A Therapeutic Vaccine for Hepatitis B Virus (HBV) using the HBV PreS1 and/or PreS2, and/or S-HBsAg regions of the HBV envelope protein

Cross-Reference

This application claims priority to U.S. Provisional Patent Application Serial No. 62/587051 filed November 16, 2017, incorporated by reference herein in its entirety.

Statement of Government Rights

This disclosure was made with government support under Grant No. HR001 1- 1 1-2-0007, awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the disclosure.

Background of the Disclosure

In spite of the availability of prophylactic Hepatitis B virus (HBV) vaccines, HBV infection remains a very significant global health problem in both industrialized and developing nations; it is second only to tobacco as a cause of cancer. There is a clear unmet need for a therapeutic HBV vaccine for patients chronically infected with HBV (CHB). 10- 30% of those vaccinated with marketed HBV vaccines do not respond either due to genetic factors, or non-compliance (failure to return for a series of 3 vaccinations). Only 37% of individuals vaccinated once with a licensed HBV vaccine are protected; even after three vaccinations, which are difficult to achieve, many people do not respond effectively. There is no effective vaccine for the 400 million people chronically infected with HBV, including asymptomatic HBV carriers. The drugs currently used to treat CHB patients are problematic. Sustained antiviral responses are rarely achieved and the currently available therapies can lead to viral resistance and produce side effects in many CHB patients.

Brief Description of the Figures

Figure 1. Schematic design of the G28-8LH-scAb-PreS1-S2-His protein.

Figure 2. Characterization of recombinant G28-8LH-scAb-PreS1-S2-His . G28-8LH- scAb-PreS1-S2-His was transiently expressed in CHO cells. Culture supernatant was passed over a Ni2+ affinity chromatography column. Bound G28-8LH-scAb-PreS1-S2-His was eluted with imidazole. Eluted protein (E) was characterized by reducing SDS-PAGE and western blotting using an anti-6x-His antibody. Figure 3. Binding of recombinant G28-8LH-scAb-PreS1-S2-His to human B cells. Direct binding to human gated CD20+ tonsillar B cells using a FITC-anti-His monoclonal antibody (bold black line). Second step only (light black line).

Figure 4. Recombinant G28-8LH-scAb-PreS1-S2-His activates human B cells. Sheep erythrocyte-binding negative blood mononuclear cells enriched for B cells were incubated at 37 C for 24 hours either with media only (light black line), or with G28-8LH- scAb-PreS1 -S2-His (bold black line). Samples were gated for CD20+ cells (Pacific blue-anti- CD20) and levels of CD40 expression measured as an indication of activation using flow cytometry. Graph shows CD40 expression of gated CD20 ⁺ B cells.

Figure 5. Immune responses in macaques immunized and boosted with recombinant G28-8LH-scAb-PreS1-S2-His recombinant protein (CD180-HBV-preS1/S2). Groups of cynomolgus macaques ( Macaca fascicularis) (N=3) were vaccinated subcutaneously with either: 1 ) 300 μg of G28-8LH-scAb-PreS1-S2-His (CD180-HBV-PreS1/S2, black circles); or 2) 300 μg of G28-8LH-scAb-PreS1 -S2-His (aCD 180-HBV-preS 1 /S2) plus 0.5 ml Addavax™ (open squares). Animals were vaccinated on days 0 and 30, and serum and heparinized blood samples were obtained on days 0, 7, 14, 30 after primary immunization and days 7,

14, and 30 after secondary immunization. (A) HBV-PreS1 -specific IgG antibody levels detected using ELISA. Mean optical densities (O.D.) at each time point ± SEM are indicated. (B) HBsAg-specific IFN-y-producing T cells detected by ELIspot assays. Statistical comparisons between the two groups for each assay were assessed at each timepoint using unpaired t test on samples with equal standard deviation. Significant differences are indicated: *P =0.01 , ***P=0.006. For all other timepoints, there was no significant difference in mean responses between the groups.

Figure 6. G28-8LH-scAb-PreS1-S2-His recombinant protein vaccine induced neutralizing antibodies (Abs) that block the production of HBV cccDNA in HBV infected liver cells. Cynomolgus macaques ( Macaca fascicularis, N=3/group) received a priming and booster as described in Fig. 11 with either 300 ug G28-8LH-scAb-PreS1-S2-His (A57, A59 and A60) or 300 μg G28-8LH-scAb-PreS1-S2-His co-formulated with 100 μg of the commercial adjuvant, AddaVax™ (A55, A58 and A68, indicated by * in figure). Sera obtained 2 weeks after the second immunization were evaluated for neutralizing antibody activity. (A) The scheme illustrates the treatment schedule with HBV inoculum (10 ³ Geq per cell) and serum samples (Neutralizing Ab). HepG2-hNTCP cells were treated with pre-bleed serum at a 1 :1000 dilution (D) or immune sera from macaques at !:300, 1 :1000 or 1 :3000 dilution for 16 hours after the time of the HBV inoculation. At 1 day post infection (dpi), the mediums containing 2.5% DMSO were replaced. The cccDNAs were extracted at 3 dpi, and analyzed by real-time PCR. (B) HepG2-hNTCP cells infected with HBV (10 ³ Geq/cell) were treated with sera as indicated (1:300 D = dilution of the original serum stock to 1/300 as vol/voi, 1 :1000 D = dilution of the original serum stock to 1/1000 as voi/vol, 1 :3000 D = dilution of the original serum stock to 1/3000 as vol/vol). Pre-bleed serums (1/1000 dilution (vol/vol)) were included as a control. At 3dpi, cccDNAs were analyzed by real-time PCR. Following digestion of T5 exonuclease, cccDNA was specifically quantified using specific primers. cccDNA was normalized as a ratio to mitochondrial DNA. Representative data are shown with quantification (means ± standard deviation) (n=2).

