DC-TARGETING VACCINE AGAINST HBV INFECTION FIELD OF THE INVENTION: The present invention is in the field of medicine, in particular virology and vaccinology. BACKGROUND OF THE INVENTION: Hepatitis B virus (HBV) is a DNA virus that belongs to the Hepadnaviridae family and causes acute and chronic liver infections in humans and animals. HBV has a circular, partially double- stranded DNA genome of about 3.2 kilobases, which encodes four main proteins: the core protein (C), the envelope proteins (L, M and S), the polymerase (P) and the X protein (X). HBV replicates by reverse transcription of a pregenomic RNA intermediate, which is encapsidated by the core protein and converted into the relaxed circular DNA (rcDNA) form by the polymerase. The rcDNA is then either recycled into new virions or transported to the nucleus, where it is converted into the covalently closed circular DNA (cccDNA) form. The cccDNA serves as the template for the transcription of all viral RNAs and is responsible for the persistence of HBV infection. HBV is a serious global health problem that affects more than 257 million people who have chronic infections and causes more than 887,000 deaths every year. In the EU and EEA, there are 4.7 million people who have HBV. W.H.O. reports that HBV is responsible for almost 40% of the cases of hepatocarcinoma (HCC), which is the second most common cause of death from cancer worldwide. HBV claims the lives of almost 900,000 people globally each year. The currently available treatments do not eliminate infection; they include nucleoside analogues (NUC) which only suppress viral replication and Interferon alpha (IFN) which induces a sustained off-treatment viral suppression in only a minority of patients. In total, among the treated patients observed for 5 years, only 10% of patients lose HBsAg and reach the endpoint of functional cure that allows treatment cessation. These treatments are not curative largely due to: (i) the failure of chronically infected patients to mount an immune response that is sufficiently robust, functional and sustained to clear the infection; and (ii) the persistence of the viral covalently closed circular DNA (cccDNA) transcriptional template in infected hepatocytes. Therefore, most patients need to be treated for life. Finding new cure methods that require shorter periods of treatment would definitely enhance the care of patients and the
prevention of HBV-related liver disease complications and help more patients in the countries where HBV is very common and lifelong therapies and their monitoring are unaffordable. The development of curative vaccines is thus very much needed. SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to antibodies specific for CD40 wherein the heavy chain and/or the light chain is fused to one or more antigenic polypeptide(s) that derive(s) from the protein C, protein S and/or protein P of HBV. DETAILED DESCRIPTION OF THE INVENTION: Definitions: As used herein, the term "subject" or "subject in need thereof", is intended for a human or non-human mammal. Typically the patient is affected or likely to be infected with HBV. As used herein, the term "HBV" refers to hepatitis B virus, a member of the Hepadnaviridae family of viruses that causes chronic and acute infections of the liver. The HBV genome is a circular, partially double-stranded DNA molecule that encodes four overlapping proteins: the core protein (C), the envelope proteins (L, M, and S), the polymerase (P), and the X protein (X). The HBV life cycle involves reverse transcription of the viral genome by the P protein and integration of a covalently closed circular DNA (cccDNA) intermediate into the host cell nucleus. The cccDNA serves as a template for the transcription of viral mRNA and pregenomic RNA, which are then exported to the cytoplasm for translation and replication, respectively. The HBV virions are enveloped particles that contain the nucleocapsid, composed of the C protein and the DNA genome, and the envelope, composed of the L, M, and S proteins. The HBV infection is transmitted through exposure to infected blood or body fluids, and can cause severe liver damage, cirrhosis, and hepatocellular carcinoma. Based on phylogenetic analysis, at least ten genotypes of HBV have been identified, designated as A to J. The HBV genotypes have distinct geographical distributions and are associated with different clinical outcomes, such as disease progression, response to antiviral therapy, and risk of hepatocellular carcinoma. The HBV genotypes also differ in their core C protein sequences, which may affect the antigenicity, stability, and assembly of the nucleocapsid.
As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. As used herein, the term "C protein" refers to the hepatitis B virus core protein, also known as the capsid protein or nucleocapsid protein. The core C protein is a 21-kDa protein that forms the icosahedral nucleocapsid of the HBV virion, enclosing the viral DNA and polymerase. The core C protein also has multiple functions in the viral replication cycle, such as pregenomic RNA encapsidation, reverse transcription, cccDNA formation, and nuclear transport. The core C protein consists of two domains: an N-terminal assembly domain that mediates capsid formation and an C-terminal domain that contains the RNA-binding and DNA-binding motifs. The core C protein can also be processed by cellular proteases to generate a smaller protein called the e antigen (HBeAg), which is secreted into the serum and serves as a marker of viral replication and infectivity. An exemplary amino acid sequence for the core C protein is represented by SEQ ID NO:1.
MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLA TWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVAFGVWIRTPPAYRPPNAP ILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC As used herein, the term "S protein" refers to the hepatitis B virus small envelope protein, also known as the surface antigen or HBsAg. The S protein is a 24-kDa protein that forms the outermost layer of the HBV virion, composed of three transmembrane domains and two extracellular loops. The S protein mediates the attachment and entry of the virus into hepatocytes by binding to cellular receptors, such as sodium-taurocholate co-transporting polypeptide (NTCP) and heparan sulfate proteoglycans (HSPGs). The S protein is also the main target of the host immune response and the basis for vaccination against HBV infection. The S protein can be classified into four major subtypes (adw, adr, ayr, and ayw) and several minor subtypes based on the amino acid variations in the extracellular loops, which may affect the
antigenicity, immunogenicity, and pathogenicity of the virus. An exemplary amino acid sequence for the S protein is represented by SEQ ID NO:2.
MGQNLSTSNPLGFFPDHQLDPASRANTANPDWDFNPNKDTWPDANKDGAGAFGLGLTPPHGGLLGWSPQ AQGILHTVPANPPPASTNRQTGRQPTPLSPPLRDTHPQAVQWNSTTFHQTLQDPRVRGLYFPAGGSSSG TVNPVPTTASPLSSIFSRIGDPVTNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFRGG TTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRGFIIFLFILLLCLIFLLVLLEYQGMLHVCPLIPGT TTTSTGPCKTCTTPAQGNSMFPSCCCTKTSDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQW FVGLSPTVWLSAIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI As used herein, the term "P protein" refers to the hepatitis B virus polymerase protein, also known as the reverse transcriptase or DNA polymerase. The P protein is a 90-kDa protein that catalyzes the synthesis of viral DNA from the pregenomic RNA template within the nucleocapsid. The P protein has four domains: a terminal protein domain that primes the DNA synthesis, a spacer domain that connects the terminal protein and the reverse transcriptase domains, a reverse transcriptase domain that has both RNA-dependent and DNA-dependent DNA polymerase activities, and an RNase H domain that degrades the RNA strand of the RNA- DNA hybrid. The P protein is essential for the replication of HBV and is a major target of antiviral drugs. An exemplary amino acid sequence for the P protein is represented by SEQ ID NO:3.
MPLSYQHFRRLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIPWTHKVGNFTGFYSSTVPV FNPHWETPSFPNIHLHQDIIKKCEQFVGPLTVNEKRRLQLIMPARFYPKVTKYLPLDKGIKPYYPEHLV NHYFQTRHYLHTLWKAGILYKRETTHSASFCGSPYSWEQDLQHGAESIHQQSSGILSRPPVGSSLQSKH RKSRLGLQSQQGHLARRQQGWSWSIRAGTHPTARRPFGVEPSGSGHTTHRASKSASCLYQSPDRKATYP SVSTFERHSSSGRAVELHNFPPNSARSQSERPIFPCWWLQFRNSKPCSDYCLSLIVNLLEDWGPCDEYG EHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGNYRVSWPKFAVPNLQSLTNLLSSNLSW LSLDVSAGFYHLPLHPAAMPHLLVGSSGVSRYVARLSSNSRNNNNQYGTMQNLHDSCSRQLYVSLMLLY QNFGWKLHLYSHPIVLGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMDDVVLGAKSVQHL ESLFTAVTNFLLSLGIHLNPNKTKRWGYSLHFMGYVIGCYGSLPQEHIIQKIKECFRKVPVNRPIDWKV CQRIVGLLGFAAPFTQCGYPALMPLYACIQFKQAFTFSPTYKAFLCKQYLNLYPVARQRPGLCQVFADA TPTGWGLGMGHQRMRGTFSAPLPIHTAELLAACFARSRSGANILGTDNSVVLSRKYTSFPWLLGCAANW ILRGTSFVYVPSALNPADDPSRGRLGLSRPLLCLPFRPTTGRTSLYADSPSVPSHLPDRVHFASPLHVA WRPP As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the
nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one). As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase “nucleotide sequence that encodes a protein or a RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
As used herein, the term “promoter/regulatory sequence” refers to a polynucleotide sequence (such as, for example, a DNA sequence) recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence, thereby allowing the expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. As used herein, the term "operably linked" or "transcriptional control" refers to functional linkage between a regulatory sequence and a heterologous polynucleotide sequence resulting in expression of the latter. For example, a first polynucleotide sequence is operably linked with a second polynucleotide sequence when the first polynucleotide sequence is placed in a functional relationship with the second polynucleotide sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame. As used herein, the term "transformation" means the introduction of a "foreign" (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA bas been "transformed". As used herein, the term "expression system" means a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can
be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology.48 (3): 443–53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or polynucleotide sequence are identical irrespective of any chemical and/or biological modification. According to the invention a first amino acid sequence having at least 80% of identity with a second amino acid sequence means that the first sequence has 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. As used herein, the term “fusion protein" indicates a protein created through the attaching of two or more polypeptides which originated from separate proteins. In particular fusion proteins can be created by recombinant DNA technology and are typically used in biological research or therapeutics. Fusion proteins can also be created through chemical covalent conjugation with or without a linker between the polypeptides portion of the fusion proteins. In the fusion protein the two or more polypeptide are fused directly or via a linker. As used herein, the term "directly" means that the first amino acid at the N-terminal end of a first polypeptide is fused to the last amino acid at the C-terminal end of a second polypeptide. This direct fusion can occur naturally as described in (Vigneron et al., Science 2004, PMID 15001714), (Warren et al., Science 2006, PMID 16960008), (Berkers et al., J. Immunol.2015a, PMID 26401000), (Berkers et al., J. Immunol.2015b, PMID 26401003), (Delong et al., Science 2016, PMID 26912858) (Liepe et al., Science 2016, PMID 27846572), (Babon et al., Nat. Med. 2016, PMID 27798614).
