in non-human hosts but not in human hosts. Antibodies of this disclosure for human use include antibodies that are not typically isolated from hosts such as mice, goats, rabbits, rats, and non-primate mammals, and in some cases, are not obtained by humanization or from xeno-mice. Variant forms of the disclosed antibodies that have been manipulated to reduce known or potential immunogenicity and / or other potential disadvantages, or to confer a desired structure and / or functionality to the antibody in non-human animals, such as mice (e.g., "moothelated" antibodies in which one or more human amino acid residues, sequences, or motifs have reduced or neutralized immunogenicity or other disadvantages, or are replaced by residues, sequences, or motifs that have a desired structure and / or function in mice; for example, for model studies using mice) are also contemplated herein.

[0145] As used herein, a "neutralizing antibody" (or antigen-binding fragment, or fusion protein) is an antibody that can neutralize, i.e., prevent, inhibit, reduce, interfere with, or disrupt, the ability of a pathogen to initiate and / or perpetuate infection in a host (e.g., a host organism or host cell). The terms "neutralizing antibody" and "antibody(ies) that neutralize" or "antibody(ies) that neutralize" are used interchangeably herein. These antibodies can be used alone or in combination (e.g., combined, with two or more of the antibodies disclosed herein, or in combination with another agent, which may or may not be an antibody agent that can neutralize HBV B and / or HBV D infection) as prophylactic or therapeutic agents, in association with active vaccination, as diagnostic tools, or as production tools, as described herein, by appropriate formulation. Thus, the antibodies or antigen-binding fragments disclosed herein can neutralize infection by HBV, HDV, or both.

[0146] As used herein, "specifically binds" or "specific for" refers to the association or binding of a binding protein (e.g., an antibody or an antigen-binding fragment thereof) or binding domain to a target molecule with an affinity or Ka equal to or greater than 10 5 M -1 and without significant association or binding to any other molecule or any component in a sample (which is equal to the ratio of the on-rate [K on to the off-rate [Koff] for this association reaction). A binding protein or binding domain can be classified as a "high-affinity" binding protein or binding domain or a "low-affinity" binding protein or binding domain. A "high-affinity" binding protein or binding domain has at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M-1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 This refers to a binding protein or binding domain having a Ka of 10. "Low affinity" binding proteins or binding domains have a maximum of 10. 7 M -1 up to 10 6 M -1 , or up to 10 5 M -1 This refers to a binding protein or binding domain having a Ka of 10. Alternatively, affinity is expressed in units of M (e.g., 10). -5 M~10 -13 It can be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction having M). The terms “binding” and “specifically binding” and similar references do not include nonspecific attachment.

[0147] The binding of the binding protein can be determined or assessed using appropriate assays, such as surface plasmon resonance (SPR), e.g., the Biacore® system; kinetic exclusion assays, e.g., KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet platform); antigen-binding ELISA (e.g., direct or indirect) using imaging, such as optical density at 450 nm or flow cytometry, including isothermal titration calorimetry (ITC).

[0148] In certain embodiments, the binding protein according to this disclosure can bind to the antigenic loop region of HBsAg. The envelope of hepatitis B virus generally contains three “HBV envelope proteins” (also known as “HBsAg” or “hepatitis B surface antigen”): the S protein (indicating “small” and also called S-HBsAg), the M protein (indicating “middle” and also called M-HBsAg), and the L protein (indicating “large” and also called L-HBsAg). S-HBsAg, M-HBsAg, and L-HBsAg correspond to the S protein (S-HBsAg) and share the same C-terminal tip (also called the “S domain,” 226 amino acids) which is important for viral assembly and infectivity. S-HBsAg, M-HBsAg, and L-HBsAg are synthesized in the endoplasmic reticulum (ER), assembled, and secreted as particles via the Golgi apparatus. The S domain contains four predicted transmembrane (TM) domains, thereby exposing both the N-terminus and C-terminus of the S domain to the lumen. Transmembrane domains TM1 and TM2 are thought to be necessary for co-translational protein integration into the ER membrane, while transmembrane domains TM3 and TM4 are located in the C-terminal third of the S domain. The "antigenic loop region" of HBsAg is located between the predicted TM3 and TM4 transmembrane domains of the HBsAg S domain, thereby containing amino acids 101–172 of the S domain, totaling 226 amino acids (Salisse J. and Sureau C., 2009, Journal of Virology 83: 9321-9328). The determinant of infectivity resides within the antigenic loop region of the HBV envelope protein. In particular, the residue between HBsAg 119 and 125 contains a CXXC motif, which is thought to be important for the infectivity of HBV and HDV (Jaoude GA, Sureau C, Journal of Virology, 2005;79:10460-6).

[0149] When the position of the S domain of HBsAg in the amino acid sequence is referred to herein, such position is relative to the amino acid sequence described in Sequence ID No. 3 (shown below) or to its natural or artificial sequence variant. [ka] (Sequence ID 3; amino acids 101-172 are underlined)

[0150] For example, the expression "amino acids 101-172 of the S domain" refers to amino acid residues derived from positions 101-172 of the polypeptide described in SEQ ID NO: 3. However, those skilled in the art will understand that mutations or variant forms (substitutions, deletions, and / or additions, including but not limited to different genotypes of HBsAg or different HBsAg variants described herein) may be naturally present in the amino acid sequence of the S domain of HBsAg or may be artificially introduced into the amino acid sequence of the S domain of HBsAg without affecting its biological properties. Therefore, as used herein, the term "S domain of HBsAg" encompasses all such polypeptides, including, for example, the polypeptide described in SEQ ID NO: 3 and its natural or artificial variants. Furthermore, where sequence fragments of the S domain of HBsAg are described herein (for example, amino acids 101-172 or amino acids 120-130 of the S domain of HBsAg), these include not only the corresponding sequence fragment of SEQ ID NO: 3 but also the corresponding sequence fragments of its natural or artificial variants. For example, the phrase "amino acid residues derived from positions 101–172 of the S domain of HBsAg" includes the amino acid residues derived from positions 101–172 of Sequence ID No. 3, and the corresponding fragments of its variants (natural or artificial variants). As used herein, the phrases "corresponding sequence fragment" and "corresponding fragment" refer to fragments located at the same position in the sequence when the sequence is subjected to optimized alignment, i.e., when the sequence is aligned to obtain the highest percentage of identity.

[0151] The M protein (M-HBsAg) corresponds to the S protein, which is extended by a 55-amino acid N-terminal domain called "pre-S2". The L protein (L-HBsAg) corresponds to the M protein, which is extended by a 108-amino acid N-terminal domain called "pre-S1" (genotype D). Both the pre-S1 and pre-S2 domains of the L protein can be located on the inner surface of the viral particle (cytoplasmic side of the ER) and are thought to play an important role in viral assembly, or they can be located on the outer surface of the viral particle (lumen side of the ER) and are available for interaction with target cells and are thought to be important for viral infectivity. Furthermore, HBV surface proteins (HBsAg) can not only be incorporated into the virion envelope but can also spontaneously bud from the ER-Golgi intermediate compartment membrane and form empty "subviral particles" (SVPs) that are released from the cell by secretion.

[0152] In some embodiments, the binding protein can bind to the antigenic loop region of HBsAg and is capable of binding to all of S-HBsAg, M-HBsAg, and L-HBsAg.

[0153] In some embodiments, the binding protein neutralizes infection by hepatitis B virus and hepatitis delta virus. In some embodiments, the binding protein reduces the viral infectivity of hepatitis B virus and hepatitis delta virus.

[0154] Standard "neutralization assays" may be used to study and quantify viral infectivity (or "neutralization") in the laboratory. For neutralization assays, animal viruses are typically grown in cells and / or cell lines. A neutralization assay may be used in which cultured cells are incubated with a fixed amount of HBV or HDV in the presence (or absence) of the antibody (or antigen-binding fragment or fusion protein) to be tested. In such assays, the levels of hepatitis B surface antigen (HBsAg) or hepatitis B e antigen (HBeAg) secreted into the cell culture supernatant may be used, and / or HBcAg staining may be assessed to provide readout. For HDV, for example, delta antigen immunofluorescence staining may be assessed.

[0155] In certain embodiments of the HBV neutralization assay, cultured cells, e.g., HepaRG cells, e.g., differentiated HepaRG cells, are incubated with a fixed amount of HBV, either in the presence or absence of the antibody to be tested. In such embodiments, incubation may be carried out, for example, at 37°C for 16 hours. This incubation may be carried out in culture medium (e.g., supplemented with 4% PEG8000). After incubation, the cells may be washed and further cultivated. To measure viral infectivity, for example, the levels of hepatitis B surface antigen (HBsAg) and / or hepatitis B e antigen (HBeAg) secreted into the culture supernatant from 7 to 11 days after infection may be determined by enzyme-linked immunosorbent assay (ELISA). Furthermore, HBcAg staining may be assessed by immunofluorescence assay. In embodiments of the HDV neutralization assay, essentially the same assay as that for HBV may be used, with the difference being that serum derived from an HDV carrier may be used as the HDV infection inoculation material for differentiated HepaRg cells (instead of HBV). For detection, delta antigen immunofluorescence staining may be used as a readout.

[0156] The embodiments of the binding proteins described herein have high neutralizing efficacy. In certain embodiments, the concentration of the antibody described herein required for 50% neutralization of hepatitis B virus (HBV) and hepatitis delta virus (HDV) is, for example, about 10 μg / ml or less. In other embodiments, the concentration of the binding protein required for 50% neutralization of HBV and HDV is about 5 μg / ml. In yet another embodiment, the concentration of the binding protein described herein required for 50% neutralization of HBV and HDV is about 1 μg / ml. In yet another embodiment, the concentration of the binding protein required for 50% neutralization of HBV and HDV is about 750 ng / ml. In yet another embodiment, the concentration of the binding protein described herein required for 50% neutralization of HBV and HDV is 500 ng / ml or less. In such embodiments, the concentration of the binding protein described herein required for 50% neutralization of HBV and HDV may be selected from 450 ng / ml or less, 400 ng / ml or less, 350 ng / ml or less, 300 ng / ml or less, 250 ng / ml or less, 200 ng / ml or less, 175 ng / ml or less, 150 ng / ml or less, 125 ng / ml or less, 100 ng / ml or less, 90 ng / ml or less, 80 ng / ml or less, 70 ng / ml or less, 60 ng / ml or less, 50 ng / ml or less, or less than 20 ng / ml, preferably 15 ng / ml or less, more preferably 10 ng / ml or less, for example, less than 7 ng / ml.

[0157] The binding proteins according to this disclosure, which can neutralize both HBV and HDV, are useful in the prevention and treatment of hepatitis B and D. Infection with HDV typically occurs concurrently with or following infection with HBV (for example, inoculation with HDV in the absence of HBV does not cause hepatitis D, because HDV requires the support of HBV for its own replication), and hepatitis D is typically observed in chronic HBV carriers.

[0158] Embodiments of the binding protein disclosed facilitate the clearance of HBsAg and HBV. In certain embodiments, the binding protein facilitates the clearance of both HBV and subviral particles (SVPs) of the hepatitis B virus. The clearance of HBsAg or subviral particles can be assessed, for example, by measuring the level of HBsAg in a blood sample derived from a hepatitis B patient. Similarly, the clearance of HBV can be assessed, for example, by measuring the level of HBV in a blood sample derived from a hepatitis B patient.

[0159] In the serum of patients infected with HBV, in addition to infectious particles (HBV), there is typically an excess (typically 1,000 to 100,000 times) of empty subviral particles (SVPs) consisting solely of HBV envelope protein (HBsAg), in the form of relatively small spheres and filaments of varying lengths. Subviral particles have been shown to strongly enhance intracellular viral replication and gene expression of HBV (Bruns M. et al. 1998 J Virol 72(2): 1462-1468). This is also relevant to the infectivity of HBV-containing serum, as infectivity depends not only on the number of viruses but also on the number of SVPs (Bruns M. et al. 1998 J Virol 72(2): 1462-1468). Furthermore, the excess subviral particles can function as decoys by absorbing neutralizing antibodies, thus delaying the clearance of infection. Achieving loss of hepatitis B surface antigen (HBsAg) is, in some cases, the ideal endpoint of treatment and is considered the closest outcome to a cure for chronic hepatitis B (CHB).

[0160] Embodiments of the binding proteins of this disclosure may facilitate the clearance of HbsAg. In certain embodiments, the binding proteins may facilitate the clearance of subviral particles of hepatitis B virus. In some embodiments, the binding proteins may be used to treat chronic hepatitis B.

[0161] In any of the embodiments disclosed herein, the binding protein of this disclosure is capable of binding to HBsAg genotypes A, B, C, D, E, F, G, H, I, and J, or any combination thereof.

[0162] In certain embodiments, the binding protein of the present disclosure is capable of binding to one, two, three, four, five, six, seven, eight, nine, or ten of the HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. Examples of different HBsAg genotypes include: GenBank accession number J02203 (HBV-D, ayw3); GenBank accession number FJ899792.1 (HBV-D, adw2); GenBank accession number AM282986 (HBV-A); GenBank accession number D23678 (HBV-B1 Japan); GenBank accession number AB117758 (HBV-C1 Cambodia); GenBank accession number AB205192 (HBV-E Ghana); GenBank accession number X69798 (HBV-F4 Brazil); GenBank accession number AF160501 (HBV-G USA); GenBank accession number AY090454 (HBV-H Nicaragua); GenBank accession number AF241409 (HBV-I Vietnam); and GenBank accession number AB486012 (HBV-J Borneo). Exemplary amino acid sequences of the antigenic loop region of the S domain of HBsAg from different genotypes are described herein (e.g., SEQ ID NOs. 5-15).

[0163] In some embodiments, the binding protein can bind to one or more of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I, and J, and in some cases, to at least six. In certain embodiments, the binding protein can bind to at least eight of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. In some embodiments, the binding protein can bind to all 10 of the 10 HBsAg genotypes A, B, C, D, E, F, G, H, I, and J. HBV is classified into several genotypes according to its genome sequence. To date, eight known genotypes (A-H) of HBV genomes have been defined. In addition, two other genotypes, I and J, have also been identified (Sunbul M., 2014, World J Gastroenterol 20(18): 5427-5434). It is well known that genotype influences disease progression, and differences between genotypes in response to antiviral treatment have been determined.

[0164] In some embodiments, the binding protein according to this disclosure can bind to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, fourteenth, fifteenth, sixteenth, seventeenth, or eighteenth HBsAg variants having mutations in the antigenic loop region, such variants include HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, and HBsAg One or more of the following variants are selected: P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A. These variants are naturally occurring variants of the S domain of HBsAg genotype D, based on Genbank accession number FJ899792 (SEQ ID NO: 4). The mutated amino acid residues in each of the variants discussed herein are indicated in the name. Sequence ID 4: [ka] (The antigenic loop region, i.e., amino acids 101-172, is underlined.)

[0165] The amino acid sequences of the antigenic loop region of the S domain of different HBsAg variants are shown in SEQ ID NOs: 16-33.

[0166] In certain embodiments, the binding proteins disclosed herein are capable of binding to one or more, and in some cases at least 12, infectious HBsAg variants selected from HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A. In some such embodiments, the binding protein is capable of binding to at least 15 infectious HBsAg variants selected from HBsAg Y100C / P120T, HBsAg P120T, HBsAg P120T / S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R, and HBsAg N146A. In some embodiments, the binding protein can bind to each of the following infectious HBsAg variants: HBsAg Y100C / P120T; HBsAg P120T; HBsAg P120T / S143L; HBsAg C121S; HBsAg R122D; HBsAg R122I; HBsAg T123N; HBsAg Q129H; HBsAg Q129L; HBsAg M133H; HBsAg M133L; HBsAg M133T; HBsAg K141E; HBsAg P142S; HBsAg S143K; HBsAg D144A; HBsAg G145R; and HBsAg N146A.

[0167] In certain embodiments, a binding protein (e.g., including an antibody or its antigen-binding fragment) can reduce the serum concentration of HBV DNA in mammals with HBV infection. In certain embodiments, a binding protein can reduce the serum concentration of HBsAg in mammals with HBV infection. In certain embodiments, a binding protein can reduce the serum concentration of HBeAg in mammals with HBV infection. In certain embodiments, a binding protein can reduce the serum concentration of HBcrAg in mammals with HBV infection. In some embodiments, a binding protein can reduce the serum concentrations of HBV DNA, HBsAg, HBeAg, and / or HbcrAg in mammals for approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days, or longer, after a single administration of the binding protein.

[0168] The term “epitope” or “antigenic epitope” includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a homozygous binding molecule, such as an immunoglobulin, chimeric antigen receptor, or other binding molecule, domain, or protein. Epitope determinants generally consist of a chemically active surface grouping of a molecule, such as an amino acid or sugar side chain, and may have specific three-dimensional structural features as well as specific charge features.

[0169] In some embodiments, the binding protein can bind to an epitope comprising at least one, at least two, at least three, or at least four amino acids in the antigenic loop region of HBsAg. In certain embodiments, the binding protein can bind to at least two amino acids selected from amino acids 115-133, amino acids 120-133, or amino acids 120-130 of the S domain of HBsAg. In certain embodiments, the binding protein can bind to at least three amino acids selected from amino acids 115-133, amino acids 120-133, or amino acids 120-130 of the S domain of HBsAg. In some embodiments, the binding protein can bind to at least four amino acids selected from amino acids 115-133, amino acids 120-133, or amino acids 120-130 of the S domain of HBsAg. As used herein, the amino acid positions (e.g., 115-133, 120-133, 120-130) refer to the S domain of HBsAg, as described above, which is present in all three HBV envelope proteins: S-HBsAg, M-HBsAg, and L-HBsAg, thereby corresponding to the S domain of HBsAg in S-HBsAg.

[0170] With respect to epitopes, the term "formed by" means, as used herein, that the epitope to which a binding protein binds can be linear (continuous) or three-dimensional (discontinuous). A linear or continuous epitope is an epitope recognized by an antibody according to its linear sequence of amino acids, or primary structure. A three-dimensional epitope can be recognized according to its three-dimensional shape and protein structure. Therefore, if an epitope is a linear epitope and contains more than one amino acid located from amino acid positions 115-133 or selected from amino acid positions 120-133 of the S domain of HBsAg, the amino acids contained by that epitope can be located in adjacent positions in the primary structure (e.g., consecutive amino acids in the amino acid sequence). In the case of a three-dimensional epitope (3D structure), the amino acid sequence typically forms the 3D structure as an epitope, and therefore, the amino acids that form the epitope may or may not be located adjacent to the primary structure (i.e., they may not be consecutive amino acids in the amino acid sequence).

[0171] In certain embodiments, the epitope to which the binding protein binds is a structural epitope. In some embodiments, the binding protein binds to an epitope containing at least two amino acids from the antigenic loop region of HBsAg, where at least two amino acids are selected from amino acids 120-133 or 120-130 of the S domain of HBsAg, and at least two of the amino acids are not located in adjacent positions (in the primary structure). In certain embodiments, the binding protein binds to an epitope containing at least three amino acids from the antigenic loop region of HBsAg, where at least three amino acids are selected from amino acids 120-133 or 120-130 of the S domain of HBsAg, and at least two of the three amino acids are not located in adjacent positions (in the primary structure). In some embodiments, the binding protein binds to an epitope comprising at least four amino acids in the antigenic loop region of HBsAg, wherein at least four amino acids are selected from amino acids 120-133 or 120-130 of the S domain of HBsAg, and at least two of the four amino acids are not located in adjacent positions (in the primary structure).