Detailed Description of the Disclosure

All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al.,

1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology ( Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), “Guide to Protein Purification" in Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al.

1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2 ^nd Ed. (R.l. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.“And" as used herein is interchangeably used with“or” unless expressly stated otherwise.

As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.

In a first aspect, the present disclosure provides compositions, comprising:

(a) a CD180 binding ligand; and

(b) Hepatitis B virus pre-S1 and/or pre-S2 regions of the HBV envelope protein (HBVpreS1-S2Ag), S-HBsAg, or antigenic fragments or mutants thereof, attached to the CD180 binding ligand. The compositions of the disclosure can be used, for example, to induce prophylactic responses in individuals at risk of HBV infection, and therapeutic responses in already infected individuals and in immunodeficient individuals who do not respond well to standard vaccines. The present disclosure is highly significant because it provides a therapeutic vaccine for one of the major causes of cancer and liver disease in the world: hepatitis B virus (HBV). HBV infection is a serious global public health problem in both industrialized and developing nations. 10-30 million people worldwide become infected with HBV each year, and more than 2 billion people worldwide have been infected with HBV. Significantly, the ability of unvaccinated individuals to mount effective immune responses against HBV is correlated with age. Infants and young children are particularly at risk, as 90% of infants and up to 50% of young children infected with HBV ultimately develop chronic infections. About 400 million are chronically infected with HBV (CHB), and in the USA there are approximately 1.4 million CHB infected people ⁵ . In the USA the prevalence of HBV while dropping in children, has changed little in adults ¹¹ such that the burden of chronic hepatitis B among adults remains large ⁶ ; in some groups it is as high as 1 %. An estimated 1 million people die each year from hepatitis and its complications, inciuding about 5,000 people in the US. Of the 5000 persons in the United States who die each year from HBV related conditions, 300 die from fulminant hepatitis; 3-4000, from cirrhosis; and 600-1000, from primary

hepatocellular carcinoma (HCC). In the US, approximately 400 health care workers are infected each year and are at risk from dying from HBV-related disease ¹⁶ .

The CD180 binding ligand may be any molecule that binds directly to CD180 present in the surface of B cells, macrophages, or dendritic cells. In various non-limiting

embodiments, the CD180 binding ligand may be a peptide mimetic or an antibody.

In a particular embodiment, the CD180 binding ligand is an antibody or antibody fragment. As used herein, "antibody" includes reference to full length and functional fragments of any of the following: an immunoglobulin molecule immunologically reactive with human CD180 (preferably selective for CD180), genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies), fully humanized antibodies, human antibodies, single chain Fv fragments (scFv), bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies, single domain molecules such as VH and VL that are capable of specifically binding to an epitope of an antigen, and camelids. Fragments with antigen-binding activity include, but are not limited to, Fab', F(ab') ₂ , Fab, Fv and rlgG and includes monoclonal antibodies. Various isotypes of antibodies exist, for example lgG1 , lgG2, !gG3, lgG4, and other Ig, e.g., IgM, IgA, IgE isotypes. The term also includes. Bivalent and bispecific molecules are described in, e.g., Kostelny et at.. (1992) J Immunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31 :1579, Hollinger et al., 1993, supra, Gruber et a/. (1994) J Immunol :5368, Zhu et al. (1997) Protein Sci 6:781 , Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301. Various antigen binding domain-fusion proteins are also disclosed, e.g., in US patent application Nos. 2003/0118592 and 2003/0133939, and are encompassed within the term "antibody" as used in this application.

An antibody immunologically reactive with human CD180 can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341 :544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or by immunizing an animal with the antigen or with DNA encoding the antigen.

In one embodiment, the CD180 binding ligand comprises a single chain (sc) recombinant protein, wherein the sc recombinant protein comprises:

(i) a variable heavy (VH) chain region of an anti-CD180 antibody; and

(ii) a variable light (VL) chain region of an anti-CD 180 antibody.

In one embodiment, the CD180 binding ligand comprises a single domain (sd) recombinant protein, wherein the sd recombinant protein comprises a variable heavy (VHH) chain region of an anti-CD180 antibody derived from a camelid species.