[0172] The amino acids to which the antibodies, antigen-binding fragments, or fusion proteins disclosed herein bind (i.e., amino acids that form an epitope), which are not located in adjacent positions on the primary structure, are, in some cases, separated by one or more amino acids to which neither the antibody, antigen-binding fragment, nor fusion protein binds. In some embodiments, at least one, at least two, at least three, at least four, or at least five amino acids may be located between two non-adjacent amino acids that are included by the epitope.

[0173] In certain embodiments, the binding protein binds to an epitope comprising at least amino acids P120, C121, R122, and C124 of the S domain of HBsAg. In other embodiments, the binding protein of this disclosure is SEQ ID NO: 115: PCRXC It binds to an epitope containing the amino acid sequence described in the formula, where X is any amino acid or not an amino acid; X is any amino acid; X is T, Y, R, S, or F; X is T, Y, or R; or X is T or R.

[0174] In other embodiments, the binding protein of this disclosure is SEQ ID NO: 107: TGPCRTC It binds to an epitope containing the amino acid sequence described in [reference], or to an amino acid sequence that shares at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 107.

[0175] In other embodiments, the binding protein of this disclosure is SEQ ID NO: 112: STTSTGPCRTC It binds to an epitope containing the amino acid sequence described in [reference], or to an amino acid sequence that shares at least 80%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 112.

[0176] In certain embodiments, the binding protein of the present disclosure is at least amino acids 145-151 of the S domain of HBsAg: GNCTCIP It binds to an epitope containing an amino acid sequence including (SEQ ID NO: 108).

[0177] In other embodiments, the binding protein of this disclosure binds to an epitope comprising the amino acid sequence described in SEQ ID NO: 107 and the amino acid sequence described in SEQ ID NO: 108.

[0178] In other embodiments, the binding protein of this disclosure binds to an epitope comprising the amino acid sequence described in SEQ ID NO: 112 and / or the amino acid sequence described in SEQ ID NO: 114.

[0179] As described above, the epitopes to which the binding proteins of this disclosure bind may be linear (continuous) or three-dimensional (discontinuous). In some embodiments, the binding proteins of this disclosure bind to three-dimensional epitopes, and in certain such embodiments, the three-dimensional epitopes exist only under non-reducing conditions.

[0180] In certain embodiments, the binding protein of this disclosure binds to a linear epitope. In certain such embodiments, the linear epitope is present under both non-reducing and reducing conditions.

[0181] In certain embodiments, the binding protein of this disclosure is SEQ ID NO: X1X2X3TCX4X5X6AX7G The amino acid sequence described above binds to an epitope in the antigenic loop of HBsAg, where X1, X2, X3, X4, X5, X6, and X7 can be any amino acid (SEQ ID NO: 1).

[0182] In some embodiments, X1, X2, X3, X4, X5, X6, and X7 are conservatively substituted amino acids compared to amino acids 120-130 of SEQ ID NO: 3. In some embodiments, X1, X2, X3, X4, X5, X6, and X7 are conservatively substituted amino acids compared to any of amino acids 20-30 of SEQ ID NOs: 5-33.

[0183] In specific embodiments, X1 in SEQ ID NO: 1 is a small amino acid. “Small” amino acid, as used herein, refers to any amino acid selected from the group consisting of alanine, aspartic acid, asparagine, cysteine, glycine, proline, serine, threonine, and valine. In certain such embodiments, X1 is proline, serine, or threonine.

[0184] In certain embodiments, X2 in SEQ ID NO: 1 is a small amino acid. In certain embodiments, X2 may be selected from cysteine ​​or threonine.

[0185] In some embodiments, X3 in SEQ ID NO: 1 is a charged amino acid or an aliphatic amino acid. “Charged” amino acid, as used herein, refers to any amino acid selected from the group consisting of arginine, lysine, aspartic acid, glutamic acid, and histidine. “Aliphatic” amino acid, as used herein, refers to any amino acid selected from the group consisting of alanine, glycine, isoleucine, leucine, and valine. In certain embodiments, X3 is selected from arginine, lysine, aspartic acid, or isoleucine.

[0186] In some embodiments, X4 in SEQ ID NO: 1 is a small amino acid and / or a hydrophobic amino acid. “Hydrophobic” amino acid, as used herein, refers to any amino acid selected from the group consisting of alanine, isoleucine, leucine, phenylalanine, valine, tryptophan, tyrosine, methionine, proline, and glycine. In certain embodiments, X4 is selected from methionine or threonine.

[0187] In some embodiments, X5 in SEQ ID NO: 1 is a small amino acid and / or a hydrophobic amino acid. In certain embodiments, X5 is selected from threonine, alanine, or isoleucine.

[0188] In some embodiments, X6 in SEQ ID NO: 1 is a small amino acid and / or a hydrophobic amino acid. In certain embodiments, X6 is selected from threonine, proline, or leucine.

[0189] In some embodiments, X7 in SEQ ID NO: 1 is a polar amino acid or an aliphatic amino acid. “Polar” amino acid, as used herein, refers to any amino acid selected from the group consisting of aspartic acid, asparagine, arginine, glutamic acid, histidine, lysine, glutamine, tryptophan, tyrosine, serine, and threonine. In certain such embodiments, X7 is glutamine, histidine, or leucine.

[0190] In some embodiments, the binding protein according to this disclosure is SEQ ID NO: X1X2X3TCX4X5X6AX7G It binds to an epitope in the antigenic loop of HBsAg formed by the amino acid sequence described above, in the formula, X1 is P, T, or S. X2 is either C or S. X3 is R, K, D, or I. The X4 is M or T. X5 is T, A, or I. X6 is T, P, or L. X7 is Q, H, or L. (Sequence ID 2).

[0191] With respect to epitopes formed by the amino acid sequences described in SEQ ID NO: 1 or 2, it should be noted that the term "formed by ~" as used herein is not intended to imply that the disclosed binding protein necessarily binds to each and all amino acids of SEQ ID NO: 1 or 2. In particular, the binding protein may bind to only some of the amino acids of SEQ ID NO: 1 or 2, thereby allowing the other amino acid residues to act as "spacers."

[0192] In certain embodiments, the binding protein according to this disclosure binds to an epitope in the antigenic loop of HBsAg, which is formed by one or more amino acid sequences selected from SEQ ID NOs. 5-33 shown below in Table 4, consisting of two or more, three or more, or four or more amino acids.

[0193] In some embodiments, the binding protein according to the present disclosure binds to the antigenic loop region of HBsAg having the amino acid sequence set forth in any one or more of SEQ ID NOs: 5-33 shown in Table 4 below, or to a sequence variant thereof. In certain embodiments, the binding protein according to the present disclosure binds to all of the antigenic loop variants of HBsAg having the amino acid sequence set forth in any of SEQ ID NOs: 5-33 shown in Table 4 below.

[0194]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

[0195] Fc portion In some embodiments, the binding protein of the present disclosure (e.g., an antibody or an antigen-binding fragment thereof) includes an Fc portion (also referred to as an Fc polypeptide). In certain embodiments, the Fc portion may be of human origin, e.g., human IgG1, IgG2, IgG3, and / or IgG4, or may be derived from another Ig class or isotype. In a specific embodiment, the antibody or antigen-binding fragment may include an Fc portion derived from human IgG1. In certain embodiments, the Fc portion includes or is derived from the IgG1m17,1 (IgHG1 * 01) allotype (e.g., includes one or more mutations compared to the IgG1m17,1 (IgHG1 * 01) allotype).

[0196] As used herein, the term “Fc moiety” refers to a sequence that includes, comprises, essentially comprises, or derives from, a portion of an immunoglobulin heavy chain, beginning in the hinge region immediately upstream of a papain cleavage site (e.g., residue 216 in native IgG according to EU numbering, with the first residue of the heavy chain constant region being 114) and ending at the C-terminus of the immunoglobulin heavy chain. Thus, an Fc moiety may be a complete Fc moiety or a portion thereof (e.g., a domain). In certain embodiments, a complete Fc moiety includes a hinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acid positions 216–446). As noted herein, an additional lysine residue (K) is sometimes present at the furthest C-terminus of the Fc moiety, which is often cleaved from the mature antibody. Amino acid positions within the Fc moiety are numbered according to Kabat’s EU numbering system. For example, see Kabat et al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983 and 1987. The amino acid positions of the Fc portion can also be numbered according to the IMGT numbering system (including its unique numbering for the C-domain and exon numbering) and the Kabat numbering system.

[0197] In some embodiments, the Fc portion comprises at least one of the following: a hinge (e.g., upper, central, and / or lower hinge regions) domain, a CH2 domain, a CH3 domain, or variants, portions, or fragments thereof. In some embodiments, the Fc portion comprises at least a hinge domain, a CH2 domain, or a CH3 domain. In further embodiments, the Fc portion is a complete Fc portion. An exemplary amino acid sequence of the Fc portion of the human IgG1 isotype is provided in SEQ ID NO: 73. The Fc portion may also include one or more amino acid insertions, deletions, or substitutions compared to the naturally occurring Fc portion. For example, at least one of the hinge domain, the CH2 domain, or the CH3 domain, or portions thereof, may be deleted. For example, the Fc portion may comprise or consist of (i) a hinge domain (or a portion thereof) fused to the CH2 domain (or a portion thereof), (ii) a hinge domain (or a portion thereof) fused to the CH3 domain (or a portion thereof), (iii) a CH2 domain (or a portion thereof) fused to the CH3 domain (or a portion thereof), (iv) a hinge domain (or a portion thereof), (v) a CH2 domain (or a portion thereof), or (vi) a CH3 domain or a portion thereof.

[0198] The Fc portion of the present disclosure can be modified such that its amino acid sequence varies from the complete Fc portion of a naturally occurring immunoglobulin molecule while retaining or enhancing at least one desirable function conferred by the naturally occurring Fc portion and / or reducing an undesirable function of the naturally occurring Fc portion. Such functions include, for example, Fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding. Portions of the naturally occurring Fc portion involved in such functions are described in the art.

[0199] For example, to activate the complement cascade, the C1q protein complex can bind to at least two IgG1 molecules or one IgM molecule when an immunoglobulin molecule(s) binds to an antigen target (Ward, ES, and Ghetie, V., Ther. Immunol. 2 (1995) 77-94). Burton, DR described that the heavy chain region containing amino acid residues 318-337 is involved in complement fixation (Mol. Immunol. 22 (1985) 161-206). Duncan, AR, and Winter, G. (Nature 332 (1988) 738-740) reported using site-directed mutagenesis that Glu318, Lys320, and Lys322 form binding sites for C1q. The roles of the Glu318, Lys320, and Lys322 residues in C1q binding were confirmed by their ability to inhibit complement-mediated lysis of short synthetic peptides containing these residues.

[0200] For example, FcR binding can be mediated by the interaction between the Fc portion (of an antibody) and the Fc receptor (FcR), a specialized cell surface receptor on cells, including hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of corresponding antibody-coated erythrocytes and various other cellular targets (e.g., tumor cells) via antibody-dependent cell-mediated cytotoxicity (ADCC; Van de Winkel, JG, and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin classes; the Fc receptor for IgG antibodies is called FcγR, the Fc receptor for IgE is called FcεR, the Fc receptor for IgA is called FcαR, and so on, with the neonatal Fc receptor being called FcRn. Fc receptor binding is described, for example, in Ravetch, JV, and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ, et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE, et al., Ann. Hematol. 76 (1998) 231-248.

[0201] Crosslinking of the receptor (FcγR) by the Fc domain of native IgG antibodies triggers a wide variety of effector functions, including phagocytosis, antibody-dependent cell-mediated cytotoxicity, and release of inflammatory mediators, as well as clearance of immune complexes and regulation of antibody production. The Fc portion that provides crosslinking of the receptor (e.g., FcγR) is intended herein. In humans, three classes of FcγR have been characterized to date: (i) FcγRI (CD64), which binds to monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils; (ii) FcγRII (CD32), which binds to complexed IgG with medium to low affinity and is widely expressed, particularly on leukocytes, and is considered a central player in antibody-mediated immunity, and can be divided into FcγRIIA, FcγRIIB, and FcγRIIC, which are involved in the immune system (iii) FcγRIII(CD16), which binds to IgG-Fc with medium to low affinity, although they perform different functions, and the extracellular domains of these receptors are highly homologous; and (iii) FcγRIII(CD16), which binds to IgG with medium to low affinity and is found in two forms: FcγRIIIA, which is found in NK cells, macrophages, eosinophils, and some monocytes and T cells and is thought to mediate ADCC; and FcγRIIIB, which is highly expressed in neutrophils.

[0202] FcγRIIA is found in many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and is thought to activate the killing process. FcγRIIB is thought to play a role in the inhibitory process and is found in B cells, macrophages, as well as mast cells and eosinophils. Importantly, it has been shown that 75% of all FcγRIIB is found in the liver (Ganesan, LP et al., 2012: "FcγRIIb on liver sinusoidal endothelium clears small immune complexes," Journal of Immunology 189: 4981-4988). FcγRIIB is highly expressed in hepatic sinusoidal endothelium called LSEC and in Kupffer cells in the liver, where LSEC is a major site for the clearance of small immune complexes (Ganesan, LP et al., 2012: FcγRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988).

[0203] In some embodiments, the antibodies and antigen-binding fragments disclosed herein include an Fc moiety for binding to FcγRIIb, particularly an Fc region such as that of an IgG-type antibody. Furthermore, as described in Chu, SY et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933, it is possible to enhance FcγRIIB binding by manipulating the Fc moiety by introducing mutations S267E and L328F. This can enhance the clearance of immune complexes (Chu, S., et al., 2014: Accelerated Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, An Fc-Engineered Antibody With Enhanced Affinity For Inhibitory Receptor FcγRIIb. Am J Respir Crit, American Thoracic Society International Conference Abstracts). In some embodiments, the antibody or antigen-binding fragment of the present disclosure includes an engineered Fc moiety having mutations S267E and L328F, in particular, as described in Chu, SY et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933.

[0204] In B cells, FcγRIIB appears to function by suppressing further immunoglobulin production and, for example, isotype switching to the IgE class. In macrophages, FcγRIIB is thought to inhibit phagocytosis mediated through FcγRIIA. In eosinophils and mast cells, the B form may help suppress the activation of these cells through the binding of IgE to its respective receptors.

[0205] Regarding FcγRI binding, at least one modification of native IgG at positions E233-G236, P238, D265, N297, A327, and P329 may reduce FcγRI binding. Substitution of the IgG2 residue at positions 233-236 to the corresponding positions of IgG1 and IgG4 reduces the FcγRI binding of IgG1 and IgG4 by 10. 3 This reduces the response by twofold, eliminating the human monocyte response to antibody-sensitized red blood cells (Armour, KL, et al. Eur. J. Immunol. 29 (1999)). 2613-2624).

[0206] Regarding FcγRII binding, for example, reduced binding to FcγRIIA is observed for at least one IgG mutation in E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414.

[0207] The two alleles of human FcγRIIA are the "H131" variant, which binds to IgG1 Fc with high affinity, and the "R131" variant, which binds to IgG1 Fc with low affinity. See, for example, Bruhns et al., Blood 113:3716-3725 (2009).

[0208] Regarding FcγRIII binding, for example, reduced binding to FcγRIIIA is found for at least one mutation in E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376. Mapping of binding sites on human IgG1 for the Fc receptor, the mutation sites mentioned above, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604.

[0209] The two alleles of human FcγRIIIA are the "F158" variant, which binds to IgG1 Fc with low affinity, and the "V158" variant, which binds to IgG1 Fc with high affinity. See, for example, Bruhns et al., Blood 113:3716-3725 (2009).

[0210] Regarding binding to FcγRII, two regions of native IgG Fc, namely (i) the lower hinge region of IgG Fc, particularly amino acid residues L, L, G, G (234-237, EU numbered), and (ii) the region adjacent to the CH2 domain of IgG Fc, particularly the loop and chain in the upper CH2 domain adjacent to the lower hinge region, for example, in the P331 region, appear to be involved in the interaction between FcγRII and IgG (Wines, BD, et al., J. Immunol. 2000; 164: 5313 - 5318). Furthermore, FcγRII appears to bind to the same site on IgG Fc, while FcRn and protein A bind to different sites on IgG Fc, which appear to be on the CH2-CH3 interface (Wines, BD, et al., J. Immunol. 2000; 164: 5313 - 5318).

[0211] Mutations of the Fc moiety of this disclosure are also intended to increase the binding affinity of the Fc receptor (i.e., to one or more) to Fcγ receptors (e.g., compared to an antibody containing it without the reference Fc moiety or mutation(s)). See, for example, Delillo and Ravetch, Cell 161(5):1035-1045 (2015) and Ahmed et al., J. Struc. Biol. 194(1):78 (2016), where these Fc mutations and techniques are incorporated herein by reference.

[0212] In any of the embodiments disclosed herein, the binding protein may include an Fc moiety (e.g., IgG1 or IgG1-derived) containing a mutation selected from G236A / S239D / A330L / I332E; or a combination of any two or more of these; for example, S239D / I332E;S239D / A330L / I332E;G236A / S239D / I332E;G236A / A330L / I332E (also referred to herein as "GAALIE"); or an Fc moiety (e.g., IgG1 or IgG1-derived) containing a mutation selected from G236A / S239D / A330L / I332E. In some embodiments, the Fc moiety does not contain S239D. In some embodiments, the Fc moiety contains a native serine at position 239.

[0213] In certain embodiments, the Fc moiety may include, or be derived from, at least a portion of the Fc moiety involved in binding to FcRn binding. In certain embodiments, the Fc moiety includes one or more amino acid modifications that improve (e.g., enhance) the binding affinity to FcRn (e.g., at pH about 6.0), and in certain embodiments, this extends the in vivo half-life of the molecule containing the Fc moiety (e.g., compared to the same antibody except that it does not contain a reference Fc polypeptide or a fragment or modification(s) therein). In certain embodiments, the Fc moiety includes or is derived from IgG Fc, and the mutations that extend the half-life include one or more of M428L;N434S;N434H;N434A;N434S;M252Y;S254T;T256E;T250Q;P257I;Q311I;D376V;T307A;E380A (EU numbering). In certain embodiments, the half-life-extending mutation includes M428L / N434S (also referred to herein as "MLNS"). In certain embodiments, the half-life-extending mutation includes M252Y / S254T / T256E. In certain embodiments, the half-life-extending mutation includes T250Q / M428L. In certain embodiments, the half-life-extending mutation includes P257I / Q311I. In certain embodiments, the half-life-extending mutation includes P257I / N434H. In certain embodiments, the half-life-extending mutation includes D376V / N434H. In certain embodiments, the half-life-extending mutation includes T307A / E380A / N434A.