The VH and VL chain regions may be from an anti-human CD180 antibody, such as an anti-human CD180 monoclonal antibody. Exemplary commercially available CD180 anti human monoclonal antibodies from which the VH and VL chains may be used include, but are not limited to, those sold by AbD Serotec™ (“MHR73-11”), BD Biosciences, Thermo Scientific, Sigma Aldrich, etc.), (“G28-8”), and LifeSpan™ (“200.1”). In one embodiment, the single chain recombinant protein does not include any other immunoglobulin domains (i.e.: a single chain variable fragment (scFv)). In an alternative embodiment, the single chain recombinant protein further comprises: CH2 and CH3 domains from an immunoglobulin (Ig), such as a human Ig, or functional mutants thereof, wherein the CH2 and CH3 domains are located C-terminal to the VH and VL domains. The CH2 and CH3 domains may be from any immunoglobulin as deemed appropriate for an intended use of the composition, including but not limited to lgA1 , lgA2, lgG1 , lgG2, lgG3, lgG4, IgM, etc. In a particular embodiment, the sc recombinant protein comprises CH2 and CH3 domains from lgG1 , such as human lgG1 , or functional mutants thereof. In a particular embodiment, such“functional mutants” comprise CH2 and/or CH3 domains that have impaired binding to human or animal Fc receptor FcγRllb and/or to human or animal complement proteins (J Biol Chem 276: 6591- 6604). The Fc domain of the recombinant molecules is an altered human lgG1 Fc domain with three amino acid changes (P238S, P331 S, K322S) that reduce the binding of the molecule to Fc receptors and C1q. Other amino acid substitutions that can reduce binding of human lgG1 to various Fc receptors include but are not limited to E233P, L234V, L235A, G236 deletion, P238A, D265A, N297A, A327Q, and P329A. Substitutions at these amino acids reduce binding to ail FcyR. Substitutions at D270A, Q295A, or A327S reduce binding to FcyR 11 and FcyRIIIA. Substitutions at S239A, E269A, E293A, Y296F, V303A, A327G, K338A, and D376A reduce binding to FcyRIIIA but not FcyRII. A combination of two of more of these substitutions can be engineered in the Fc domains of human lgG1 to achieve the desired effects on inhibiting Fc-FcyR interaction between CD180 targeted vaccines and FcgR expressing cells. Similarly, modifying the glycosylation profile of human lgG1 , for example, substitution of the N-linked glycosylation site at Asn-297 of human lgG1 , eliminates N-linked glycosylation of human lgG1 , thereby eliminating its binding to Fc receptors as well as complement fixation functions (John S. Axford (ed.), Glycobiology and Medicine, 27-43; 2005 Springer).

In these various embodiments of the compositions of the disclosure, the VL chain region may be located N-terminal to the VH chain region, or the VH chain region may be located N-terminal to the VL chain region, as disclosed in the examples that follow.

In one embodiment, the CD 180-antibody comprises mAb G28-8, which is commercially available from a number of sources, (BD Biosciences, Thermo Scientific,

Sigma Aldrich, etc.), a F(ab’)2 fragment of mAb G28-8, or a single chain recombinant protein having the VL and VH domains of G28-8, and optionally further comprising CH2 and CH3 domains from an immunoglobulin (Ig), such as a human Ig, or functional mutants thereof.

In another embodiment, the CD180 binding ligand competes for binding to CD180 with monoclonal antibody G28-8. As used herein, competing CD 180 binding ligands are those binding proteins that bind to about, substantially or essentially the same, or even the same, epitope as G28-8. Competing binding proteins, such as competing antibodies or derivatives thereof, include binding proteins with overlapping epitope specificities.

Competing binding proteins are thus able to effectively compete with G28-8 antibody, such as the G28-8 antibody obtained from Thermo Scientific (the“reference antibody”) for binding to CD180. A binding protein that competes with the reference G28-8 antibody for binding to CD180 will be able to effectively or significantly reduce (i.e.: reduce by at least 10%;

preferably by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) reference G28- 8 antibody binding to CD180, as evidenced by a reduction in bound label. In one embodiment, the reference G28-8 antibody is pre-mixed with varying amounts of the test binding proteins (e.g., 1 :10, 1 :100 or 1 :1000) for a period of time prior to applying to a CD180 composition. In other embodiments, the reference G28-8 antibody and varying amounts of test binding proteins can simply be admixed during exposure to the CD180 composition. By using species or isotype secondary antibodies one will be able to detect only the bound reference G28-8 antibody, the binding of which will be reduced by the presence of a test binding protein that "competes" for binding. Examples of detection of such binding events are provided herein. As the identification of competing binding proteins is determined in comparison to the reference G28-8 antibody, it will be understood that actually determining the epitope to which the binding proteins bind is not in any way required in order to identify a competing binding proteins. However, epitope mapping can be performed, if desired.