[0214] In some embodiments, the binding protein includes an Fc moiety containing the substitution mutation M428L / N434S. In some embodiments, the binding protein includes an Fc moiety containing the substitution mutation G236A / A330L / I332E. In certain embodiments, the binding protein includes an Fc moiety containing the G236A mutation, the A330L mutation, and the I332E mutation (GAALIE), but not the S239D mutation (e.g., containing native S at position 239) (e.g., IgG). In certain embodiments, the binding protein includes an Fc moiety containing the substitution mutations: M428L / N434S and G236A / A330L / I332E, and optionally, not containing S239D (e.g., may contain native S at position 329). In certain embodiments, the binding protein includes an Fc moiety containing the substitution mutations: M428L / N434S and G236A / S239D / A330L / I332E. In certain further embodiments, the binding protein contains substitutional mutations in the Fc portion, and these substitutional mutations consist of or essentially consist of M428L / N434S, G236A / S239D / A330L / I332E, or G236A / S239D / A330L / I332E / M428L / N434S.

[0215] In any of the embodiments disclosed herein, the binding protein of this disclosure comprises an Fc moiety containing the GAALIE mutation and has enhanced binding to human FcγRIIa and / or human FcγRIIIa compared to a reference polypeptide (i.e., a polypeptide that may be a binding protein and contains an Fc moiety that does not contain the GAALIE mutation).

[0216] In certain embodiments, the reference polypeptide comprises an Fc moiety that is either a wild-type Fc moiety (e.g., of the same isotype) or an Fc moiety containing one or more substitutional mutations (or insertions or deletions), provided that the substitutional mutation is not GAALIE or does not contain it. In certain embodiments, the reference polypeptide does not contain substitutional mutations that are known or thought to affect binding to human FcγRIIa and / or human FcγRIIIa.

[0217] The binding between polypeptides, such as between an Fc portion (or a binding protein containing the same) and a human Fcγ receptor, such as human FcγRIIA, human FcγRIIIA, or human FcγRIIB, or between a complement protein, such as C1q, can be determined or detected using methods known in the art. For example, a Biolayer Interference (BLI) assay can be performed using an Octet® RED96 (ForteBio, Fremont, California USA) instrument according to the manufacturer's instructions to determine the real-time association and dissociation between a first polypeptide of interest (e.g., an Fc portion containing a GAALIE mutation) and a second polypeptide of interest captured on a sensor chip (e.g., FcγRIIA(H131), FcγRIIA(R131), FcγRIIIA(F158), FcγRIIIA(V158), or FcγRIIb).

[0218] In certain embodiments, the binding protein comprises an Fc moiety containing the GAALIE mutation and has enhanced binding to human FcγRIIA(H131), human FcγRIIA(R131), human FcγRIIIA(F158), human FcγRIIIA(V158), or any combination thereof, compared to a reference polypeptide comprising an Fc moiety without the GAALIE mutation. In certain embodiments, enhanced binding is determined by an increase in the signal shift compared to the reference binding protein in the BLI assay (e.g., a higher peak signal; a faster association rate; a slower dissociation rate; or a larger area under the curve, one or more of these). In certain embodiments, the BLI assay involves the use of an Octet® RED96 (ForteBio, Fremont, California, USA) instrument. In further embodiments, the BLI assay comprises tagged human FcγR captured on an anti-penta-tag sensor and exposed to the binding protein. In some embodiments, the binding protein comprises IgG Fab, and the BLI assay further comprises exposing the binding protein to captured human FcγR in the presence of an anti-IgG Fab binding fragment in order to crosslink the binding protein via the Fab fragment.

[0219] In certain embodiments, the binding protein comprises an Fc moiety containing the GAALIE mutation and has enhanced binding to human FcγRIIA(H131), human FcγRIIA(R131), human FcγRIIIA(F158), and / or human FcγRIIIA(V158) compared to the reference polypeptide, and this enhanced binding may include a signal shift (nanometers) in the BLI assay that is 1.5 times, 2 times, 2.5 times, 3 times, or greater than the signal shift observed using the reference binding protein.

[0220] In certain embodiments, the binding protein comprises an Fc moiety containing the GAALIE mutation and has enhanced binding to human FcγRIIA(H131), human FcγRIIA(R131), human FcγRIIIA(F158), and human FcγRIIIA(V158) compared to the reference polypeptide.

[0221] In any of the embodiments disclosed herein, the binding protein comprises an Fc moiety containing the GAALIE mutation and has reduced binding to human FcγRIIB compared to the reference polypeptide. In certain embodiments, the binding protein comprises an Fc moiety containing the GAALIE mutation and does not bind to human FcγRIIB, as determined, for example, by the absence of a statistically significant signal shift compared to baseline in a BLI assay.

[0222] In any of the embodiments disclosed herein, the binding protein comprises an Fc moiety containing the GAALIE mutation and has reduced binding to human C1q (complement protein) compared to the reference polypeptide. In certain embodiments, the binding protein comprises an Fc moiety containing the GAALIE mutation and does not bind to human C1q, as determined by the absence of a statistically significant signal shift compared to baseline in the BLI assay.

[0223] In any of the embodiments disclosed herein, the binding protein comprises an Fc moiety containing the GAALIE mutation and activates human FcγRIIA, human FcγRIIIA, or both to a higher degree than the reference polypeptide (i.e., a polypeptide that may be an HBsAg-specific binding protein comprising an Fc moiety that does not contain the GAALIE mutation). In certain embodiments, the reference polypeptide comprises either a wild-type Fc moiety or an Fc moiety containing one or more substitutional mutations, wherein the substitutional mutation is not GAALIE.

[0224] Activation of human FcγR can be determined or detected using methods known in the art. For example, well-validated commercially available bioreporter assays involve incubating HBsAg-specific binding protein with recombinant HBsAg (Engerix B, GlaxoSmithKline) in the presence of Jurkat effector cells (Promega; catalog no.: G9798) that stably express (i) the FcγR of interest and (ii) the firefly luciferase reporter under the control of an NFAT response element. Binding of Fc to the cell surface-expressed FcγR drives NFAT-mediated expression of the luciferase reporter gene. Luminescence is then measured using a luminometer (e.g., Bio-Tek) with the Bio-Glo-(trademark) luciferase assay reagent (Promega) according to the manufacturer's instructions for use. Activation is expressed as the average relative luminescence units (RLU) above the background by applying the following formula: (RLU of the bound protein (e.g., mAb) at concentration [x] - RLU of the background).

[0225] In certain embodiments, the binding protein comprises an Fc moiety containing the GAALIE mutation and activates human FcγRIIA(H131), human FcγRIIA(R131), human FcγRIIIA(F158), and / or human FcγRIIIA(V158) to a higher degree than the reference polypeptide. In certain embodiments, the higher degree of activation refers to a higher peak emission and / or a larger area under the emission curve, as determined using a luminescence bioreporter assay as described herein. In certain embodiments, the binding protein comprises an Fc moiety containing the GAALIE mutation and activates human FcγRIIA(H131), human FcγRIIA(R131), and human FcγRIIIA(F158) to a higher degree than the reference polypeptide, and the higher degree of activation includes a peak RLU that is 1.5 times, 2 times, 2.5 times, 3 times, or greater than the peak RLU observed using the reference binding protein.

[0226] In any of the embodiments disclosed herein, the binding protein comprises an Fc moiety containing the GAALIE mutation and does not activate human FcγRIIB, as determined by the absence of a statistically significant and / or measurable RLU in the luminescence bioreporter assay described above.

[0227] In any of the embodiments disclosed herein, the binding protein comprises an Fc moiety containing the GAALIE mutation and activates human natural killer (NK) cells in the presence of HBsAg to a higher degree than the reference polypeptide. In certain embodiments, NK cell activation is determined by CD107a expression (e.g., by flow cytometry). In certain embodiments, the NK cells include cells comprising the FcγRIIIa genotype that is V158 / V158 homozygous, F158 / F158 homozygous, or V158 / F158 heterozygous.

[0228] Any binding protein containing an Fc moiety with the GAALIE mutation as disclosed herein is understood to be able to perform or have any or more of the features described herein; for example, enhanced binding to human FcγRIIA and / or human FcγRIIIA compared to a reference polypeptide; reduced binding to human FcγRIIB (and / or no binding to human FcγRIIB) compared to a reference polypeptide; reduced binding to human C1q (and / or no binding to human C1q) compared to a reference polypeptide; activation of FcγRIIA, human FcγRIIIA, or both to a higher degree than the reference polypeptide; no activation of human FcγRIIB; and / or activation of human natural killer (NK) cells in the presence of HBsAg to a higher degree than the reference polypeptide (e.g., an antibody that is specific to HBsAg and contains an Fc moiety without the GAALIE mutation).

[0229] In certain embodiments, the binding protein of the Disclosure comprises an Fc moiety containing the GAALIE mutation and has enhanced binding to human FcγRIIA, human FcγRIIIA, or both, compared to a reference polypeptide comprising an Fc moiety not containing G236A / A330L / I332E, wherein human FcγRIIA is optionally H131 or R131, and / or human FcγRIIIA is optionally F158 or V158; (ii) has reduced binding to human FcγRIIB compared to a reference polypeptide comprising an Fc moiety not containing G236A / A330L / I332E; (iii) does not bind to human FcγRIIB; (iv) is a reference polypeptide comprising an Fc moiety not containing G236A / A330L / I332E. (v) Has reduced binding to human C1q compared to the reference polypeptide; does not bind to human C1q; (vi) Activates FcγRIIA, human FcγRIIIA, or both to a higher degree than the reference polypeptide containing an Fc moiety that does not contain G236A / A330L / I332E, where human FcγRIIA is H131 or R131 as necessary, and / or human FcγRIIIA is F158 or V158 as necessary; (vii) Does not activate human FcγRIIB; (viii) Activates human natural killer (NK) cells in the presence of HBsAg to a higher degree than the reference polypeptide containing an Fc moiety that does not contain G236A / A330L / I332E, where the reference polypeptide is HB as necessary Ag is an antibody that binds to HBsAg as needed; (ix) It can bind to HBsAg variants including HBsAg-Y100C / P120T, HBsAg-P120T, HBsAg-P120S / S143L, HBsAg-C121S, HBsAg-R122D, HBsAg-R122I, HBsAg-T123N, HBsAg-Q129H, HBsAg-Q129L, HBsAg-M133H, HBsAg-M133L, HBsAg-M133T, HBsAg-K141E, HBsAg-P142S, HBsAg-S143K, HBsAg-D144A, HBsAg-G145R, HBsAg-N146A, or any combination thereof;(x) Compared to a reference antibody or antigen-binding fragment that binds to HBsAg and contains an Fc portion that does not include G236A / A330L / I332E, HBsAg-Y100C / P120T, HBsAg-P120T, HBsAg-P120S / S143L, HBsAg-C121S, HBsAg-R122D, HBsAg-R122I, HBsAg-T123N, HBsAg-Q It has improved binding to HBsAg variants, including 129H, HBsAg-Q129L, HBsAg-M133H, HBsAg-M133L, HBsAg-M133T, HBsAg-K141E, HBsAg-P142S, HBsAg-S143K, HBsAg-D144A, HBsAg-G145R, HBsAg-N146A, or any combination thereof.

[0230] Alternatively, the Fc portion of the binding protein of the present disclosure may include at least a portion known in the art as required for protein A binding; and / or the Fc portion of the antibody of the present disclosure may include at least a portion of the Fc molecule known in the art as required for protein G binding. In some embodiments, the retained function includes clearance of HBsAg and HBVg. Thus, in certain embodiments, the Fc portion may include at least a portion known in the art as required for FcγR binding. Therefore, as outlined above, the Fc portion may include at least (i) the lower hinge region of native IgG Fc, particularly amino acid residues L, L, G, G (234-237, EU numbered), and (ii) the region adjacent to the CH2 domain of native IgG Fc, particularly in the upper CH2 domain adjacent to the lower hinge region, for example, the loop and chain in the P331 region, for example, the region of at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids in the upper CH2 domain of native IgG Fc, for example, between amino acids 320 and 340 (EU numbered) of native IgG Fc, around P331.

[0231] In some embodiments, the binding proteins according to this disclosure include an Fc region. As used herein, the term “Fc region” refers to a portion of immunoglobulin formed by two or more Fc parcels of an antibody heavy chain. For example, an Fc region may be a monomer or a “single-chain” Fc region (i.e., an scFc region). A single-chain Fc region consists of Fc parcels linked within a single polypeptide chain (e.g., encoded in a single consecutive nucleic acid sequence). An exemplary scFc region is disclosed in WO2008 / 143954A2, which is incorporated herein by reference. An Fc region may be or may include a dimeric Fc region; it is understood that a dimeric Fc region is not the same as an undesirable dimer (e.g., antibody:antibody, antibody:antigen-binding fragment, or antigen-binding fragment:antigen-binding fragment), such as shown in Figure 7 in one embodiment, as described above. In certain preferred embodiments, the antibody or antigen-binding fragment includes a dimeric Fc region while producing a small antibody-containing dimer or antigen-binding fragment-containing dimer.

[0232] A “dimeric Fc region” or “dcFc” refers to a dimer formed by the Fc portions of two separate immunoglobulin heavy chains. A dimeric Fc region may be a homodimer of two identical Fc portions (e.g., the Fc regions of naturally occurring immunoglobulins) or a heterodimer of two non-identical Fc portions (e.g., one Fc monomer of the dimeric Fc region contains at least one amino acid modification (e.g., substitution, deletion, insertion, or chemical modification) that is not present in the other Fc monomer, or one Fc monomer may be shortened compared to the other).

[0233] The Fc portions disclosed herein may comprise Fc sequences or regions of the same or different classes and / or subclasses. For example, an Fc portion may originate from an immunoglobulin of the IgG1, IgG2, IgG3, or IgG4 subclass (e.g., human immunoglobulin), or any combination thereof. In certain embodiments, the Fc portions of an Fc region belong to the same class and subclass. However, an Fc region (or one or more Fc portions of an Fc region) may also be chimeric, thereby a chimeric Fc region may comprise Fc portions derived from different immunoglobulin classes and / or subclasses. For example, at least two of the Fc portions of a dimer or single-chain Fc region may originate from different immunoglobulin classes and / or subclasses. In certain embodiments, the dimeric Fc region may contain sequences derived from two or more different isotypes or subclasses; see, for example, SEEDbody ("strand-exchanged domain"), Davis et al., Protein Eng. Des. Sel. 23(4):195 (2010).

[0234] Furthermore, or alternatively, a chimeric Fc region may comprise one or more chimeric Fc moieties. For example, a chimeric Fc region or moiety may comprise one or more moieties derived from an immunoglobulin of a first subclass (e.g., IgG1, IgG2, or IgG3 subclass), while the remainder of the Fc region or moiety is from a different subclass. For example, an Fc region or moiety of an Fc polypeptide may comprise CH2 and / or CH3 domains derived from an immunoglobulin of a first subclass (e.g., IgG1, IgG2, or IgG4 subclass) and a hinge region derived from an immunoglobulin of a second subclass (e.g., IgG3 subclass). For example, an Fc region or moiety may comprise a hinge and / or CH2 domain derived from an immunoglobulin of a first subclass (e.g., IgG4 subclass) and a CH3 domain derived from an immunoglobulin of a second subclass (e.g., IgG1, IgG2, or IgG3 subclass). For example, a chimeric Fc region may include an Fc portion (e.g., a complete Fc portion) derived from an immunoglobulin of a first subclass (e.g., an IgG4 subclass) and an Fc portion derived from an immunoglobulin of a second subclass (e.g., an IgG1, IgG2, or IgG3 subclass). For example, an Fc region or portion may include a CH2 domain derived from IgG4 immunoglobulin and a CH3 domain derived from IgG1 immunoglobulin. For example, an Fc region or portion may include CH1 and CH2 domains derived from an IgG4 molecule and a CH3 domain derived from an IgG1 molecule. For example, an Fc region or portion may include a portion of a CH2 domain derived from a particular subclass of the antibody, e.g., EU positions 292-340 of the CH2 domain. For example, the Fc region or portion may include amino acid positions 292-340 of CH2 derived from the IgG4 portion and the remainder of CH2 derived from the IgG1 portion (or, CH2 292-340 may be derived from the IgG1 portion, and the remainder of CH2 may be derived from the IgG4 portion).

[0235] It is understood that any antibody, antigen-binding fragment, or Fc region or portion of the present disclosure may be of any allotype and / or haplotype. For example, human immunoglobulin G allotypes include those disclosed in Jefferis and LeFranc, mAbs 1(4):1-7 (2009), and these allotypes (including G1m(1(a);2(x);3(f);and 17(z));G2m(23(n));G3m(21(g1);28(g5);11(b0);5(b2);13(b3);14(b4);10(b5);15(s);16(t);6(c3);24(c5);26(u);and 27(v));A2m(1 and 2);and Km(1;2;and 3)) as well as haplotypes, the resulting amino acid sequences, and combinations thereof are incorporated herein by reference. In certain embodiments, the antibody, antigen-binding fragment, or Fc region or portion of the present disclosure comprises IgG1 allotype g1m17,k1.

[0236] Furthermore, the Fc region or portion may (furthermore or otherwise) include, for example, a chimeric hinge region. For example, the chimeric hinge may be partially derived from, for example, an IgG1, IgG2, or IgG4 molecule (e.g., upper and lower central hinge sequences) and partially derived from an IgG3 molecule (e.g., a central hinge sequence). In another example, the Fc region or portion may include a chimeric hinge partially derived from an IgG1 molecule and partially derived from an IgG4 molecule. In yet another example, the chimeric hinge may include upper and lower hinge domains derived from an IgG4 molecule and a central hinge domain derived from an IgG1 molecule. Such a chimeric hinge may be created, for example, by introducing a proline substitution (Ser228Pro) at EU position 228 in the central hinge domain of the IgG4 hinge region. In another embodiment, the chimeric hinge may include amino acids at EU positions 233-236 and / or the Ser228Pro mutation derived from the IgG2 antibody, with the remaining amino acids of the hinge derived from the IgG4 antibody (e.g., the chimeric hinge of sequence ESKYGPPCPPCPAPPVAGP (SEQ ID NO: 74)). Further chimeric hinges that may be used in the Fc portion of the antibody according to this disclosure are described in US2005 / 0163783A1.

[0237] In some embodiments of the binding proteins disclosed herein, the Fc moiety or Fc region comprises or consists of an amino acid sequence derived from a human immunoglobulin sequence (e.g., derived from an Fc region or Fc moiety from a human IgG molecule). However, the polypeptide may contain one or more amino acids from another mammalian species. For example, a primate Fc moiety or primate binding site may be included in the polypeptide in question. Alternatively, one or more mouse amino acids may be present in the Fc moiety or Fc region.

[0238] In some embodiments, an antibody is provided comprising a heavy chain (HC) comprising or derived from the amino acid sequence described in SEQ ID NO: 75, with the C-terminal lysine removed as necessary, and a light chain (LC) comprising or derived from (i) the VL amino acid sequence described in any one of SEQ ID NOs. 58 to 72 and (ii) the CL amino acid sequence described in SEQ ID NO: 79.