In another embodiment, the CD180 binding ligand activates antigen-presenting cells, e.g., to increase expression of CD40, as shown in the examples that follow.

The compositions of the disclosure comprise a Hepatitis B virus preS1 and/or pre-S2 region of the envelope antigen (HBVpreS1/S2Ag), an S-HBsAg, or antigenic fragments or mutants thereof (collectively referred to as HBVpreS1/S2Ag or S-HBsAg) attached to the CD180 binding ligand. Thus, the compositions may comprise one or more

HBVpreS1/S2Ags and/or S-HBsAgs. In all embodiments, one or more copies of

HBVpreS1/S2Ags and/or S-HBsAgs may be present at the N-terminus or the C-terminus of the single chain recombinant protein. PreS1 and PreS2 are present in HBV surface antigens L-HBsAg and M-HBsAg and thus the HBVpreS1/S2Ag may comprise isolated PreS1 and/or PreS2, or may comprise HBV L-HBsAg or M-HBsAg. The HBVpreS1/S2Ag, L-HBsAg, M- HBsAg, and S-HBsAg may be from any HBV genotype, serotype, variant, mutant, or isolate.

In various embodiments, the composition may comprise an HBVpreS1/S2Ag or S- HBsAg at least 90% identical over the length of the amino acid sequence of one or more of the following:

P31873 Hepatitis B virus genotype A1 subtype adw2 (isolate Southern- Africa/Cai)

P03141 Hepatitis B virus genotype A2 subtype adw2 (strain Rutter 1979)

Q4R1R8 Hepatitis B virus genotype A3 (isolate Cameroon/CMR711/1994)

Q8JXB9 Hepatitis B virus genotype B1 (isolate Japan/Ry30/2002)

Q9PWW3 Hepatitis B virus genotype B2 (isolate Vietnam/ 16091/1992)

Q76R62 Hepatitis B virus genotype C subtype ayr (isolate Human/ Japan/Okamoto/-)

P03138 Hepatitis B virus genotype D subtype ayw (isolate France/Tiollais/1979)

Q69603 Hepatitis B virus genotype E subtype ayw4 (isolate Kou) GN=S PE=1 SV=2

Q99HS3 Hepatitis B virus genotype Fl (isolate Argentina/sall/2000)

Q99HR4 Hepatitis B virus genotype F2 (isolate Argentina/sal 6/2000)

Q9IBI3 Hepatitis B virus genotype G (isolate IG29227/2000)

Q8JMY6 Hepatitis B virus genotype H (isolate United States/LAS2523/2002)

In various further embodiments, the composition may comprise an HBVpreS1/S2Ag or S-HBsAg polypeptide at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the length of the amino acid sequence of the sequences shown above in various embodiments, additional HBVpreS1/S2Ag mutations may be included (alone or in combination). These mutations may include, but are not limited to, the preS1 S98T substitution (PLOS One 9: e110012, 2014), the preS1 F53L substitution (J Med Virol 85: 1698, 2013), or the preS1 A39R and preS1 S96A/T substitutions (Clin Microbiol Infect 18: E412, 2012).

In all of these embodiments, the composition may further comprise an amino acid linker position between the CD180 binding ligand and the HBVpreS1/S2Ag or S-HBsAg.

The linker may be of any suitable length and amino acid composition, depending on the intended use. In one embodiment, the linker is between about 2-40 amino acids in length.

In other embodiments, the linker may be between 10-30 or 15-25 amino acids in length. In another embodiment, the linker may be a linker rich in glycine and serine residues. In one specific embodiment, the linker may comprise the amino acid sequence

GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:69).

In various further embodiments, the composition may comprise or consist of a polypeptide at least 90% identical over its length to the following amino acid sequence of G28-8LH-scAb-PreS1-S2-His protein _(expressed)

The compositions of any embodiment or combination of embodiments of the disclosure may be provided as a stand-alone composition, or may be provided as part of a molecular scaffold. In various embodiments, the composition may be attached to molecular scaffold. Any suitable scaffold can be used, including but not limited to a VNAR single domain antibody (shark variable new antigen receptor), a lamprey variable lymphocyte receptor, a Im 7(colicin immunity 7 protein), an anticalin (lipocalin transport proteins), an FN3 (fibronectin 3) monobody, a DARPin (designed ankyrin repeat proteins), an affibody (Z domain of protein A), a single domain antibody, e.g, isolated from camelids or antibody libraries, and aptamer, etc., with CD180-binding polypeptide loops.