[0239] In some embodiments, an antibody is provided comprising a heavy chain (HC) comprising or derived from the amino acid sequence described in SEQ ID NO: 76, with the C-terminal lysine removed as necessary, and a light chain (LC) comprising or derived from (i) the VL amino acid sequence described in any one of SEQ ID NOs. 58 to 72 and (ii) the CL amino acid sequence described in SEQ ID NO: 79.

[0240] In some embodiments, an antibody is provided comprising a heavy chain (HC) comprising or derived from the amino acid sequence described in SEQ ID NO: 77, with the C-terminal lysine removed as necessary, and a light chain (LC) comprising or derived from (i) the VL amino acid sequence described in any one of SEQ ID NOs. 58 to 72 and (ii) the CL amino acid sequence described in SEQ ID NO: 79.

[0241] In some embodiments, an antibody is provided comprising a heavy chain (HC) comprising or derived from the amino acid sequence described in SEQ ID NO: 78, with the C-terminal lysine removed as necessary, and a light chain (LC) comprising or derived from (i) the VL amino acid sequence described in any one of SEQ ID NOs. 58 to 72 and (ii) the CL amino acid sequence described in SEQ ID NO: 79.

[0242] Nucleic acid molecules / polynucleotides In another embodiment, the Disclosure provides nucleic acid molecules comprising polynucleotides encoding an antibody, antigen-binding fragment, or fusion protein according to the Disclosure. For example, a first nucleic acid molecule may encode the heavy chain of an antibody, and a second nucleic acid molecule may encode the light chain of an antibody; it is understood that these first and second nucleic acid molecules may still be referred to as “polynucleotides” or “nucleic acid molecules” encoding an antibody. In other words, embodiments of polynucleotides or nucleic acid molecules include those in which a portion (e.g., a chain) of an antibody or antigen-binding fragment is encoded by separate nucleic acid molecules and / or separate portions of nucleic acid molecules. Exemplary polynucleotide sequences are provided in SEQ ID NOs: 80–99. In some embodiments, a polynucleotide encoding an antibody heavy chain comprises or comprises the polynucleotide sequence described in SEQ ID NOs: 81, and a polynucleotide encoding an antibody VL or LC comprises the polynucleotide sequence described in any one of SEQ ID NOs: 85–99. In other embodiments, the polynucleotide encoding the antibody heavy chain includes or comprises the polynucleotide sequence described in SEQ ID NO: 83, and the polynucleotide encoding the antibody VL or LC includes the polynucleotide sequence described in any one of SEQ ID NOs.85 to 99. In other embodiments, the polynucleotide encoding the antibody heavy chain includes or comprises the polynucleotide sequence described in SEQ ID NO: 84, and the polynucleotide encoding the antibody VL or LC includes the polynucleotide sequence described in any one of SEQ ID NOs.85 to 99.

[0243] Due to the degeneracy of the genetic code, this disclosure also includes sequence variants of these nucleic acid sequences, in particular, such sequence variants that encode the same amino acid sequence.

[0244] In certain embodiments, the polynucleotide or nucleic acid molecule comprises a nucleotide sequence that shares at least 50% (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity with any nucleotide sequence described in any one of SEQ ID NOs.

[0245] In certain embodiments, the nucleic acid molecule according to this disclosure comprises or consists of any one of the nucleic acid sequences described in SEQ ID NOs: 80 to 99.

[0246] In a particular embodiment, the polynucleotide has at least 50% identity with the amino acid sequence described in SEQ ID NO: 81. H The coding nucleotide sequence has at least 50% identity with the amino acid sequence described in any one of sequence numbers 85-97. L Includes a coding nucleotide sequence.

[0247] In any of the embodiments disclosed herein, the polynucleotide may include the VH-CH1-hinge-CH2-CH3 coding nucleotide sequence described in SEQ ID NO: 84. In some embodiments, the polynucleotide includes a CL coding nucleotide sequence having at least 50% identity to the amino acid sequence described in SEQ ID NO: 98 or 99.

[0248] vector Vectors, such as expression vectors containing nucleic acid molecules as disclosed herein, are further included within the scope of this disclosure.

[0249] The term "vector" refers to a construct containing a nucleic acid molecule. For the purposes of this disclosure, a vector is suitable for incorporating or holding a desired nucleic acid sequence. Such a vector may be a storage vector, expression vector, cloning vector, or import vector. A storage vector is a vector that enables the convenient storage of a nucleic acid molecule. Therefore, a vector may, for example, contain a sequence corresponding to a desired antibody or a fragment of that antibody as described herein.

[0250] As used herein, “expression vector” refers to a DNA construct containing a nucleic acid molecule operably ligated to appropriate regulatory sequences capable of producing expression of the nucleic acid molecule in a suitable host. Such regulatory sequences include a promoter that produces transcription (e.g., a heterologous promoter), an operator sequence as needed to control such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences that control the termination of transcription and translation. Any of the elements of the expression vector contributing to the transcription of the nucleic acid molecule of interest may be heterologous to the vector. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, be incorporated into the genome itself. In this specification, “plasmid,” “expression plasmid,” “virus,” and “vector” are often used interchangeably.

[0251] A cloning vector is typically a vector containing a cloning site that can be used to incorporate a nucleic acid sequence into the vector. Cloning vectors can be, for example, plasmid vectors or bacteriophage vectors.

[0252] An import vector may be a vector suitable for introducing nucleic acid molecules into a cell or organism, such as a viral vector. In relation to this disclosure, a vector may be, for example, an RNA vector or a DNA vector. A vector may be a DNA molecule. For example, a vector in the sense of this application includes a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for vector replication, such as an origin of replication.

[0253] In certain embodiments, the vector includes plasmid vectors or viral vectors (e.g., lentiviral vectors or γ-retroviral vectors). Examples of viral vectors include negative-strand RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), and paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canary pox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis viruses. Examples of retroviruses include bird leukemia sarcoma, mammalian type C, type B, and type D viruses, HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0254] A "retrovirus" is a virus that has an RNA genome that is reverse-transcribed into DNA using reverse transcriptase, and then this reverse-transcribed DNA is incorporated into the host cell genome. "Gamma retrovirus" refers to a genus of the family Retroviridae. Examples of gamma retroviruses include mouse stem cell virus, mouse leukemia virus, feline leukemia virus, feline sarcoma virus, and reticuloendotheliosis virus.

[0255] Lentiviral vectors include HIV-based lentiviral vectors for gene delivery, which can be integrated or non-integrated, have relatively large packaging capacity, and can transduce into a range of different cell types. Lentiviral vectors are typically produced after transient transfection into plasmid-producing cells containing three (packaging, envelope, and transduction) or more plasmids. Similar to HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. Once inside, the viral RNA undergoes reverse transcription mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is the substrate for viral integration into the infected cell's DNA.

[0256] In certain embodiments, the viral vector may be a gamma retrovirus, such as a Moloney mouse leukemia virus (MLV) vector. In other embodiments, the viral vector may be a more complex retrovirus vector, such as a lentivirus vector. HIV-1 vectors belong to this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedivisna virus (sheep lentivirus). Methods using retroviral vectors, lentiviral vectors, and packaging cells to transduce mammalian host cells with viral particles containing transgenes are known in the art and have been previously described, for example, in U.S. Patent No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Constructs and expression systems for retroviral and lentiviral vectors are also commercially available. Other viral vectors can also be used for polynucleotide delivery, including, for example, DNA viral vectors such as adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; amplicon vectors, and vectors derived from herpes simplex virus (HSV), including replication-deficient HSV and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0257] Other vectors that can be used in conjunction with the compositions and methods of this disclosure include vectors derived from baculoviruses and alpha-viruses (Jolly, D J. 1999. Emerging Viral Vectors. pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).

[0258] If a viral vector genome contains multiple polynucleotides expressed in a host cell as separate transcripts, the viral vector may also contain additional sequences between the two (or more) transcripts to enable bicistronic or polycistronic expression. Examples of such sequences used in a viral vector include intrasequence ribosome entry sites (IRESs), furin cleavage sites, viral 2A peptides, or any combination thereof.

[0259] Plasmid vectors comprising plasmid vectors encoding DNA-based antibodies or antigen-binding fragments for direct administration to a subject are further described herein.

[0260] cell In a further embodiment, the Disclosure also provides cells (also referred to as “host cells”) that express antibodies, antigen-binding fragments, or fusion proteins according to the Disclosure; or vectors or polynucleotides according to the Disclosure.

[0261] Examples of such cells include, but are not limited to, eukaryotic cells such as yeast cells, animal cells, insect cells, and plant cells; and prokaryotic cells, including E. coli. In some embodiments, the cells are mammalian cells. In certain such embodiments, the cells are mammalian cell lines, e.g., CHO cells (e.g., DHFR-CHO cells (Urlaub et al., PNAS 77:4216 (1980), CHO-KSV, ExpiCHO)), human fetal kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, Sp2 / 0 cells, NS0 cells), human hepatocytes (e.g., Hepa RG cells), myeloma cells, or hybridoma cells. Other examples of mammalian host cell lines include mouse Sertoli cells (e.g., TM4 cells); monkey kidney CV1 line transformed with SV40 (COS-7); baby hamster kidney cells (BHK); African green monkey kidney cells (VERO-76); monkey kidney cells (CV1); human cervical cancer cells (HELA); human lung cells (W138); human hepatocytes (Hep G2); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); and mouse mammary tumor cells (MMT). Examples include TRI cells, MRC 5 cells, and FS4 cells. Suitable mammalian host cell lines for antibody production include, for example, those described in Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0262] In certain embodiments, the host cell is a prokaryotic cell such as E. coli. Peptide expression in prokaryotic cells such as E. coli has been well-established (see, for example, Pluckthun, A. Bio / Technology 9:545-551 (1991)). For example, antibodies can be generated in bacteria, particularly when glycosylation and Fc effector function are not required. For example, regarding the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523.

[0263] Insect cells useful for expressing the binding proteins of this disclosure are known in the art and include, for example, Spodoptera frugipera Sf9 cells, Trichoplusia ni BTI-TN5B1-4 cells, and Spodoptera frugipera SfSWT01 "Mimic" cells. See, for example, Palmberger et al., J. Biotechnol. 153(3-4):160-166 (2011). A very large number of baculovirus strains have been identified, which can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0264] Eukaryotic microorganisms, such as filamentous fungi or yeasts, are also suitable hosts for the cloning or expression of protein-coding vectors, including fungal and yeast strains that possess a “humanized” glycosylation pathway that results in the production of antibodies with partially or completely human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006).

[0265] Plant cells can also be used as hosts for the expression of the binding proteins of this disclosure. For example, PLANTIBODIES® technology (as described in U.S. Patents No. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429, for example) uses transgenic plants to generate antibodies.

[0266] In some embodiments, the fusion protein is expressed on the cell surface by immune cells, such as T cells, NK cells, or NK-T cells, or any subtype thereof.

[0267] The binding proteins of this disclosure can be produced using any protein expression system conforming to this disclosure. Suitable expression systems include the transgenic animals described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.

[0268] In certain embodiments, cells may be transfected with the vector described herein along with an expression vector. The term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, such as eukaryotic cells. For the purposes of this description, the term “transfection” encompasses any method known to those skilled in the art for the introduction of nucleic acid molecules into cells, such as into mammalian cells, or into eukaryotic cells. Such methods include, for example, electroporation, lipofection, such as lipofection based on cationic lipids and / or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethyleneimine. In certain embodiments, the introduction is not virus-based.

[0269] Furthermore, for example, cells of the disclosed invention can be stably or transiently transfected with the vector described herein to express the antibody or its antigen-binding fragment. In such embodiments, cells are stably transfected with the vector described herein that encodes the binding protein. Alternatively, cells can be transiently transfected with the vector described herein that encodes the binding protein. In any of the embodiments disclosed herein, the polynucleotide may be heterogeneous to the host cell.

[0270] In a relevant embodiment, the Disclosure provides a method for producing an antibody, antigen-binding fragment, or fusion protein, comprising culturing host cells of the Disclosure under conditions sufficient for producing the antibody, antigen-binding fragment, or fusion protein, for a sufficient amount of time for that purpose.

[0271] Accordingly, the Disclosure also provides recombinant host cells heterologously expressing the antibodies, antigen-binding fragments, or fusion proteins of the Disclosure. For example, the cells may be of a different species from the species from which the antibody is obtained completely or partially (e.g., CHO cells expressing human antibodies or engineered human antibodies). In some embodiments, the cell type of the host cell does not natively express the antibody or antigen-binding fragment. Furthermore, the host cell may confer post-translational modifications (PTMs; e.g., glycosylation or fucosylation) to the binding protein that are not present in the native binding protein (or the native parent binding protein from which the target binding protein is engineered or derived). Such PTMs may result in functional differences (e.g., reduced immunogenicity). Therefore, the binding proteins of this disclosure produced by host cells disclosed herein may include one or more posttranslational modifications that are distinctly different from the native binding protein or parent binding protein (for example, human antibodies produced by CHO cells may include posttranslational modifications that are distinctly different from antibodies isolated from humans and / or produced by native human B cells or plasma cells).

[0272] Further features of antibodies, antigen-binding fragments, and fusion proteins as needed. The antibodies, antigen-binding fragments, and fusion proteins of this disclosure can be coupled, for example, with a drug for delivery to a treatment site, or with a detectable label to facilitate imaging of a site containing cells of interest. Methods for coupling antibodies with drugs and detectable labels are well known in the art, as are methods for imaging using detectable labels. Labeled antibodies can be used in a wide variety of assays, and a wide variety of labels can be used. Detection of antibody-antigen complex formation between the antibody (or antigen-binding fragment or fusion protein) of this disclosure and a target epitope on HBsAg, particularly the antigenic loop region of HBsAg, can be facilitated by attaching a detectable substance to the antibody. Suitable detection means include, for example, the use of labels such as radionuclides, enzymes, coenzymes, fluorescent agents, chemiluminescent materials, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, and dyes. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansilchloride, or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include 125I, 131I, 35S, or 3H. Such labeled reagents can be used in various well-known assays, such as radioimmunoassays, enzyme immunoassays, such as ELISA and fluorescence immunoassays. Accordingly, the labeled antibodies, antigen-binding fragments, and fusion proteins according to this disclosure can be used in assays as described, for example, in US3,766,162;US3,791,932;US3,817,837; and US4,233,402.

[0273] The antibodies, antigen-binding fragments, or fusion proteins according to this disclosure can be conjugated with a therapeutic moiety, such as a cytotoxin, therapeutic agent, or radioactive metal ion or radioisotope. Examples of radioisotopes, but not limited to, include I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, and In-111. Such conjugates can be used to modify a given biological response; the drug moiety should not be interpreted as being limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, toxins, such as abrin, lysine A, Pseudomonas exotoxin, or diphtheria toxin.

[0274] Techniques for conjugating such therapeutic portions with antibodies are well known. For example, Arnon et al. (1985) "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy", in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp. 243-256;ed. Hellstrom et al. (1987) "Antibodies for Drug Delivery," in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker, Inc.), pp. 623-653;Thorpe (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al. pp. 475-506 (Editrice Kurtis, Milano, Italy, 1985);"Analysis, Results, and See "Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy," in Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al. (1982) Immunol. Rev. 62: 119-158.

[0275] Alternatively, as described, for example, in US4,676,980, an antibody, antibody fragment, or fusion protein can be conjugated with a second antibody or its antibody fragment (or second fusion protein) to form a heteroconjugate. Furthermore, a linker can be used between the label and the antibody described herein, as described, for example, in US4,831,175. Antibodies, antigen-binding fragments, and fusion proteins can be directly labeled with radioactive iodine, indium, yttrium, or other radioactive particles known in the art, as described, for example, in US5,595,721. Treatment may consist of a combination of treatments using conjugated and unconjugated antibodies, antigen-binding fragments, and / or fusion proteins, administered simultaneously or subsequently, as described, for example, in WO00 / 52031;WO00 / 52473.

[0276] The antibodies, antigen-binding fragments, and fusion proteins described herein can also be attached to solid supports. Furthermore, the antibodies, their functional antibody fragments, or fusion proteins of this disclosure can be chemically modified, for example, by covalent conjugation with polymers to increase their circulating half-life. Examples of polymers and methods for attaching them to peptides are shown in US4,766,106;US4,179,337;US4,495,285 and US4,609,546. In some embodiments, the polymers can be selected from polyoxyethylated polyols and polyethylene glycol (PEG). PEG is water-soluble at room temperature and has the general formula: R(O-CH2-CH2) nThe formula has OR, where R may be hydrogen or a protecting group, such as an alkyl group or an alkanol group. In certain embodiments, the protecting group may have between 1 and 8 carbon atoms. For example, the protecting group may be methyl. The sign n is a positive integer. In one embodiment, n is between 1 and 1,000. In another embodiment, n is between 2 and 500. In some embodiments, the average molecular weight of PEG is selected from between 1,000 and 40,000, between 2,000 and 20,000, and between 3,000 and 12,000. Furthermore, PEG may have at least one hydroxyl group, for example, PEG may have a terminal hydroxyl group. For example, the terminal hydroxyl group is a terminal hydroxyl group activated to react with the free amino group of the inhibitor. However, it will be understood that the type and amount of the reactive group may be varied so that the covalently conjugated PEG / antibody described herein is realized.

[0277] With respect to the antibodies and antigen-binding fragments described herein, water-soluble polyoxyethylated polyols may also be used. Examples of water-soluble polyoxyethylated polyols include polyoxyethylated sorbitol, polyoxyethylated glucose, and polyoxyethylated glycerol (POG). In one embodiment, POG is used. Without being bound by any theory, the glycerol backbone of polyoxyethylated glycerol is the same backbone that naturally exists in animals and humans, for example, as monoglycerides, diglycerides, and triglycerides; therefore, this branch is not necessarily found as an exogenous drug in the body. POG may have the same molecular weight range as PEG. Another drug delivery system that can be used to increase the circulating half-life is liposomes. Methods for preparing liposome delivery systems are known to those skilled in the art. Other drug delivery systems are known in the art and are described and mentioned, for example, in Poznansky et al. (1980) and Poznansky (1984).

[0278] The antibodies, antigen-binding fragments, and fusion proteins of this disclosure can be provided in purified form. Typically, the antibodies, antigen-binding fragments, or fusion proteins are present in a composition substantially free of other polypeptides, for example, less than 90% (by weight), usually less than 60%, and more typically less than 50% of the composition consisting of other polypeptides.

[0279] The antibodies, fusion proteins, or antigen-binding fragments of this disclosure may be immunogenic in non-human (or heterologous) hosts, for example, in mice. In particular, the antibodies, antigen-binding fragments, or fusion proteins may have idiotopes that are immunogenic in non-human hosts but not in human hosts. In particular, such molecules of this disclosure intended for human use include those that cannot be readily isolated from hosts, such as mice, goats, rabbits, rats, or non-primate mammals, and cannot generally be obtained by humanization or from xenomuse.