In another embodiment, the composition of any embodiment or combination of embodiments of the disclosure further comprises an adjuvant. While adjuvant is not required to induce rapid activation of HBVpreS1/S2Ag or S-HBsAg, addition of adjuvant to the compositions can result in additional enhancement of the immune response when the compositions are used in the methods of the disclosure. Any suitable adjuvant can be used, including but not limited to inorganic compounds (aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, beryllium, etc.), mineral oil, detergents, cytokines, toll-like receptor agonists, Freund’s complete adjuvant, Freund's incomplete adjuvant, squalene, etc. In a preferred embodiment, the adjuvant comprises or consists of a toll-like receptor 4 (TLR4) agonist, a toll-like receptor 7 (TLR7) agonist, a toll-like receptor 8 (TLR8) agonist, a toll-like receptor 9 (TLR9) agonist, alum-containing adjuvant, monophosphoryl lipid A, oil-in- water emulsion, and a-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion. The adjuvant may be present in the composition as an unlinked component or a linked component, depending on the adjuvant used.

In another embodiment, the compositions of the disclosure can be modified to extend half-life, such as by attaching at least one molecule to the composition for extending serum half-life, including but not limited to a polyethlyene glycol (PEG) group, serum albumin, a serum albumin binding domain, transferrin, transferrin receptor or the transferrin-binding portion thereof, or combinations thereof. As used herein, the word“attached" refers to a covalently or noncovalently conjugated substance. The conjugation may be by genetic engineering or by chemical means.

The compositions of the present disclosure may be stored in any suitable buffer. In a second aspect, the present disclosure provides isolated nucleic acids encoding the composition of any embodiment of the first aspect of the disclosure. The isolated nucleic acid sequence may comprise RNA or DNA. Such isolated nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. In one non-limiting embodiment, the isolated nucleic acids encode a polypeptide of the disclosure noted herein. In other embodiments, the isolated nucleic acids comprise or consist of the nucleotide sequence shown below.

G28-8LH-scAb-PreS1-S2-His 2142 bp

5’ end Hindlll and 3’ end BamHI sites for directional cloning into appropriate expression vector • Kozak consensus, GCCACC, right before 5' ATG start codon

• One 5’ in frame stop codon after 5’ end Hindiil site

• Two in frame stop codons before 3’ end BamHI site

In a third aspect, the present disclosure provides nucleic acid vectors comprising the isolated nucleic acid of the second aspect of the disclosure. "Recombinant expression vector" includes vectors that operatively link a nucleic acid coding region or gene to any promoter capable of effecting expression of the gene product. The promoter sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The expression vector must be replicable in the host organisms either as an episome or by integration into host

chromosomal DNA. In a preferred embodiment, the expression vector comprises a plasmid. However, the disclosure is intended to include other expression vectors that serve equivalent functions, such as viral vectors.

The nucleic acids and vectors of the disclosure can be used not only for production of large quantities of the compositions of the disclosure, but also for use as a nucleic acid (such as a DNA) vaccine administered by gene gun or other methods.

In a fourth aspect, the present disclosure provides recombinant host cells comprising the nucleic acid vector of the third aspect of the disclosure. The host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected. Such transfection of expression vectors into prokaryotic and eukaryotic cells (including but not limited to Chinese hamster ovary (CHO) cells) can be accomplished via any suitable means, including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.

The recombinant host cells can be used, for example in methods for producing antibody (when the binding protein is an antibody), comprising:

(a) culturing the recombinant host cell of the disclosure under conditions suitable for expression of the nucleic-acid encoded antibody composition; and

(b) isolating the antibody composition from the cultured cells.

Suitable conditions for expression of the nucleic-acid encoded antibody composition can be determined by those of skill in the art based on the teachings herein and the specific host cells and vectors used.

The term“recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, e.g., recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. By the term "recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operable linkage of different sequences is achieved. Thus an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes disclosed herein. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes disclosed herein.

In a fifth aspect, the present disclosure provides pharmaceutical compositions, comprising:

(a) the composition, isolated nucleic acid, or recombinant expression vector of any embodiment or combination of embodiments disclosed herein; and

(b) a pharmaceutically acceptable carrier.

In this embodiment, the compositions of the disclosure are present in a

pharmaceutical formulation. In this embodiment, the compositions are combined with a pharmaceutically acceptable carrier. Suitable acids which are capable of forming such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like. Suitable bases capable of forming such salts include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol-amines (e.g., ethanolamine, diethanolamine and the like).

The pharmaceutical composition may comprise in addition to the composition of the disclosure (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooieate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The

pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood.

Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

The pharmaceutical compositions of the disclosure may be made up in any suitable formulation, preferably in formulations suitable for administration by injection. Such pharmaceutical compositions can be used, for example, in methods of use as vaccines, prophylactics, or therapeutics.

The pharmaceutical compositions may contain any other components as deemed appropriate for a given use, such as additional therapeutics or vaccine components. In one embodiment, the pharmaceutical compositions further comprise toll-like receptor 4 (TLR4) agonist, a toll-like receptor 7 (TLR7) agonist, a toll-like receptor 8 (TLR8) agonist, a toll-like receptor 9 (TLR9) agonist, alum-containing adjuvant, monophosphoryl lipid A, oil-in-water emulsion, and a-tocopherol, squalene and polysorbate 80 in an oil-in-water emulsion.

In a sixth aspect, the present disclosure provides methods for treating or limiting development of an HBV infection or a hepatitis-B virus (HBV)-related disorder, comprising administering to an individual in need thereof an amount effective to treat or limit development of the disorder of the composition, isolated nucleic acid, recombinant expression vector, or pharmaceutical composition, or a pharmaceutical salt thereof, of any embodiment or combination of embodiments of the present disclosure. In one embodiment, the compositions are used prophylactically as vaccines to limit development of HBV infection disease/severity of infectious disease, such as in individuals that have not been exposed to an infectious agent but are at risk of such exposure. In other embodiments, the methods can be used therapeutically to treat people exposed to or chronically infected with HBV.

The methods of the disclosure target antigen to CD 180, a surface protein expressed on B cells, macrophages, and dendritic cells, that to produce antigen-specific IgG in the absence of T cell costimulation (such as CD40 deficiency) or the complete absence of T cells (such as TCR b/d deficiency). Thus, the methods can be used in any therapeutic or prophylactic treatment for HBV infection or vaccination. This approach also finds use, for example, for neonates, the elderly, the immunocompromised, and the immunodeficient, both in specifically targeting cellular populations enriched in underdeveloped or otherwise deficient immune systems and by improving responses to antigens that require linked recognition (carbohydrate epitopes, etc.).

As used herein, "treat" or "treating" means accomplishing one or more of the following in an individual that already has a disorder or has already been exposed to a disorder-causing substance/pathogen: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated (ex: immune deficiencies in cancer patients or other patients) undergoing chemotherapy and/or radiation therapy); (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s).

As used herein, "limiting" or "limiting development of" means accomplishing one or more of the following in an individual that does not have the disorder to be limited: (a) preventing the disorder; (b) reducing the severity of the disorder; and (c) limiting or preventing development of symptoms characteristic of the disorder.

As used herein, an“amount effective” refers to an amount of the composition that is effective for treating and/or limiting the relevant disorder.

While the methods of the disclosure do not require use of an adjuvant, the methods may further comprise administering an adjuvant for possible additional enhancement of the immune response Any suitable adjuvant can be used, including but not limited to toll-like receptor 4 (TLR4) agonist, a toll-like receptor 7 (TLR7) agonist, a toll-like receptor 8 (TLR8) agonist, a toll-like receptor 9 (TLR9) agonist, alum-containing adjuvant, monophosphoryl lipid A, oil-in-water emulsion, and a-tocopherol, squalene and poiysorbate 80 in an oil-in- water emulsion.

The individual may be any suitable individual, including but not limited to mammals. Preferably the individual is a human. In one embodiment, the individual has a T-cell deficiency and/or a defect in co-stimulation between B cells and T cells, or is immuno compromised by chronic infections or from acute or chronic taking of immunosuppressive drugs for treatment of autoimmune diseases, or other inflammatory disease . In another embodiment, the individual is less than one month old or is elderly (i.e.: at least 65 years old).

In various other embodiments, the individual has a hepatitis B-related disease, such as hepatitis, hepatitis-related disease, fulminant hepatitis, cirrhosis, and/or hepatocellular carcinoma, and the methods are used to treat the a hepatitis B-related disease, such as hepatitis, hepatitis-related disease, fulminant hepatitis, cirrhosis, and/or hepatocellular carcinoma.

Example 1

Generation and characterization of G28-8LH-scAb-PreS1-S2-His recombinant protein molecules.

G28-8LH-scAb-PreS1-S2-His_protein_(expressed)

G28-8 (anti-human CD180)-scAb-PreS1/S2 recombinant protein molecules. The inventors have demonstrated that for the specific anti-CD180 antibody, G28-8, a single chain antibody (scAb) in the form of VLVH-human lgG1 Fc retains both the efficient binding as well as the biological properties of its parent G28-8 IgG. The G28-8LH scAb is used to create G28-8LH-scAb-PreS1-S2-His recombinant protein constructs. It is anticipated that scFv generated from other anti-CD180 antibodies may retain the antibody characteristics in either the VLVH, VHVL, or only the VHVL configuration.

Production of recombinant the G28-8LH-scAb-PreS1-S2-His protein.

Complementary DNAs (cDNAs) encoding the G28-8LH-scAb-PreS1-S2-His recombinant proteins (Fig. 1 , G28-8LH-scAb-PreS1-S2-His protein) were cloned into the mammalian expression vector pTT5 that harbors a CMV promoter to drive protein expression. Transient transfection of these plasmids into Chinese hamster ovary (CHO) cells was done using Lipofectamine™ reagents (Invitrogen Carlsbad, CA) or polyethyleninmine (PEI). Small-scaie transfection optimization using 5%, 20% and 80% ratios of expression plasmid in the lipofection reagent was conducted to identify to optimal plasmid to lipofection reagent ratios for larger scale expression. Once optimized transfection conditions are established, a large- scale transfection will be conducted for each of the plasmids for recombinant protein production. Nickel affinity chromatography, e.g., using the HisPurNi-NTA™ resin (Thermo Fisher Scientific Inc., Rockford IL), was used to purify the recombinant proteins. The cDNA sequences for the G28-8LH-scAb-HBV-PreS1/S2 protein predicts a polypeptide size of ~ 75 kDa. The expressed dimeric form of the recombinant protein is predicted to have a molecular weight of 150 kDa.