[0280] Production of antibodies, antigen-binding fragments, and fusion proteins The antibodies, antigen-binding fragments, and fusion proteins described herein can be prepared by any method known in the art. For example, a general set of methods for preparing monoclonal antibodies using hybridoma technology is well known (Kohler, G. and Milstein, C., 1975; Kozbar et al. 1983). In one embodiment, the EBV immortalization method described in WO2004 / 076677 is used.

[0281] In one embodiment, antibodies are produced using the method described in WO2004 / 076677. In this method, antibody-producing B cells are transformed with EBV and polyclonal B cell activator. To further enhance efficiency, additional stimulants for cell proliferation and differentiation may be added as needed during the transformation step. These stimulants may be cytokines, such as IL-2 and IL-15. In one embodiment, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, although this use is not essential. The immortalized B cells produced using these methods can then be cultured using methods known in the art, and antibodies can be isolated from them.

[0282] Another method for producing antibodies is described in WO2010 / 046775. In this method, plasma cells are cultured in a limited number or as single plasma cells in a microwell culture plate. Antibodies can be isolated from the plasma cell culture. Furthermore, RNA can be extracted from the plasma cell culture and PCR can be performed using methods known in the art. The VH and VL regions of the antibody can be amplified by RT-PCR (reverse transcriptase PCR), sequenced, cloned into an expression vector, and then the expression vector can be transfected into HEK293T cells or other host cells. Cloning of nucleic acids into an expression vector, transfection of host cells, culture of transfected host cells, and isolation of the produced antibodies can be carried out using any method known to those skilled in the art.

[0283] If desired, antibodies can be further purified by filtration, centrifugation, and various chromatographic methods, such as HPLC or affinity chromatography. Techniques for purifying antibodies, such as monoclonal antibodies, including techniques for producing pharmaceutical-grade antibodies, are well known in the art.

[0284] DNA sequences encoding the antibodies, antigen-binding fragments, or fusion proteins described herein can be prepared using standard molecular biology techniques. The desired DNA sequences can be synthesized entirely or partially using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as needed.

[0285] Any suitable host cell / vector system can be used to express the DNA sequences encoding the antibody or fusion protein molecules or fragments thereof of this disclosure. Bacterial, e.g., E. coli, and other microbial systems can be partially used to express antibody fragments, e.g., Fab and F(ab')2 fragments, particularly the Fv fragment and single-chain antibody fragments, e.g., single-chain Fv. Eukaryotic, e.g., mammalian host cell expression systems can be used to produce larger antibody molecules, including complete antibody molecules. Suitable mammalian host cells include, but are not limited to, exemplary host cells and cell systems disclosed herein.

[0286] The Disclosure also provides a process for producing an antibody, antigen-binding fragment, or fusion protein molecule according to the Disclosure, which includes culturing a host cell containing a vector encoding the nucleic acid of the Disclosure under conditions suitable for expressing a protein from DNA encoding the antibody molecule described herein, and isolating the antibody molecule.

[0287] The antibody molecule or antibody fragment may contain only heavy chain or light chain polypeptides, in which case it is necessary to transfect the host cell using only the heavy chain or light chain polypeptide coding sequence. To produce a product containing both heavy and light chains, the cell line can be transfected with two vectors: a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector can be used, which contains sequences encoding both light and heavy chain polypeptides.

[0288] Alternatively, the antibodies, antigen-binding fragments, and fusion proteins according to this disclosure can be produced by (i) expressing the nucleic acid sequence according to this disclosure in host cells, for example, by using the vector according to this disclosure, and (ii) isolating the desired expressed product. Furthermore, this method may include (iii) purifying the isolated antibody, antigen-binding fragment, or fusion protein. Transformed B cells and cultured plasma cells can be screened for those that produce antibodies, antigen-binding fragments, or fusion proteins of desired specificity or function.

[0289] Screening can be carried out by any immunoassay, such as ELISA, by staining of tissues or cells (including transfected cells), by a neutralization assay, or by one of many other methods known in the art for identifying desired specificity or function. The assay may be based on the simple recognition of one or more antigens, or it may be based on additionally desired function, for example, to select a neutralizing antibody rather than simply an antigen-binding antibody, to select an antibody that can alter the characteristics of the targeted cells, such as their signaling cascade, their morphology, their growth rate, their ability to influence other cells, their response to influence by other cells, other reagents, or changes in conditions, or their differentiation state.

[0290] Subsequently, individual transformed B cell clones can be generated from the positively transformed B cell culture. The cloning step for isolating individual clones from a mixture of positive cells can be carried out using limiting dilution, micromanipulation, single-cell deposition by cell sorting, or other methods known in the art.

[0291] Nucleic acids can be isolated from cultured plasma cells, cloned, and expressed in HEK293T cells or other known host cells using methods known in the art.

[0292] The immortalized B cell clones or transfected host cells described herein can be used in various ways, for example, as a source of monoclonal antibodies, as a source of nucleic acids (DNA or mRNA) encoding the monoclonal antibody of interest, for research purposes, etc.

[0293] HBV protein expression inhibitors and delivery systems This disclosure also provides HBV protein expression inhibitors for use in combination therapies and compositions for treating HBV, wherein the combination therapy includes a binding protein provided herein. In certain embodiments, the HBV gene expression inhibitor is an RNAi agent. As used herein, the terms “RNA interferant” or “RNAi agent” refer to agents containing RNA as defined herein and mediating targeted cleavage of RNA transcripts by the RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agents described herein result in inhibition of HBV gene expression.

[0294] In one embodiment, RNA interference agents include single-stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by a specific theory, long double-stranded RNA (dsRNA) introduced into plant and invertebrate cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp, et al., Genes Dev. 15: 485 (2001)). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into small interfering RNA (siRNA) of 19-23 base pairs with a characteristic 2-base 3' overhang (Bernstein, et al., Nature 409: 363 (2001)). Subsequently, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where the siRNA double helix is ​​unwound by one or more helicases, thereby enabling the complementary antisense strand to guide target recognition (Nykanen, et al., Cell 107: 309 (2001)). Upon binding to a suitable target mRNA, the target is cleaved by one or more endonucleases within the RISC, inducing silencing (Elbashir, et al, Genes Dev. 15: 188 (2001)). Thus, in one embodiment, the technique described herein relates to single-stranded RNA that facilitates the formation of the RISC complex, resulting in the silencing of a target gene.

[0295] The terms "silencing," "inhibiting expression," "downregulating expression," and "suppressing expression," insofar as they refer to the HBV gene, mean, in this specification, at least a partial reduction in HBV gene expression, where the HBV gene is transcribed and treated with an HBV gene expression inhibitor, and therefore the amount of HBV mRNA isolated from or detected in a first group of cells or cells in which HBV gene expression is inhibited is substantially the same as that of the first group of cells or cells, but is reduced compared to a second group of cells or cells (control cells) that have been or have not been treated in this way. The degree of inhibition can be measured, for example, by the difference obtained by subtracting the degree of mRNA expression in treated cells from the degree of mRNA expression in control cells. Alternatively, the degree of inhibition can be expressed in terms of parameters functionally linked to HBV gene expression, such as the amount of protein encoded by the HBV gene, or a specific phenotype, such as a reduction in the number of cells exhibiting an HBV infection phenotype, such as HBV infection, HBV protein expression (e.g., hepatitis B surface antigen, HBsAg, etc.), or changes in gene expression reflecting HBV gene expression in cells (e.g., Smc5 / 6 expression and localization). The degree of inhibition can also be measured using cells engineered to express a reporter gene that reflects HBV RNA expression. In principle, HBV gene silencing can be determined by any suitable assay in any cell expressing an HBV gene engineered to express the HBV gene, such as HBV-infected cells or cells.

[0296] The level of HBV RNA expressed by a cell or group of cells, or the level of circulating HBV RNA, can be determined using any method known in the art for assessing mRNA expression, for example, the rtPCR method provided in Example 2 of International Patent Application Publication WO2016 / 077321A1 and U.S. Patent Application US2017 / 0349900A1, the method of which is incorporated herein by reference. In some embodiments, the level of HBV gene expression in a sample (e.g., total HBV RNA, HBV transcript, e.g., HBV 3.5kb transcript) is determined by detecting the transcribed polynucleotide or a portion thereof, e.g., RNA of the HBV gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, the use of acidic phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen®), or PAXgene (PreAnalytix, Switzerland). Typical assay methods utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12: 7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating HBV mRNA can be detected using the methods described in International Patent Application Publication WO2012 / 177906A1 and U.S. Patent Application US2014 / 0275211A1, the methods of which are incorporated herein by reference.

[0297] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the HBV gene, including mRNA, which is the product of RNA processing of the primary transcript. The target portion of the sequence is at least long enough to serve as a substrate for RNAi-directed cleavage, either in or near that portion. For example, a target sequence is generally 9–36 nucleotides long, including all subranges in between, e.g., 15–30 nucleotides. As a non-limiting example, a target sequence could be 15–30 nucleotides, 15–26 nucleotides, 15–23 nucleotides, 15–22 nucleotides, 15–21 nucleotides, 15–20 nucleotides, 15–19 nucleotides, 15–18 nucleotides, 15–17 nucleotides, 18–30 nucleotides, 18–26 nucleotides, 18–23 nucleotides, 18–22 nucleotides, 18–21 nucleotides, 18–20 nucleotides, 19–30 nucleotides, 19–26 nucleotides Otide may consist of 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

[0298] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a chain of nucleotides described by a sequence, which is named using standard nucleotide nomenclature.

[0299] As used herein, unless otherwise specified, the term “complementary” means, as will be understood by those skilled in the art, that an oligonucleotide or polynucleotide containing a first nucleotide sequence can hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions to form a double-chain structure. Such conditions may be, for example, stringent conditions, which may include 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50°C or 70°C for 12–16 hours, followed by washing. Other conditions, such as physiologically relevant conditions that may occur in living organisms, can be applied. Those skilled in the art can determine the optimal set of conditions for testing the complementarity of the two sequences, depending on the final application of the hybridized nucleotides.

[0300] In RNAi agents, for example, in siRNAs described herein, complementary sequences include base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence, over the full length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary” to each other. However, where the first sequence is referred to herein as “substantially complementary” to the second sequence, the two sequences may be fully complementary, forming one or more, but generally five or fewer, four or fewer, three or fewer, or two or fewer mismatched base pairs when hybridized into a double helix of up to 30 base pairs, while still retaining their ability to hybridize under conditions most relevant to their final application, such as inhibition of gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs when hybridized, such overhangs shall not be considered mismatches for the purpose of determining complementarity. For example, if one oligonucleotide constituting an siRNA is 21 nucleotides long and the other oligonucleotide is 23 nucleotides long, and the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, then for the purposes described herein, it can still be referred to as “perfectly complementary.”

[0301] When used herein, “complementary” sequences may include, or be entirely formed from, non-Watson-Crick base pairs and / or non-natural and modified nucleotides, insofar as they satisfy the above requirements regarding the ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuation base pairings or Hoogsteen base pairings.

[0302] The terms “complementary,” “fully complementary,” and “substantially complementary” may be used herein in reference to base matching between the sense strand and antisense strand of an siRNA, or between the antisense strand and target sequence of an RNAi agent, as understood from the context in which they are used.

[0303] As used herein, a polynucleotide "substantially complementary" to at least a portion of messenger RNA (mRNA) means a polynucleotide substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding the HBV protein). For example, a polynucleotide is complementary to at least a portion of HBV mRNA if its sequence is substantially complementary to an uninterrupted portion of HBV mRNA.

[0304] a.siRNA In some embodiments, the RNAi agent includes siRNA. The term “siRNA,” as used herein, refers to an RNAi comprising an RNA molecule or complex of molecules having a hybridized double-stranded region containing two antiparallel, substantially complementary nucleic acid strands, which are called having “sense” and “antisense” orientations to the target RNA. The double-stranded region may be of any length that allows for the specific degradation of the desired target RNA via the RISC pathway, but typically ranges from 9 base pairs to 36 base pairs, for example, 15–30 base pairs. Considering double-stranded regions between 9 and 36 base pairs, the double-stranded region can be of any length within this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, or 36, and, but not limited to, 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19- The lengths may include 26 base pairs, 19–23 base pairs, 19–22 base pairs, 19–21 base pairs, 19–20 base pairs, 20–30 base pairs, 20–26 base pairs, 20–25 base pairs, 20–24 base pairs, 20–23 base pairs, 20–22 base pairs, 20–21 base pairs, 21–30 base pairs, 21–26 base pairs, 21–25 base pairs, 21–24 base pairs, 21–23 base pairs, and any sub-range between these, including 21–22 base pairs. siRNA produced in cells by processing with dicers and similar enzymes is generally in the 19–22 base pair length range. The terms “double-stranded RNA” or “dsRNA” are also used herein synonymously to refer to the above-mentioned siRNA.

[0305] One strand of the siRNA duplex region contains a sequence substantially complementary to a region of the target RNA. The two strands forming the duplex structure may originate from a single RNA molecule having at least one self-complementary region, or they may be formed from two or more separate RNA molecules. When the duplex region is formed from two strands of a single molecule, the molecule may have a duplex region separated by a single strand of nucleotides (referred to herein as a “hairpin loop”) between the 3’ end of one strand forming the duplex structure and the 5’ end of the other strand. The hairpin loop may contain at least one unpaired nucleotide; in some embodiments, the hairpin loop may contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. When the two substantially complementary strands of siRNA are composed of separate RNA molecules, those molecules do not need to be covalently linked, but may be. When two chains are connected by covalent bonds through means other than hairpin loops, the connecting structure is called a "linker."

[0306] The terms “antisense strand” or “guide strand” refer to a strand of RNAi agent, such as siRNA, that contains a region substantially complementary to the target sequence. As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to the sequence, such as the target sequence as defined herein. If the complementary region is not perfectly complementary to the target sequence, a mismatch may exist within the internal or terminal regions of the molecule.

[0307] Generally, the most acceptable mismatches are within the terminal region, for example, within 5, 4, 3, or 2 nucleotides from the 5' and / or 3' end.

[0308] When used herein, the terms “sense strand” or “passenger strand” refer to a strand of RNAi that contains a region substantially complementary to the antisense strand region as defined herein.

[0309] In another embodiment, the drug is a single-stranded antisense RNA molecule. The antisense RNA molecule may have 15 to 30 nucleotides complementary to the target. For example, the antisense RNA molecule may have a sequence of at least 15, 16, 17, 18, 19, 20, 21, or more consecutive nucleotides from one of the antisense sequences disclosed herein.

[0310] Those skilled in the art will understand that the terms “RNA molecule” or “ribonucleic acid molecule” encompass not only naturally expressed or found RNA molecules but also RNA analogs and derivatives, including one or more ribonucleotide / ribonucleoside analogs or derivatives described herein or known in the art. Strictly speaking, “ribonucleoside” contains a nucleoside base and a ribose sugar, and “ribonucleotide” is a ribonucleoside having one, two, or three phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” may be considered equivalent when used herein. RNA may be modified to a nucleic acid base structure or a ribose-phosphate backbone structure, for example, as described in more detail below. However, siRNA molecules containing ribonucleoside analogs or derivatives retain the ability to form double helixes. As a non-limiting example, an RNA molecule may also include, but is not limited to, at least one modified ribonucleoside, including 2'-O-methyl modified nucleosides, nucleosides containing a 5'-phosphorothioate group, terminal nucleosides linked to a cholesteryl derivative or bisdecylamide dodecanoate group, locked nucleosides, baseless nucleosides, 2'-deoxy-2'-fluoro modified nucleosides, 2'-amino-modified nucleosides, 2'-alkyl-modified nucleosides, morpholino nucleosides, nucleosides containing phosphoramidates or non-natural bases, or any combination thereof. Alternatively, an RNA molecule may include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more, modified ribonucleosides totaling the full length of the largest siRNA molecule. The modifications do not need to be the same for each of the multiple modified ribonucleosides in the RNA molecule. In some embodiments, the modified RNA intended for use in the methods and compositions described herein is a peptide nucleic acid (PNA) that has the ability to form the required double-stranded structure and enables or mediates the specific degradation of the target RNA via the RISC pathway.

[0311] In some embodiments, the modified ribonucleoside comprises a deoxyribonucleoside. For example, an RNAi agent may comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang or one or more deoxynucleosides within the double-stranded portion of an siRNA. However, as used herein, the term “RNAi agent” does not encompass a complete DNA molecule.

[0312] As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide protruding from the double-stranded structure of an RNAi agent, such as siRNA. For example, a nucleotide overhang exists if the 3' end of one strand of siRNA extends beyond the 5' end of the other strand, or vice versa. siRNA may contain an overhang of at least one nucleotide; or an overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain or consist of a nucleotide / nucleoside analog, including a deoxyribonucleotide / nucleoside. Overhangs may be located on the sense strand, on the antisense strand, or in any combination thereof. Furthermore, the nucleotides of an overhang may be located at the 5' end, 3' end, or both ends of either the antisense or sense strand of the siRNA.

[0313] In some embodiments, the antisense strand of the siRNA has 1 to 10 nucleotide overhangs at its 3' and / or 5' ends. In some embodiments, the sense strand of the siRNA has 1 to 10 nucleotide overhangs at its 3' and / or 5' ends. In some other embodiments, one or more nucleotides within the overhangs are replaced with nucleoside thiophosphates.

[0314] In some embodiments, at least one end of the siRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. siRNAs with at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts.

[0315] When the term “blunt” or “blunt-ended” is used herein in relation to siRNA, it means that the siRNA does not contain any nucleotides or nucleotide analogs that are not paired to a given end; in other words, it does not have nucleotide overhangs. One or both ends of siRNA may be blunt. If both ends of siRNA are blunt, then siRNA can be said to be “blunt-ended.” “Blunt-ended” siRNA is siRNA in which both ends are blunt, i.e., has no nucleotide overhangs at either end of the molecule. Such molecules are often double-stranded along their entire length.

[0316] In certain embodiments, the combination therapy described herein comprises one or more RNAi agents that inhibit HBV gene expression. In some embodiments, the RNAi agent comprises a small interfering ribonucleic acid (siRNA) molecule for inhibiting HBV gene expression in a mammal, e.g., an HBV-infected human, wherein the siRNA comprises an antisense strand having a complementary region complementary to at least a portion of the mRNA formed in HBV gene expression, the complementary region being 30 nucleotides or less in length, generally 19 to 24 nucleotides, and the siRNA, upon contact with cells expressing the HBV gene, inhibits HBV gene expression by at least 10% when assayed, e.g., by PCR or a branched DNA (bDNA) based method, or by a protein-based method, e.g., by Western blotting. The expression of the HBV gene in cell cultures, or the expression of intracellular genes (e.g., Smc5 / 6) as a surrogate for HBV gene expression in, for example, COS cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells, or subject-derived biological samples, can be assayed, for example, by measuring HBV mRNA levels by bDNA or TaqMan assays, or by measuring protein levels using, for example, immunofluorescence analysis, or by, for example, Western blotting or flow cytometry techniques.