Fig. 2 shows the results from a 2-iiter expression run. The plasmid encoding the G28-8LH-scAb-PreS1-S2-Fiis protein was transiently expressed in CHO cells for 8 days. Culture supernatants (~ 2 liters) were collected and cellular debris was removed by centrifugation. Clarified culture supernatants were then loaded on to a column containing HisPurNi-NTA™ resin. After washing the column with the wash buffer (50 mM phosphate buffer, pH 7.0, 300 mM NaCI, 1 mM imidazole), bound recombinant protein was eluted with the elution buffer (50 mM phosphate buffer, pH 7.0, 300 mM NaCI, 150 mM imidazole). Protein containing fractions as monitored by absorbance at 280 nM were collected, pooled, and dialyzed against phosphate-buffer saline at pH 7.0. Purified G28-8LH-scAb-PreS1-S2- His and unbound flow through materials from HisPurNi-NTA™ chromatography was analyzed on SDS-PAGE (4-15% gradient under reducing conditions) stained with

Coomassie blue. Fig. 2, left panel shows a major protein band migrating at the MW of ~85 kDa, suggesting that G28-8LH-scAb-PreS1-S2-His protein was in fact expressed by CHO cells as an intact protein and secreted into the culture supernatants. A duplicate gel was then transferred onto a nylon membrane and immuno-blotted with an anti-6x-His antibody. Intense anti-6xHis signals were only observed at ~85 kDa (Fig. 2, right panel), at the identical MW G28-8LH-scAb-PreS1-S2-His migrated to on the Coomassie blue stained gel (Fig. 2, left panel).

Example 2. Characterization of G28-8LH-scAb-PreS1-S2-His

Fig. 3 shows that human CD20+ tonsillar B cells (10 ⁶ ) were incubated in 96 well round bottom plates with PBSA (PBS w / 0.2% BSA + 0.2% NaN3) media only (gray) or with the His tagged recombinant protein containing the light and heavy chains of G28-8 antihuman CD180 (LH) G28-8LH-scAb-PreS1 -S2-His (black), at 10 μg/ml. After a 40 min incubation on ice, the cells were washed twice (centrifuged at 1200 rpm, 4 min). Then 100 μI of PBSA + 5 μI a fluorescein (FITC)-conjugated anti 6XHis (FITC-6x-His epitope tag ThermoScientific MA1 -81891 ) were added to the wells, and after a 40 min incubation on ice, cells were washed twice and the level of fluorescence measured by flow cytometry shown on abscissa (log scale). Unstained cells are shown in black. The recombinant protein bound to B cells as shown by binding being above the FITC control, demonstrating binding to CD180 expressed on B cells.

Ligation of CD180 on B cells has been shown to upregulate CD40 expression ⁵¹ . The ability of G28-8LH-scAb-PreS1-S2-His to upregulate CD40 expression was then tested to evaluate its functional activity (Fig. 4). Er- blood mononuclear cells enriched for B cells were incubated for 24 hrs at 37C with either media (gray line) or 10 μg/ml of G28-8LH-scAb-HBV- PreS1/S2-His (black line). Samples were washed twice with PBSA, stained with mAb specific for CD20 (Pacific Blue Biolegend™) and CD40 (FITC BD BioSciences) and evaluated for CD40 and CD20 expression using flow cytometry. Graph shows CD40 expression of gated CD20 ⁺ B cells. G28-8LH-scAb-PreS1-S2-His upregulated CD40 expression, confirming that G28-8LH-scAb-PreS1-S2-His was functionally active (Fig. 4).

Example 3.

Induction in macaques of preS1-S2-specific IgG antibody responses by G28-8LH- scAb-PreS1-S2-His recombinant protein

The ability of G28-8LH-scAb-PreS1-S2-His to induce humoral and cellular immune responses was examined in a vaccination experiment in cynomolgus macaques ( Macaca fascicularis ). Groups of macaques (N=3) were vaccinated subcutaneously with either: 1 ) 300 μg of G28-8LH-scAb-PreS1 -S2-His (αCD180-HBV-PreS1/S2) in 1 ml; or 2) 300 μg of G28- 8LH-scAb-PreS1-S2-His co-formulated with 100 μg of the commercial adjuvant AddaVax™ (InVivoGen, San Diego, CA) in a total of 1 ml. Animals were vaccinated on days 0 and 30 and serum and heparinized blood samples were obtained on days 0, 7, 14, 30 (time-points after first dose), 37, 44 and 60 (time-points after second dose). Serum samples were assessed for IgG antibody responses to HBV preS1 by ELISA as follows: a) coating 96 well plates with 200 ng/well purified recombinant preS1 peptide (115 amino acids, Cosmo Bio. Japan cat# BCL-AGS1 -01 ); b) adding serial dilutions of serum samples (100 μI diluted in TBS + 0.05% tween-20) starting with a 1 :1000 dilution, followed by washing and adding HRP-anti-macaque IgG second step (Rockland, 1 :5000 dilution). Both groups produced IgG after immunizations (Fig. 5A). The antibody titers increased after each boost. The group receiving recombinant protein with AddaVax™ adjuvant had higher IgG antibody responses compared to group not given AddaVax™ at two time points after the second immunization.