[0317] siRNA contains two RNA strands that are complementary and hybridize to form a double-stranded structure under the conditions in which siRNA is used. One strand of siRNA (the antisense strand) contains a complementary region that is substantially complementary to, and generally fully complementary to, the target sequence. The target sequence may originate from the mRNA sequence formed during HBV gene expression. The other strand (the sense strand) contains a region complementary to the antisense strand, and therefore, these two strands hybridize when combined under appropriate conditions to form a double-stranded structure. Generally, the length of the double-stranded structure is between 15 and 30 base pairs including both ends, more commonly between 18 and 25 base pairs including both ends, even more commonly between 19 and 24 base pairs including both ends, and most commonly between 19 and 21 base pairs including both ends. Similarly, complementary regions to the target sequence are between 15 and 30 nucleotides, including both ends; more commonly between 18 and 25 nucleotides, including both ends; even more commonly between 19 and 24 nucleotides, including both ends; and most commonly between 19 and 21 nucleotides, including both ends. In some embodiments, the siRNA is between 15 and 20 nucleotides, including both ends, and in other embodiments, the siRNA is between 25 and 30 nucleotides, including both ends. As will be understood by those skilled in the art, the targeted region of the RNA targeted for cleavage is often part of a larger RNA molecule, often a part of an mRNA molecule. Where appropriate, the “part” of the mRNA target is a contiguous sequence of mRNA targets long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). siRNAs with double helix as short as 9 base pairs can also, under certain circumstances, mediate RNAi-directed cleavage. The target is often at least 15 nucleotides long. In certain embodiments, the target is 15 to 30 nucleotides long.

[0318] Those skilled in the art will also understand that the double-stranded region, e.g., 9–36 base pairs, e.g., 15–30 base pairs, is the primary functional portion of siRNA. Therefore, in some embodiments, RNA molecules or complexes of RNA molecules having a double-stranded region larger than 30 base pairs are also siRNA, insofar as they are processed into a functional double-stranded region of, e.g., 15–30 base pairs, targeting the desired RNA for cleavage. Thus, in some embodiments, those skilled in the art will understand that miRNA is siRNA. In some other embodiments, siRNA is not a naturally occurring miRNA. In some embodiments, RNAi agents useful for targeting HBV gene expression are not produced by cleavage from larger double-stranded RNA in target cells.

[0319] The siRNAs described herein can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0320] In some embodiments, the RNAi agent includes an siRNA that targets HBV mRNA and inhibits its expression. In some embodiments, the RNAi agent includes an siRNA that targets mRNA encoded by the HBV genome according to NCBI Reference Sequence NC_003977.2 (GenBank accession number GI:21326584) (SEQ ID NO: 116) and inhibits its expression. Transcription of the HBV genome results in polycistronic, duplicated RNA, and therefore, in some embodiments, siRNA in combination therapy targeting a single HBV gene can result in significant inhibition of the expression of the majority or all HBV transcripts. In some embodiments, the mRNA target of the siRNA may be mRNA encoded by the P gene, nucleotides 2309-3182 and 1-1625 of NC_003977.1; the S gene (encoding L, M, and S proteins), nucleotides 2850-3182 and 1-837 of NC_003977; the X protein, nucleotides 1376-1840 of NC_003977; and / or the C gene, nucleotides 1816-2454 of NC_003977.

[0321] In some embodiments, the siRNA targets mRNA encoded by the HBV X gene and inhibits its expression. In some embodiments, the RNAi agent or siRNA targets mRNA encoded by a portion of the HBV genome, including the sequence GTGTGCACTTCGCTTCAC (Sequence ID 117), which corresponds to nucleotides 1579-1597 of NC_003977.2 (GenBank accession number GI:21326584) (Sequence ID 116).

[0322] In yet another embodiment, the siRNA has a sense strand containing 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 118) and an antisense strand containing 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 119).

[0323] In a particular embodiment, the HBV gene expression inhibitor comprises an siRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises sequence number 118, or a sequence that differs from sequence number 118 by 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, or 1 nucleotide or less, and the antisense strand comprises sequence number 119, or a sequence that differs from sequence number 119 by 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, or 1 nucleotide or less.

[0324] In one embodiment, the siRNA comprises at least two nucleotide sequences, a sense sequence and an antisense sequence, wherein the sense sequence comprises SEQ ID NO: 118 and the corresponding antisense sequence comprises SEQ ID NO: 119. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of these sequences is substantially complementary to the mRNA sequence produced in HBV gene expression. Thus, in this embodiment, the siRNA comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described elsewhere in this specification and as is known in the art, the complementary sequences of the siRNA may also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being located on separate oligonucleotides.

[0325] In yet another embodiment, the siRNA has a sense strand containing 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 120) and an antisense strand containing 5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 121).

[0326] In a particular embodiment, the HBV gene expression inhibitor comprises an siRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises sequence number 120, or a sequence that differs from sequence number 120 by 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, or 1 nucleotide or less, and the antisense strand comprises sequence number 121, or a sequence that differs from sequence number 121 by 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, or 1 nucleotide or less.

[0327] In one embodiment, the siRNA comprises at least two nucleotide sequences, a sense sequence and an antisense sequence, wherein the sense sequence comprises SEQ ID NO: 120 and the corresponding antisense sequence comprises SEQ ID NO: 121. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of these sequences is substantially complementary to the mRNA sequence produced in HBV gene expression. Thus, in this embodiment, the siRNA comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described elsewhere in this specification and as is known in the art, the complementary sequences of the siRNA may also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being located on separate oligonucleotides.

[0328] Those skilled in the art are well aware that siRNAs having a double-stranded structure of 21 base pairs, between 20 and 23 base pairs, are recognized as particularly effective in inducing RNA interference (Elbashir, et al., EMBO 20: 6877-88 (2001)). However, others have found that shorter or longer RNA double-stranded structures can be equally effective. In the embodiments described above, the siRNAs described herein may contain at least one strand with a minimum length of 21 nucleotides. In some embodiments, shorter double-stranded sequences having a sequence obtained by subtracting just a few nucleotides from one or both ends of one of SEQ ID NOs: 118, 119, 120, or 121 are equally effective compared to the siRNAs described above. Accordingly, siRNAs having at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotide subsequences from one or both of SEQ ID NOs: 118 and SEQ ID NOs: 119, and whose ability to inhibit HBV gene expression differs from siRNA containing the complete sequence by only 5% or less inhibition, 10% or less inhibition, 15% or less inhibition, 20% or less inhibition, 25% or less inhibition, or 30% or less inhibition, are contemplated in accordance with the art described herein. siRNAs having at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotide subsequences from one or both of SEQ ID NOs: 120 and SEQ ID NOs: 121, and whose ability to inhibit HBV gene expression differs from siRNA containing the complete sequence by only 5% or less inhibition, 10% or less inhibition, 15% or less inhibition, 20% or less inhibition, 25% or less inhibition, or 30% or less inhibition, are also within the scope of this disclosure.

[0329] Furthermore, the siRNAs provided herein identify sites within the HBV gene transcript that are susceptible to RISC-mediated cleavage. Thus, the techniques described herein further feature RNAi agents that target within one of such sequences. As used herein, an RNAi agent can be said to target within a particular site of an RNA transcript if RNAi facilitates cleavage of the transcript at any site within that particular site. In some embodiments, the RNAi agent comprises at least 15 consecutive nucleotides from one or both of the sequences of SEQ ID NO: 118 and SEQ ID NO: 119, coupled with an additional nucleotide sequence obtained from a region adjacent to a selected sequence within the HBV gene. In some embodiments, the RNAi agent comprises at least 15 consecutive nucleotides from one or both of the sequences of SEQ ID NO: 120 and SEQ ID NO: 121, coupled with an additional nucleotide sequence obtained from a region adjacent to a selected sequence within the HBV gene.

[0330] Target sequences are generally 15–30 nucleotides long, but the suitability of specific sequences within this range for directing the cleavage of any given target RNA varies widely. While various software packages and guidelines provided herein offer guidance for identifying the optimal target sequence for any given gene target, empirical methods can also be employed, placing a "window" or "mask" of a given size (e.g., 21 nucleotides) literally or figuratively (including in silico) around the target RNA sequence to identify sequences within that size range that can function as a target sequence. The next potential target sequence can be identified by gradually shifting the sequence "window" one nucleotide upstream or downstream of the initial target sequence until a complete set of possible sequences for any chosen given target size is identified. This process can be coupled with systematic synthesis and testing of the identified sequences (using assays described herein or known in the art) to identify the optimally functioning sequence, thereby identifying the RNA sequence that best mediates the inhibition of target gene expression when targeted by an RNAi agent. The aim is to further optimize inhibition efficiency by gradually "advancing the window" one nucleotide at a time upstream or downstream of a given sequence to identify sequences with equal or better inhibitory characteristics.

[0331] Furthermore, it is intended that further optimization can be achieved by systematically adding or removing nucleotides from any identified sequence, e.g., SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, or SEQ ID NO: 121, to generate longer or shorter sequences, and then testing those and the sequences generated by advancing from that point upstream or downstream of the target RNA to longer or shorter size windows. Again, this method for generating new candidate targets can be coupled with testing the efficacy of RNAi agents based on target sequences in inhibitory assays known in the art or described herein to lead to further improvements in the efficiency of inhibition. Furthermore, such optimized sequences can be further optimized as expression inhibitors by modifying them, for example, by introducing modified nucleotides described herein or known in the art, adding or altering overhangs, or by other modifications known in the art and / or considered herein (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting specific sites or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.).

[0332] The RNAi agents described herein may contain one or more mismatches with the target sequence. In some embodiments, the RNAi agents described herein contain three or fewer mismatches. In some embodiments, if the antisense strand of the RNAi agent contains a mismatch with the target sequence, the mismatch region is not located in the center of the complementary region. In certain embodiments, if the antisense strand of the RNAi agent contains a mismatch with the target sequence, the mismatch is limited to within the last five nucleotides from either the 5' or 3' end of the complementary region. For example, with respect to the RNA strand complementary to the HBV gene region of a 23-nucleotide RNAi agent, the RNA strand may not contain any mismatches within the central 13 nucleotides. Using the methods described herein or methods known in the art, it is possible to determine whether an RNAi agent containing a mismatch with the target sequence is effective in inhibiting HBV gene expression. Considering the effectiveness of RNAi agents with mismatches in inhibiting HBV gene expression is important, especially when it is known that certain complementary regions within the HBV gene have polymorphic sequence variations.

[0333] b. Chemically modified RNAi agents In some embodiments, the RNA of the RNAi agent, such as siRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids that characterize the techniques described herein can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL, et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, whose methods are incorporated herein by reference.

[0334] Modifications include, for example, (a) terminal modifications, e.g., 5' terminal modifications (phosphorylation, conjugation, reverse linking, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse linking, etc.), (b) base modifications, e.g., stabilizing bases, destabilizing bases, or bases that base-pair with the partner's extended repertoire, base removal (baseless nucleotides), or substitution with conjugated bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) skeletal modifications, including modifications or substitutions of phosphate diester linkages. Specific examples of RNA compounds useful in the embodiments described herein, but not limited to, include RNAs containing a modified skeleton or lacking natural internucleoside linkages. RNAs having a modified skeleton include, in particular, those lacking a phosphorus atom in the skeleton. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs lacking a phosphorus atom in the internucleoside skeleton can also be considered oligonucleosides. In certain embodiments, the modified RNA has a phosphorus atom in the internucleoside skeleton.

[0335] Not all positions within a given compound need to be uniformly modified; in fact, more than one of the modifications described above may be incorporated into a single compound, or even a single nucleoside within an RNAi agent. The techniques described herein also include RNAi agent compounds that are chimeric compounds. A “chimeric” RNAi agent compound or “chimera” is, for the purposes of this disclosure, an RNAi agent compound such as an siRNA that contains two or more chemically distinct regions, each composed of at least one monomeric unit, i.e., nucleotides in the case of an siRNA compound. These RNAi agents typically contain at least one region, the RNA, which has been modified to confer to the RNAi agent increased resistance to nuclease degradation, increased intracellular uptake, and / or increased binding affinity to a target nucleic acid. The additional region of the RNAi agent may function as a substrate for an enzyme capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is an intracellular endonuclease that cleaves the RNA strand of an RNA:DNA double helix. Therefore, activation of RNase H leads to cleavage of the RNA target, thereby significantly enhancing the efficiency of gene expression inhibition by RNAi agents. Consequently, when using chimeric siRNA, comparable results can often be obtained with shorter RNAi agents compared to phosphorothioate deoxysiRNAs that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0336] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, and boranophosphates with the usual 3'-5' linkage, their 2'-5' linked analogues, and those with reverse polarity where the linkage of adjacent pairs of nucleoside units has changed from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0337] Representative U.S. patents teaching the preparation of the phosphorus-containing linkage described above include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286, No. 717; No. 5,321,131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5, No. 476,925; No. 5,519,126; No. 5,536,821; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. 5,571,799; Same No. 5,587,361; Same No. 5,625,050; Same No. 6,028,188; Same No. 6,124,445; Same No. 6,160,109; Same No. 6,169,170; Same No. 6,172,2 No. 09; No. 6, 239,265; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,444,423; No. 6,531,590; No. 6,5 Examples include U.S. Patent Nos. 34,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent No. RE 39464; each of these is incorporated herein by reference.

[0338] Modified RNA skeletons that do not contain a phosphorus atom have skeletons formed by short alkyl or cycloalkyl nucleoside linkages, mixtures of heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short heteroatoms or heterocyclic nucleoside linkages. These include morpholino linkages (partially formed by the sugar moiety of the nucleoside); siloxane skeletons; sulfides, sulfoxides, and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; alkene-containing skeletons; sulfamic acid skeletons; methyleneimino and methylenehydrazino skeletons; sulfonic acid and sulfonamide skeletons; those having an amide skeleton; and others having mixtures of N, O, S, and CH2 components.

[0339] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489, Nos. 677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439 are examples; each of these is incorporated herein by reference with respect to teachings relating to such preparation methods.

[0340] In other embodiments, suitable RNA mimeographs are intended for use in RNAi agents in which both sugar and nucleoside linkages, i.e., the nucleotide unit backbone, are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimeograph that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bind directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents in which the preparation of PNA compounds is taught include, but are not limited to, U.S. Patents 5,539,082; 5,714,331; and 5,719,262; each of which is incorporated herein by reference with respect to teachings relating to such preparation methods. Further teachings regarding PNA compounds can be found, for example, in Nielsen, et al. (Science, 254: 1497-1500 (1991)).

[0341] Some embodiments of the techniques described herein include RNA having a phosphorothioate backbone and oligonucleosides having a heteroatom backbone, in particular -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as methylene(methylimino) or MMI backbone], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [where the native phosphate diester backbone is represented as -OPO-CH2-], and the amide backbone of U.S. Patent No. 5,602,240. In some embodiments, the RNA characteristic of the present invention has a morpholino backbone structure of U.S. Patent No. 5,034,506.

[0342] Modified RNA may also contain one or more substituted sugar moieties. A characteristic RNAi agent of the present invention, such as siRNA, may contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl; where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 They can be alkenyls and alkinyls. An exemplary suitable modification is O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)2 is an example, where n and m are from 1 to about 10. In other embodiments, the siRNA includes one of the following at the 2' position: C1~C 10Lower alkyl groups, substituted lower alkyl groups, alkaryl groups, aralkyl groups, O-alkaryl or O-aralkyl groups, SH, SCH3, OCN, CI, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of RNAi agents, or groups for improving the pharmacodynamic properties of RNAi agents, and other substituents having similar properties. In some embodiments, modifications include 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin, et al., Helv. Chim. Acta 78: 486-504 (1995)), i.e., alkoxy-alkoxy groups. Another exemplary modification involves 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (2 in the art). * -O-dimethylaminoethoxyethyl or 2 * -Also known as DMAEOE), that is, 2 * It is -O-CH2-O-CH2-N(CH2)2.

[0343] Other exemplary modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can be made to other parts of the RNAi agent's RNA, particularly the 3' portion of the sugar in the 3' terminal nucleotide or the 2'-5' ligated siRNA and the 5' portion of the 5' terminal nucleotide. The RNAi agent may also have sugar mimes, such as a cyclobutyl moiety in place of pentofuranosyl sugar.

[0344] Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; Nos. 5,118,800; Nos. 5,319,080; Nos. 5,359,044; Nos. 5,393,878; Nos. 5,446,137; Nos. 5,466,786; Nos. 5,514,785; Nos. 5,519,134; Nos. 5,567,811; and Nos. 5,576,427; Nos. 5,591,722; Nos. 5,597,909; Nos. 5,610,300; Nos. 5,627,053; Nos. 5,639,873; Nos. 5,646,265; Nos. 5,658,873; Nos. 5,670,633; and Nos. 5,700,920 are examples; each of these is incorporated herein by reference with respect to teachings relating to such preparation methods.

[0345] RNAi agents may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, such as 5-methylcytosine (5-me-C) and 5-hydroxymethyl Cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, Examples include 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines; 5-halo, in particular 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and 3-deazaadenine.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine (Herdewijn, P. ed. Wiley-VCH, (2008)); those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering (pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons (1990)); those disclosed by Englisch et al. (Angewandte Chemie, International Edition, 30, 613 (1991)); and those disclosed by Sanghvi, Y S. (Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press (1993)). Certain of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds that characterize the techniques described herein. These include 5-substituted pyrimidines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines. The 5-methylcytosine substitution has been shown to increase nucleic acid double-chain stability by 0.6–1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, pp. 276–278 (1993)), and is an exemplary base substitution, even more remarkable when combined with 2'-O-methoxyethyl sugar modifications.

[0346] Representative U.S. patents teaching the preparation of certain modified nucleic acid bases and other modified nucleic acid bases include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; and 5,594,121. Nos. 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088 are examples; each of which is incorporated herein by reference with respect to teachings relating to such preparation methods.

[0347] The RNA in RNAi agents can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide that has a modified ribose moiety in which the ribose moiety has an additional bridge connecting the 2' and 4' carbon atoms. This structure effectively "locks" the 3' terminal ribose into a structural conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J., et al., Nucleic Acids Research 33 (1): 439-47 (2005); Mook, OR, et al., Mol Cane Ther 6 (3): 833-43 (2007); Grunweller, A., et al, Nucleic Acids Research 31 (12): 3185-93 (2003)).

[0348] Representative U.S. patents teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845; each of which is incorporated herein by reference with respect to teachings relating to such preparation methods.

[0349] In certain embodiments, the combination therapy includes siRNA modified to contain one or more adenosine-glycol nucleic acids ("GNAs"). A description of adenosine-GNAs can be found, for example, in Zhang, et al. (JACS 127 (12): 4174-75 (2005)).

[0350] In some embodiments, this disclosure provides methods and related compositions in which the RNAi is an siRNA comprising an oligonucleotide sequence having one or more modified nucleotides. Abbreviations for nucleotide monomers in modified nucleic acid sequences used herein are provided in Table 5.

[0351] [Table 5-1] [Table 5-2] [Table 5-3]

[0352] In some embodiments, the HBV gene expression inhibitor comprises an siRNA, where the siRNA has a sense strand comprising 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 122) and an antisense strand comprising 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 123).

[0353] In yet another embodiment, the siRNA has a sense strand containing 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 124) and an antisense strand containing 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 125).

[0354] In a particular embodiment, the HBV gene expression inhibitor comprises an siRNA including a sense strand and an antisense strand, wherein the sense strand includes sequences that differ from SEQ ID NO: 122 or SEQ ID NO: 124 by 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, or 1 nucleotide or less, respectively from SEQ ID NO: 122 or SEQ ID NO: 124.

[0355] In a particular embodiment, the HBV gene expression inhibitor comprises an siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises sequences that differ from SEQ ID NO: 123 or SEQ ID NO: 125, or from SEQ ID NO: 123 or SEQ ID NO: 125 by 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, or 1 nucleotide or less, respectively.

[0356] In some embodiments, the HBV gene expression inhibitor comprises siRNA, where the siRNA has a sense strand comprising 5'-gsgsuggaCfuUfCfUfcucaAfUfuuuaL96-3' (SEQ ID NO: 126) and an antisense strand comprising 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 127).

[0357] In a particular embodiment, the HBV gene expression inhibitor comprises an siRNA including a sense strand and an antisense strand, wherein the sense strand includes sequence number 126, or a sequence that differs from sequence number 126 by 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, or 1 nucleotide or less.

[0358] c. Ligand-conjugated RNAi agents In some embodiments, the RNAi agent includes modifications that involve chemically linking one or more ligands, moieties, or conjugates to RNA that enhance the activity, cell distribution, or intracellular uptake of the RNAi agent. These are not limited to these, but also include lipid portions, such as cholesterol (Letsinger, et al., Proc. Natl. Acid. Sci. USA 86: 6553-56 (1989)), cholic acid (Manoharan, et al., Biorg. Med. Chem. Let. 4: 1053-60 (1994)), thioethers, such as beryl-S-tritylthiol (Manoharan, et al., Ann. NY Acad. Sci. 660: 306-9 (1992); Manoharan, et al., Biorg. Med. Chem. Let. 3: 2765-70 (1993)), and thiocholesterol (Oberhauser, et al., Nucl. Acids Res. 20: 533-38) (1992)), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras, et al., EMBO J 10: 1111-18 (1991); Kabanov, et al., FEBS Lett. 259: 327-30 (1990); Svinarchuk, et al., Biochimie 75: 49-54 (1993)), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan, et al., Tetrahedron Lett. 36: 3651-54 (1995); Shea, et al., Nucl. Acids Res. 18: 3777-83 (1990)), polyamine or polyethylene glycol chain (Manoharan, et al., Nucleosides & Nucleotides 14: 969-73 (1995)), or adamantane acetate (Manoharan, et al., Tetrahedron Lett.Examples include the palmityl moiety (36: 3651-54 (1995)), the palmityl moiety (Mishra, et al., Biochim. Biophys. Acta 1264: 229-37 (1995)), or the octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke, et al., J. Pharmacol. Exp. Ther. 277: 923-37 (1996)).

[0359] In some embodiments, the ligand alters the distribution, targeting, or lifespan of the RNAi agent into which it is incorporated. In some embodiments, the ligand results in enhanced affinity for selected targets, e.g., molecules, cells, or cell types; compartments, e.g., intracellular or intraorganic compartments; tissues, organs, or regions of the body, compared to species in which such ligand is absent. In such embodiments, the ligand does not participate in the double-stranding of the double-stranded nucleic acid.

[0360] Ligands may include naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethyl acrylate), N-isopropylacrylamide polymer, or polyphosphatidine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptide-mimicking polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helix peptides.

[0361] Ligands may include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids or proteins, or antibodies that bind to specific cell types, such as hepatocytes. Targeting groups may include thyroid-stimulating hormone, melanotropin, lectins, glycoproteins, surfactant protein A, mucin-type glycans, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartate, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimics. Other examples of ligands include dyes, inserts (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl) Examples include (yl)cholenic acid, dimethoxytrityl, or phenoxazine), peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraaza macrocyclic compounds), dinitrophenyl, HRP, and AP.

[0362] Ligands can be proteins, such as glycoproteins or peptides, such as molecules having a specific affinity for a co-ligand, or antibodies, such as antibodies that bind to a specific cell type, such as hepatocytes. Ligands may also include hormones and hormone receptors. Ligands may also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, and polyvalent fucose. Ligands may also be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-KB.

[0363] A ligand can be a drug that can increase the uptake of a substance, such as an RNAi agent, into a cell, for example, by disrupting the cytoskeleton of the cell, for example, by disrupting the cellular microtubules, microfilaments, and / or intermediate filaments. The drug may be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

[0364] In another embodiment, the ligand is a portion taken up by target cells, such as hepatocytes, e.g., a vitamin. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by target cells, e.g., hepatocytes. HSA and low-density lipoprotein (LDL) are also included.

[0365] In some embodiments, the ligands attached to the RNAi agents described herein act as pharmacokinetic (PK) modulators. As used herein, “PK modulator” refers to a pharmacokinetic modulator. Examples of PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, and vitamins. Examples of PK modulators, but not limited to, include cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing several phosphorothioate linkages have also been shown to bind to serum proteins, and therefore, short oligonucleotides, such as oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in their backbone, can also be applied as ligands (e.g., as PK-modulating ligands) in the techniques described herein. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0366] (i) Lipid conjugate. In some embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Lipid or lipid-based ligands can be used to (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, such as HSA. Such lipid or lipid-based molecules can bind to serum proteins, such as human serum albumin (HSA). Ligands that bind to HSA make it possible to distribute the conjugate to target tissues in the body, such as target tissues other than the kidney. For example, the target tissue could be the liver, including the parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, neproxine or aspirin can be used.

[0367] Lipid-based ligands can be used to inhibit or control the binding of conjugates to target tissues. For example, lipids or lipid-based ligands that bind strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be removed from the body. To target the conjugate to the kidneys, lipids or lipid-based ligands that do not bind very strongly to HSAs can be used.

[0368] In some embodiments, lipid-based ligands bind to HSA. Lipid-based ligands can bind to HSA with sufficient affinity, and therefore the conjugate is distributed to tissues other than the kidney. In some specific embodiments, HSA-ligand binding is reversible.

[0369] In some other embodiments, lipid-based ligands bind weakly to HSA or not at all, and therefore the conjugate is distributed to the kidney. Other parts that target kidney cells can also be used instead of, or in addition to, lipid-based ligands.

[0370] (ii) Cell-permeable peptides and agents. In another embodiment, the ligand is a cell-permeable agent, e.g., a helix cell-permeable agent. In some embodiments, the agent is amphiphilic. Exemplary agents are peptides such as tat or antennopedia. If the agent is a peptide, it may be modified, including the use of peptidyl mimes, inverted isomers, non-peptide or pseudopeptide linkages, and D-amino acids. In some embodiments, the helix agent is an alpha-helix agent. In some specific embodiments, the helix agent has an oleophilic phase and an oleophobic phase.

[0371] "Cell-permeable peptides" are capable of permeating cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-permeable peptides may be, for example, alpha-helix linear peptides (e.g., LL-37 or Ceropin PI), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bactenesin), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidine).

[0372] Ligands can be peptides or peptide mimes. Peptide mimes (also referred to herein as oligopeptide mimes) are molecules capable of folding into a defined three-dimensional structure similar to that of natural peptides. By attaching peptides and peptide mimes to RNAi agents, the pharmacokinetic distribution of the RNAi can be influenced, for example, by enhancing cellular recognition and absorption. The peptide or peptide mime portion may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0373] Peptides or peptide mimeographs may be, for example, cell-permeable peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety may be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane-transfer sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 128). RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 129)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide capable of carrying large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences derived from HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 130)) and Drosophila Antennapedia protein (RQIKIWFQNRRMKWK (SEQ ID NO: 131)) have been found to be capable of functioning as delivery peptides. Peptides or peptide mimes can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam, et al., Nature 354: 82-84 (1991)).

[0374] Cell-permeable peptides may also include nuclear localization signals (NLS). For example, a cell-permeable peptide may be an amphiphilic peptide consisting of two parts, such as an MPG derived from the fusion peptide domain of the NLS of HIV-1 gp41 and SV40 large T antigen (Simeoni, et al., Nucl. Acids Res. 31: 2717-24 (2003)).

[0375] (iii) Carbohydrate conjugates. In some embodiments, the RNAi agent oligonucleotides described herein further comprise carbohydrate conjugates. Carbohydrate conjugates may be advantageous for the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use. As used herein, “carbohydrate” means either the carbohydrate itself, which has at least six carbon atoms (and may be linear, branched, or cyclic), and which consists of one or more monosaccharide units to which an oxygen, nitrogen, or sulfur atom is bonded to each carbon atom; or a compound which has as part a carbohydrate portion, each of which has at least six carbon atoms (and may be linear, branched, or cyclic), and which consists of one or more monosaccharide units to which an oxygen, nitrogen, or sulfur atom is bonded to each carbon atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4 to 9 monosaccharide units), and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include C5 and higher (C5-C8 in some embodiments) sugars, while disaccharides and trisaccharides include sugars having two or three monosaccharide units (C5-C8 in some embodiments).

[0376] In some embodiments, the carbohydrate conjugate is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka]

[0377] Other representative carbohydrate conjugates for use in the embodiments described herein include, but are not limited to, [ka] (Formula XXII), (wherein if one of X or Y is an oligonucleotide, the other is hydrogen) These are some examples.

[0378] In some embodiments, the carbohydrate conjugate further comprises another ligand, such as, but is not limited to, a PK modulator, an endosomal lytic ligand, or a cell-permeable peptide.

[0379] (iv) Linker. In some embodiments, the conjugate described herein can be attached to an RNAi agent oligonucleotide using a variety of linkers that may be cleavable or incleavable.

[0380] The term "linker" or "linking group" refers to an organic part that connects two parts of a compound. Linkers are typically directly bonded or atom, e.g., oxygen or sulfur, unit, e.g., NR8, C(O), C(O)NH, SO, SO2, SO2NH, or, for example, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkynyl Alkenyl heteroaryl alkenyl, alkenyl heteroaryl alkinyl, alkinyl heteroaryl alkyl, alkinyl heteroaryl alkenyl, alkinyl heteroaryl alkinyl, alkyl heterocyclyl alkyl, alkyl heterocyclyl alkenyl, alkyl heterocyclyl alkinyl, alkenyl heterocyclyl alkyl, alkenyl heterocyclyl alkenyl, alkenyl heterocyclyl alkinyl, alkinyl heterocyclyl alkyl, alkinyl heterocyclyl alkenyl, alkinyl hetero A chain of atoms such as cyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, and alkynylheteroaryl, in which one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic groups, where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic, is included.In certain embodiments, the linker is composed of 1 to 24 atoms, 4 to 24 atoms, 6 to 18 atoms, 8 to 18 atoms, or 8 to 16 atoms.

[0381] The cleavable linking group is sufficiently stable outside the cell, but upon entering the target cell, it is cleaved, releasing the two parts that were held by the linker. In certain embodiments, the cleavable linking group is cleaved at least 10 times, or at least 100 times, faster inside the target cell or under a first reference condition (which may be selected to mimic or represent intracellular conditions, for example) than in the subject's blood or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum, for example).

[0382] Cleavable linking groups are susceptible to the influence of cleavage factors, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage factors are found inside cells, more widely distributed, or at higher levels or activity than in serum or blood. Examples of such degradable agents include redox agents that are selected for or not substrate-specific to specific substrates, and these include oxidizing or reductases or reducing agents, such as intracellular mercaptans; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of 5 or less; and enzymes that hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which may be substrate-specific), and phosphatases. Cleavable linking groups, such as disulfide bonds, may be susceptible to pH influence. The pH of human serum is 7.4, while the mean intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, while lysosomes have an even more acidic pH, approximately 5.0. Some linkers have cleavable linking groups that are cleaved at specific pH levels, thereby releasing cationic lipids from their intracellular ligands or into desired compartments within the cell.

[0383] A linker may contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, a ligand targeting the liver can be linked to a cationic lipid via a linker containing an ester group. Hepatocytes are rich in esterases, and therefore, the linker is cleaved more efficiently in hepatocytes than in esterase-inriched cell types. Other esterase-rich cell types include lung, renal cortex, and testicular cells.

[0384] When targeting peptidase-rich cell types, such as hepatocytes and synovial cells, linkers containing peptide bonds can be used.

[0385] Generally, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradable agent (or condition) to cleave the candidate linker. It may also be desirable to test the candidate cleavable linker's resistance to cleavage in blood or in contact with other non-target tissues. Thus, the relative ease of cleavage between a first and second condition can be determined, where the first condition is selected to demonstrate cleavage in target cells, and the second condition is selected to demonstrate cleavage in other tissues or bodily fluids, such as blood or serum. Evaluations can be performed in cell-free systems, in cells, in cell cultures, in organ or tissue cultures, or in whole animals. It may be useful to perform an initial evaluation in cell-free or culture conditions and confirm it with further evaluation in whole animals. In certain embodiments, a useful candidate compound is cleaved in cells (or under in vitro conditions selected to mimic intracellular conditions) at a rate at least 2 times, at least 4 times, at least 10 times, or at least 100 times faster than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0386] One class of cleavable linking groups is redox-cleavable linking groups, which are cleaved upon reduction or oxidation. An example of a redox-cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable “redox-cleavable linking group” or, for example, suitable for use with a particular RNAi moiety and a particular targeting agent, one can rely on the methods described herein. For example, a candidate can be evaluated by incubating it with dithiothreitol (DTT) or another reducing agent using a reagent known in the art that mimics the cleavage rate observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In some embodiments, at most 10% of candidate compounds are cleaved in blood. In certain embodiments, a useful candidate compound is degraded at least twice, at least four times, at least ten times, or at least 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of candidate compounds can be determined using a standard enzyme kinetics assay under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0387] Phosphate-based cleavable linking groups are cleaved by agents that degrade or hydrolyze the phosphate group. Examples of agents that cleave phosphate groups in cells include enzymes, such as intracellular phosphatases. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(O)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. In certain embodiments, the phosphoric acid-based linking group is selected from -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -O-Ρ(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In certain embodiments, the phosphoric acid linking group is -OP(O)(OH)-O-. These candidates can be evaluated using a method similar to that described above.

[0388] Acid-cleavable linking groups are those that are cleaved under acidic conditions. In some embodiments, acid-cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less), or by agents such as enzymes that can act as general acids. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups, but not limited to them, include hydrazones, esters, and amino acid esters. Acid-cleavable groups may have the general formula -C=N-, C(O)O, or -OC(O). In some embodiments, the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0389] Ester-based cleavable linking groups are cleaved by enzymes, such as intracellular esterases and amidases. Examples of ester-based cleavable linking groups, but not limited to them, include esters of alkylene, alkenylene, and alkynylene groups. Ester-based cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0390] Peptide-based cleavable linking groups are cleaved by enzymes, such as intracellular peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not contain amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to produce peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) that do not contain the entire amide functional group formed between amino acids to produce peptides and proteins. Peptide-based cleavable linking groups have the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0391] While not limited to these, these are representative carbohydrate conjugates using linkers. [ka] [ka] (In the formula, if either X or Y is an oligonucleotide, the other is hydrogen.) These are some examples.

[0392] In certain embodiments of the composition and method, the ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached via a divalent or trivalent branched linker. For example, in some embodiments, the siRNA is conjugated with the GalNAc ligand as shown in the following structure: [ka] (Here, X is either O or S).

[0393] In some embodiments, the siRNA sense strand and ligand are conjugated as shown in the following structure, with the ligand attached to the 3' end of the sense strand via a linker: [ka] (Here, X is either O or S).

[0394] In some embodiments, the combination therapy includes siRNA conjugated with a divalent or trivalent branched linker selected from the group of structures represented by formulas (XXXI) to (XXXIV): [ka] (In the formula: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each represent independently, for each occurrence, a range from 0 to 20, where the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , and T 5C For each occurrence, independently, it is either nonexistent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B , and Q 5Cis, independently for each occurrence, absent, alkylene, or substituted alkylene, where one or more methylenes may be interrupted or terminated by one or more of O, S, S(O), SO2, N(R N ), C(R’)=C(R’’), C≡C or C(O); R 2A 、R 2B 、R 3A 、R 3B 、R 4A 、R 4B 、R 5A 、R 5B 、and R 5C are, independently for each occurrence, absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a ), C(O)-CH(R a )-NH-, CO, CH=N-O,

Chemical formula

Chemical formula

[0395] Examples of GalNAc derivatives conjugated with appropriate divalent and trivalent branched linker groups include, but are not limited to, the structures listed above as formulas I, VI, X, IX, and XII.

[0396] Representative US patents teaching the preparation of RNA conjugates include U.S. Patents No. 4,828,979; No. 4,948,882; No. 5,218,105; No. 5,525,465; No. 5,541,313; No. 5,545,730; No. 5,552,538; No. 5,578,717, No. 5,580,731; No. 5,591,584; No. 5,109,124; No. 5,118,802; No. 5,138,045; No. 5,414,077; and No. 5,486,603. No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737; No. 4,824,9 No. 41; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013; No. 4,958,013; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112, No. 963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,37 No. 1,241, No. 5,391,723; No. 5,416,203, No. 5,451,463; No. 5,510,475 ; Same No. 5,512,667; Same No. 5,514,785; Same No. 5,565,552; Same No. 5,567,810; Same No. 5,5 Examples include Nos. 74,142; Nos. 5,585,481; Nos. 5,587,371; Nos. 5,595,726; Nos. 5,597,696; Nos. 5,599,923; Nos. 5,599,928 and Nos. 5,688,941; Nos. 6,294,664; Nos. 6,320,017; Nos. 6,576,752; Nos. 6,783,931; Nos. 6,900,297; and Nos. 7,037,646; each of which is incorporated herein by reference with respect to teachings relating to such preparation methods.

[0397] In certain cases, the RNA of an RNAi agent can be modified with a non-ligand group. Numerous non-ligand molecules have been conjugated with RNAi agents to enhance their activity, cell distribution, or intracellular uptake, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand portions include lipid portions, such as cholesterol (Kubo, T., et al., Biochem. Biophys. Res. Comm. 365 (1): 54-61 (2007); Letsinger, et al., Proc. Natl. Acad. Sci. USA 86: 6553 (1989)), cholic acid (Manoharan, et al., Bioorg. Med. Chem. Lett. 4: 1053 (1994)), thioethers, such as hexyl-S-tritylthiol (Manoharan, et al., Ann. NY Acad. Sci. 660: 306 (1992); Manoharan, et al., Bioorg. Med. Chem. Let. 3: 2765 (1993)), and thiocholesterol (Oberhauser, et al., Nucl. Acids Res. 20: 533 (1992)), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras, et al., EMBO J. 10: 111 (1991); Kabanov, et al., FEBS Lett. 259: 327 (1990); Svinarchuk, et al., Biochimie 75: 49 (1993)), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan, et al., Tetrahedron Lett. 36: 3651 (1995); Shea, et al., Nucl. Acids Res. 18: 3777 (1990)), polyamine or polyethylene glycol chains (Manoharan, et al.This includes , Nucleosides & Nucleotides 14: 969 (1995) or adamantane acetate (Manoharan, et al., Tetrahedron Lett. 36: 3651 (1195)), palmityl moiety (Mishra, et al., Biochim. Biophys. Acta 1264: 229 (1995)), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke, et al., J. Pharmacol. Exp. Ther. 277: 923 (1996)).