Example 4.

Induction in macaques of HBV-specific T cell responses by G28-8LH-scAb-PreS1-S2- His

To determine the frequency of HBV-PreS1/S2-specific, intracellular cytokine- producing T cells after vaccination of the macaques, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples obtained from immunized macaques at the times before and after immunization as noted in Example 3. PBMCs were separated using gradient centrifugation and stimulated in vitro for 18 hours with HBsAg peptide pools (15mers overlapping by 11 amino acids). HBs-specific T cells secreting IFN-γ were detected using paired anti-macaque IFN-g monoclonal antibodies (U-cytech-BV). Spot forming cells (SFC) were enumerated using an Immunospot™ Analyzer with CTL

Immunospot™ Profession Software (Cellular Technology Ltd.). Results shown in Figure 5B are mean SFC per 1 million PBMC time point ± SEM. Tests were run in replicate wells. Net responses shown were determined by subtracting the number of spots from DMSO stimulated control wells from the same animal. Statistical comparisons between the two groups for each assay were assessed at each time point using unpaired t test on samples with equal standard deviation and resuspended in growth media at defined concentrations (~1.2 million cells/condition). The number of HBsAg-specific IFN-y-producing T cells increased within 14 days after primary immunization with G28-8LH-scAb-PreS1-S2-His only and the addition of the AddaVax™ adjuvant did not increase HBsAg-specific T cell levels.

Example 5.

Induction in macaques of HBV-specific neutralizing antibodies (Abs) by G28-8LH- scAb-PreS1 -S2-His

To determine the frequency neutralizing antibodies (Abs) to HBV after vaccination of the macaques, sera were from macaques before (pre-bieed) and 14 days after the second immunization as noted in Example 3. HBV particles were obtained from the culture supernatants of HepAD38 ceils as an HBV-productive ceil line. For HBV infection, HepG2 liver cells expressing the NTCP receptor for HBV (HepG2-hNTCP cells) were seeded in 60- mm dishes or 6-well plates coated with collagen type 1. After one day, cells were inoculated with HBV virions at 10 ³ genome equivalents (Geq) per cell in completed DMEM containing 4% polyethylene glycol (PEG) 8000 for 16h. Then, cells were maintained in completed DMEM containing 2.5% DMSO for additional days. For the virus neutralizing experiment, the sera being tested for neutralizing Abs were added during the inoculation (16h) as indicated in Fig. 6A.

For analysis of HBV cccDNAs, viral cccDNAs were isolated with the Hirt Extraction Method, for protein-free DNA extraction from HBV-infected cells. Briefly, cells from 60-mm dishes were lysed in 1 ml of lysis buffer containing 50 mM Tris-HCI (pH 7.4), 150 mM NaCI, 10 mM EDTA, and 1% SDS. After 1 h of incubation at room temperature, the lysates were transferred into a 2-mi tube, and this step was followed by the addition of 0.25 ml of 2.5 M KCI, then incubation at 4°C overnight. The lysates were clarified by centrifugation and extracted with phenol-chloroform. DNA was precipitated with isopropanol overnight and dissolved in Nuclease-free water. The extracted DNA was treated with Plasmid-Safe ATP- dependent Dnase for southern blot analysis or T5 exonuclease for real-time PCR. For realtime PCR, total DNAs were purified from infected cells using DNeasy™ Blood & Tissue Kit (Qiagen). cccDNA levels were expressed as a normalized ratio to mitochondrial DNA, and cccDNA were detected using specific PCR primers.

The sera tested for neutralizing Ab activity included the pre-bleed controls (1 :1000 dilution) for each animal and sera obtained from immunized macaques day 14 after a second immunization. The immune sera were tested for neutralizing Ab activity at either a 1 :300 dilution (D), a 1 :1000 D or a 1 :3000 D. Three sera from immunized macaques (day 14 boost) had neutralizing Ab activity that prevented HBV from expressing cccDNA in hepatocytes in vitro, two animals from group 1 , and one from group 2 (Fig. 6B). Group 1 : G28-8LH-scAb-PreS1-S2-His (300 ug) Monkey ID: A16157, A16159, A16160. Group 2: G28-8LH-scAb-PreS1 -S2-His (300 ug) + AddaVax (100 μg) Monkey ID: A16155, A16158, A16168

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