[0398] A typical conjugation protocol involves the synthesis of RNA having aminolinkers at one or more positions in its sequence. The amino groups are then reacted with the conjugated molecule using appropriate coupling or activating reagents. The conjugation reaction can be carried out either with RNA still bound to a solid support or in the liquid phase after RNA cleavage. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0399] d. RNAi agent delivery "Introducing into cells," as understood by those skilled in the art, means facilitating or bringing about the uptake or absorption into cells when referring to RNAi agents...

Claims

1. An antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and the VL are (i) Sequence IDs 38 and 62, respectively; (ii) Sequence IDs 38 and 58, respectively; (iii) Sequence IDs 38 and 59, respectively; (iv) Sequence IDs 38 and 60, respectively; (v) Sequence IDs 38 and 61, respectively; (vi) Sequence IDs 38 and 63, respectively; (vii) Sequence IDs 38 and 64, respectively; (viiii) Sequence IDs 38 and 65, respectively; (ix) Sequence IDs 38 and 66, respectively; (x) Sequence IDs 38 and 67, respectively; (xi) Sequence IDs 38 and 68, respectively; (xi) Sequence IDs 38 and 71 respectively; or (xiiii) Sequence IDs 38 and 72, respectively It contains or consists of the amino acid sequences described in The antibody or antigen-binding fragment is capable of binding to the antigenic loop region of HBsAg and neutralizing infection by hepatitis B virus (HBV) of genotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof.

2. When a sample containing multiple antibodies or antigen-binding fragments is incubated at 40°C for 120 to 168 hours, the sample contains less than 12%, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less of the multiple antibodies or antigen-binding fragments as dimers, and the presence of dimers is determined, if necessary, by absolute size exclusion chromatography; and / or Incubation of multiple antibodies or antigen-binding fragments results in a reduction in dimer formation compared to incubation of multiple reference antibodies or antigen-binding fragments. The reference antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs: 34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence described in SEQ ID NO: 38 and the VL amino acid sequence described in SEQ ID NO:

57. If necessary, the presence of antibody dimers is determined by absolute size exclusion chromatography; and / or The antibody or antigen-binding fragment is (i) In incubation at 4°C for 5 days, 15 days, and / or 32 days; (ii) In incubation at 25°C for 5 days, 15 days, and / or 32 days; and / or (iii) In incubation at 40°C for 5 days, 15 days, and / or 32 days, Compared to the reference antibody, it forms a lower amount of dimers, and / or forms dimers at a reduced frequency, and / or as a lower percentage of the total antibody or antigen-binding fragment molecules in the sample or composition. The reference antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences described in SEQ ID NOs: 34, 35, 37, 41, 44, and 55, respectively, and optionally comprises the VH amino acid sequence described in SEQ ID NO: 38 and the VL amino acid sequence described in SEQ ID NO:

57. The antibody or antigen-binding fragment according to claim 1.

3. The antibody or its antigen-binding fragment comprises a human antibody, a monoclonal antibody, a purified antibody, a single-chain antibody, Fab, Fab', F(ab')2, Fv, or scFv; and / or The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody or antigen-binding fragment is a multispecific antibody, a bispecific antibody, or an antigen-binding fragment.

4. The antibody or antigen-binding fragment includes an Fc portion; and / or The Fc portion includes a mutation that enhances binding to FcRn compared to the reference Fc portion, and the reference Fc portion does not include the mutation; The Fc portion contains a mutation that enhances binding to FcγR compared to the reference Fc portion, and the reference Fc portion does not contain said mutation; and / or The Fc portion is an IgG isotype or derived from an IgG isotype, and if necessary, the IgG isotype is IgG1m17,1(IgG1 ＊ An antibody or antigen-binding fragment according to any one of claims 1 to 3, comprising or derived from 01).

5. The mutation that enhances binding to FcRn, (i) M428L / N434S; (ii) M252Y / S254T / T256E; (iii) T250Q / M428L; (iv) P257I / Q311I; (v) P257I / N434H; (vi) D376V / N434H; (vii) T307A / E380A / N434A; or (viiii) Any combination of (i) to (vii) Includes, The amino acid numbering of the Fc portion conforms to the EU numbering system; and / or The mutations that enhance binding to FcRn include M428L / N434S; and / or The mutations that enhance binding to FcγR include S239D;I332E;A330L;G236A; or any combination thereof, and the amino acid numbering of the Fc portion conforms to the EU numbering system; and / or The mutation that enhances binding to FcγR is (i) S239D / I332E; (ii) S239D / A330L / I332E; (iii) G236A / S239D / I332E; or (iv) G236A / A330L / I332E Including; and / or The mutation that enhances binding to FcγR comprises or consists of G236A / A330L / I332E, and optionally the antibody or antigen-binding fragment does not contain S239D, and optionally the antibody or antigen-binding fragment further contains native S at position 239; and / or The antibody or antigen-binding fragment according to claim 4, wherein the Fc portion comprises the amino acid substitution mutations: M428L; N434S; G236A; A330L; and I332E, and optionally does not contain S239D.

6. A light chain constant region (CL) comprising or having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 79; and / or An antibody or antigen-binding fragment according to any one of claims 1 to 5, comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 73, or comprising a CH1-CH2-CH3 sequence thereof, or a variant thereof comprising one or more of the following amino acid substitutions (EU numbering): G236A; A330L; I332E; M428L; N434S, wherein the C-terminal lysine is optionally removed from the CH1-CH2-CH3 sequence.

7. An antibody comprising a heavy chain (HC) containing or comprising the amino acid sequence described in SEQ ID NO: 75, with the C-terminal lysine removed as necessary, and a light chain (LC) containing (i) the VL amino acid sequence described in any one of SEQ ID NOs: 62, 58-61, 63-68, 71, and 72, and (ii) the CL amino acid sequence described in SEQ ID NO: 79, or comprising these.

8. The antibody according to claim 7, wherein the LC comprises a VL amino acid sequence described in any one of SEQ ID NOs. 62, 66, 67, and 72.

9. A polynucleotide comprising a nucleotide sequence encoding an antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the nucleotide sequence encoding the antibody or antigen-binding fragment is optionally codon-optimized for expression in a host cell.

10. A polynucleotide encoding a light chain variable region (VL) and optionally a light chain constant domain (CL) of an antibody or antigen-binding fragment according to any one of claims 1 to 8.

11. A nucleotide sequence having at least 90% identity with any one of the nucleotide sequences described in Sequence ID No. 89, 85-88, and 90-99; and / or (i) a polynucleotide sequence described in SEQ ID NO: 81 or SEQ ID NO: 82, and (ii) a polynucleotide sequence described in any one or more of SEQ ID NOs: 89, 85-88, and 90-99; and / or (i) a polynucleotide sequence described in SEQ ID NO: 83, and (ii) a polynucleotide sequence described in any one or more of SEQ ID NOs: 89, 85-88, and 90-99; and / or (i) the polynucleotide sequence described in Sequence ID No. 84, and (ii) the polynucleotide sequence described in any one or more of Sequence ID Nos. 89, 85-88, and 90-99, according to claim 9 or claim 10.

12. A vector comprising a polynucleotide according to any one of claims 9 to 11, which optionally comprises a lentiviral vector or a retroviral vector.

13. A host cell comprising the polynucleotide according to any one of claims 9 to 11 and / or the vector according to claim 12.

14. (i) The antibody or antigen-binding fragment according to any one of claims 1 to 8; (ii) The polynucleotide according to any one of claims 9 to 11; (iii) The vector according to claim 12; (iv) The host cell according to claim 13; or (v) Any combination of (i) to (iv), and Pharmacologically acceptable excipients, diluents, or carriers A pharmaceutical composition containing the following:

15. (a) (i) The antibody or antigen-binding fragment according to any one of claims 1 to 8; (ii) The polynucleotide according to any one of claims 9 to 11; (iii) The vector according to claim 12; (iv) The host cell according to claim 13; (v) The pharmaceutical composition according to claim 14; or (vi) Any combination of (i) to (v) Components selected from; and (b) (1) Instructions for use of the components for the prevention, treatment, attenuation, and / or diagnosis of hepatitis B infection and / or hepatitis D infection, and / or (2) Means for administering the above-mentioned components to a subject A kit that includes this.

16. A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 8, comprising culturing a host cell according to claim 13 under conditions and for a period of time sufficient to produce the antibody or antigen-binding fragment.

17. A composition for treating, preventing, and / or attenuating hepatitis B and / or hepatitis D infection in a subject, comprising an effective amount of (i) an antibody or antigen-binding fragment according to any one of claims 1 to 8; (ii) a polynucleotide according to any one of claims 9 to 11; (iii) a vector according to claim 12; (iv) a host cell according to claim 13; and / or (v) the pharmaceutical composition according to claim 14.

18. (1) The composition is intended to be administered to the subject together with (vi) a polymerase inhibitor, optionally comprising lamivudine, adefovir, entecavir, terbivudine, tenofovir, or any combination thereof; (vii) an interferon, optionally comprising IFN beta and / or IFN alpha; (viiii) a checkpoint inhibitor, optionally comprising an anti-PD-1 antibody or its antigen-binding fragment, an anti-PD-L1 antibody or its antigen-binding fragment, and / or an anti-CTLA4 antibody or its antigen-binding fragment; (ix) an agonist of a stimulating immune checkpoint molecule; or any combination of (x)(vi) to (ix); and / or (2) The hepatitis B virus infection is a chronic hepatitis B virus infection; and / or (3) The subject has received a liver transplant; and / or (4) The subject is not immune to the hepatitis B virus; and / or (5) The subject is a newborn; and / or (6) The composition for use according to claim 17, wherein the subject is undergoing or has undergone hemodialysis.

19. The composition is intended to be administered to the subject as a single dose of a pharmaceutical composition containing the antibody or antigen-binding fragment; and / or The single dose of the pharmaceutical composition contains the antibody in the range of 2 to 18 mg / kg (subject body weight); and / or The single dose of the pharmaceutical composition contains up to 6 mg, up to 10 mg, up to 15 mg, up to 18 mg, up to 25 mg, up to 30 mg, up to 35 mg, up to 40 mg, up to 45 mg, up to 50 mg, up to 55 mg, up to 60 mg, up to 75 mg, up to 90 mg, up to 300 mg, up to 900 mg, or up to 3000 mg of the antibody, or The single dose of the pharmaceutical composition contains the above-mentioned assay in an amount within the range of 1 mg to 3000 mg, or 5 mg to 3000 mg, or 10 mg to 3000 mg, or 25 mg to 3000 mg, or 30 mg to 3000 mg, or 50 mg to 3000 mg, or 60 mg to 3000 mg, or 75 mg to 3000 mg, or 90 mg to 3000 mg, or 100 mg to 3000 mg, or 150 mg to 3000 mg, or 2000 mg to 3000 mg, or 300 mg to 3000 mg, or 500 mg to 3000 mg, or 750 mg to 3000 mg, or 900 mg to 3000 mg, or 1500 mg to 3000 mg, or 2000 mg to 3000 mg, or The single dose of the pharmaceutical composition contains the antibody in an amount within the range of 1 mg to 900 mg, or 5 mg to 900 mg, or 10 mg to 900 mg, or 25 mg to 900 mg, or 30 mg to 900 mg, or 50 mg to 900 mg, or 60 mg to 900 mg, or 75 mg to 900 mg, or 90 mg to 900 mg, or 100 mg to 900 mg, or 150 mg to 900 mg, or 200 mg to 900 mg, or 300 mg to 900 mg, or 500 mg to 900 mg, or 750 mg to 900 mg, or The single dose of the pharmaceutical composition contains the antibody in an amount within the range of 1 mg to 500 mg, or 5 mg to 500 mg, or 10 mg to 500 mg, or 25 mg to 500 mg, or 30 mg to 500 mg, or 50 mg to 500 mg, or 60 mg to 500 mg, or 75 mg to 500 mg, or 90 mg to 500 mg, or 100 mg to 500 mg, or 150 mg to 500 mg, or 200 mg to 500 mg, or 300 mg to 500 mg, or 400 mg to 500 mg, or the single dose of the pharmaceutical composition contains the antibody in such an amount, or The single dose of the pharmaceutical composition contains the kinase in an amount within the range of 1 mg to 300 mg, or 5 mg to 300 mg, or 10 mg to 300 mg, or 25 mg to 300 mg, or 30 mg to 300 mg, or 50 mg to 300 mg, or 60 mg to 300 mg, or 75 mg to 300 mg, or 90 mg to 300 mg, or 100 mg to 300 mg, or 150 mg to 300 mg, or 200 mg to 300 mg, or The single dose of the pharmaceutical composition contains the antibody in an amount within the range of 1 mg to 200 mg, or 5 mg to 200 mg, or 10 mg to 200 mg, or 25 mg to 200 mg, or 30 mg to 200 mg, or 50 mg to 200 mg, or 60 mg to 200 mg, or 75 mg to 200 mg, or 90 mg to 200 mg, or 100 mg to 200 mg, or 150 mg to 200 mg, or The single dose of the pharmaceutical composition contains the antibody in an amount within the range of 1 mg to 100 mg, or 5 mg to 100 mg, or 10 mg to 100 mg, or 25 mg to 100 mg, or 30 mg to 100 mg, or 50 mg to 100 mg, or 60 mg to 100 mg, or 75 mg to 100 mg, or 75 mg to 100 mg, or 90 mg to 100 mg, or The single dose of the pharmaceutical composition contains the antibody in an amount within the range of 1 mg to 25 mg, or 5 mg to 25 mg, or 10 mg to 25 mg, or 15 mg to 25 mg, or 20 mg to 25 mg, or The single dose of the pharmaceutical composition contains the antibody in an amount within the range of 1 mg to 50 mg, or 1 mg to 25 mg, or 5 mg to 50 mg, or 5 mg to 25 mg, or 10 to 50 mg, or 10 to 25 mg, or 1 to 15 mg, or 5 mg to 15 mg, or 10 mg to 15 mg, or The single dose of the pharmaceutical composition is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, Contains 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 mg or more of the aforementioned antibodies, or The single dose of the pharmaceutical composition is less than 3000 mg, less than 2500 mg, less than 2000 mg, less than 1500 mg, less than 1000 mg, less than 900 mg, less than 500 mg, less than 300 mg, less than 200 mg, less than 100 mg, less than 90 mg, less than 75 mg, less than 50 mg, less than 25 mg, or less than 10 mg, but contains the antibody in an amount greater than 1 mg, greater than 2 mg, greater than 3 mg, greater than 4 mg, or greater than 5 mg; and / or The composition for use according to claim 17 or claim 18, wherein the single dose of the pharmaceutical composition contains the antibody at a concentration in the range of 100 mg / mL to 200 mg / mL.

20. An antibody or antigen-binding fragment according to any one of claims 1 to 8, having a concentration in the range of 100 mg / mL to 200 mg / mL, and Pharmacologically acceptable carriers, excipients, or diluents A pharmaceutical composition containing the following:

21. The pharmaceutical composition comprises water, and optionally USP water; and / or The pharmaceutical composition contains histidine, optionally at a concentration of 10 mM to 40 mM; and / or The pharmaceutical composition optionally contains a disaccharide in an amount of 5%, 6%, 7%, 8%, or 9% (w / v); and / or The pharmaceutical composition comprises a surfactant, optionally a polysorbate, optionally the polysorbate present in an amount of 0.01% to 0.05% (w / v); and / or The pharmaceutical composition according to claim 20, wherein the pharmaceutical composition has a pH in the range of 5.8 to 6.2, in the range of 5.9 to 6.1, or 5.8, 5.9, 6.0, 6.1, or 6.

2.

22. The aforementioned pharmaceutical composition (i) 150 mg / mL of the antibody; (ii) USP Water; (iii) 20 mM histidine; (iv) 7% sucrose; and (v) Contains 0.02% PS80, The pharmaceutical composition according to claim 21, wherein the pharmaceutical composition contains a pH of 6.

23. The pharmaceutical composition according to any one of claims 20 to 22, wherein the pharmaceutical composition is a liquid solution; and / or the pharmaceutical composition is formulated for injection via a pre-filled syringe.

24. A method for providing detected results for use in in vitro diagnosis of hepatitis B infection, (i) Contacting a sample derived from a subject with an antibody or antigen-binding fragment according to any one of claims 1 to 8; and (ii) Detection of a complex comprising an antigen and the antibody, or a complex comprising an antigen and the antigen-binding fragment. Methods that include...

25. A method for detecting the presence or absence of an epitope in the precise three-dimensional structure of an anti-hepatitis B vaccine, (i) Contacting the vaccine with the antibody or antigen-binding fragment described in any one of claims 1 to 8; and (ii) Determining whether a complex comprising the antigen and the antibody, or a complex comprising the antigen and the antigen-binding fragment, has been formed. Methods that include...

26. A composition for treating chronic HBV infection and / or HDV infection in a subject requiring treatment for chronic HBV infection and / or HDV infection, wherein the composition comprises an antibody or antigen-binding fragment according to any one of claims 1 to 8. The subjects will also be administered drugs that reduce HBV antigen load and / or inhibit HBV gene expression; The agent that reduces the HBV antigen load and / or inhibits HBV gene expression is an RNAi agent. The RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that are 3 nucleotides or less different from nucleotides 1579 to 1597 of SEQ ID NO: 116; and / or, The agent that reduces the HBV antigen load and / or inhibits HBV gene expression is an RNAi agent, wherein the RNAi agent comprises a sense strand and an antisense strand, and the sense strand comprises nucleotides 1579 to 1597 of SEQ ID NO:

116.

27. An antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL each comprise or consist of the amino acid sequences described in SEQ ID NOs. 38 and 62, respectively; The antibody or antigen-binding fragment is capable of binding to the antigenic loop region of hBsAg and is capable of neutralizing infection by hepatitis B virus (HBV) of genotype D, A, B, C, E, F, G, H, I, or J, or any combination thereof, and / or is capable of neutralizing infection by hepatitis D virus (HDV).

28. A composition for treating chronic HBV infection and / or HDV infection in a subject requiring treatment of chronic HBV infection and / or HDV infection, comprising the anti-HBV antibody described in claim 27.

29. A composition for treating chronic HBV infection and / or HDV infection in a subject requiring treatment for chronic HBV infection and / or HDV infection, wherein the composition comprises the anti-HBV antibody described in claim 27 and is for use in combination therapy in which the subject is administered siRNA. The siRNA has a sense strand containing 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 124) and an antisense strand containing 5'-usGfsuga(Agn)gCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 125), In the formula, a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively; s is a phosphorothioate linkage; L96 is 【Chemistry 1】 That is, composition.

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