As used herein, the term “linker” has its general meaning in the art and refers to an amino acid sequence of a length sufficient to ensure that the proteins form proper secondary and tertiary structures. In some embodiments, the linker is a peptidic linker which comprises at least one, but less than 30 amino acids e.g., a peptidic linker of 2-30 amino acids, preferably of 10-30 amino acids, more preferably of 15-30 amino acids, still more preferably of 19-27 amino acids, most preferably of 20-26 amino acids. In some embodiments, the linker has 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30 amino acid residues. Typically, linkers are those which allow the compound to adopt a proper conformation. The most suitable linker sequences (1) will adopt a flexible extended conformation, (2) will not exhibit a propensity for developing ordered secondary structure which could interact with the functional domains of fusion proteins, and (3) will have minimal hydrophobic or charged character which could promote interaction with the functional protein domains. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. In natural antibodies of rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. In typical IgG antibodies, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans- placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate in the antibody binding site, or influence the overall
domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H- CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDRs set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. Accordingly, the variable regions of the light and heavy chains typically comprise 4 framework regions and 3 CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereafter “Kabat et al.”). The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31- 35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. For the agonist antibodies described hereafter, the CDRs have been determined using CDR finding algorithms from www.bioinf.org.uk - see the section entitled « How to identify the CDRs by looking at a sequence » within the Antibodies pages. As used herein, the term “immunoglobulin domain” refers to a globular region of an antibody chain (such as e.g., a chain of a heavy chain antibody or a light chain), or to a polypeptide that essentially consists of such a globular region. As used herein, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residue
from position C226 or from P230 to the carboxyl-terminus of the IgG antibody. The numbering of residues in the Fc region is that of the EU index of Kabat. The C-terminal lysine (residue K447) of the Fc region may be removed, for example, during production or purification of the antibody. Accordingly, a composition of antibodies of the invention may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. As used herein, the term "chimeric antibody" refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody. In one embodiment, a “chimeric antibody” is an antibody molecule in which (a) the constant region (i.e., the heavy and/or light chain), or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, an agonist molecule, e.g., CD40 Ligand, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. Chimeric antibodies also include primatized and in particular humanized antibodies. Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593- 596 (1992). (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). As used herein, the term “humanized antibody” includes antibodies which have the 6 CDRs of a murine antibody, but humanized framework and constant regions. More specifically, the term "humanized antibody", as used herein, may include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. As used herein the term "human monoclonal antibody", is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by
human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, in one embodiment, the term "human monoclonal antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. As used herein, the term “immune response” refers to a reaction of the immune system to an antigen in the body of a host, which includes generation of an antigen-specific antibody and/or cellular cytotoxic response. The immune response to an initial antigenic exposure (primary immune response) is typically, detectable after a lag period of from several days to two weeks; the immune response to subsequent stimulus (secondary immune response) by the same antigen is more rapid than in the case of the primary immune response. An immune response to a transgene product may include both humoral (e.g., antibody response) and cellular (e.g., cytolytic T cell response) immune responses that may be elicited to an immunogenic product encoded by the transgene. The level of the immune response can be measured by methods known in the art (e.g., by measuring antibody titre). As used herein the term “APCs” or "Antigen Presenting Cells" denotes cells that are capable of activating T-cells, and include, but are not limited to, certain macrophages, B cells and dendritic cells. As used herein, the term "Dendritic cells" or “DCs” refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology, high levels of surface MHC-class II expression (Steinman, et al., Ann. Rev. Immunol. 9:271 (1991); incorporated herein by reference for its description of such cells). As used herein, the term “CD40” has its general meaning in the art and refers to human CD40 polypeptide receptor. In some embodiments, CD40 is the isoform of the human canonical sequence as reported by UniProtKB-P25942 (also referred as human TNR5). As used herein, the term “CD40 agonist antibody” refers to antibody that binds and activates the CD40 receptor, thereby enhancing antigen presentation, cytokine production and immune
cell activation. By binding to FcReceptor, an agonistic antibody activates bystander cells through Fc clustering leading to cytokines release. As used herein, the term “CD40 partial agonist antibody” refers to antibody that binds CD40 receptor and triggers partial activation of CD40 signalling, thereby enhancing antigen presentation, cytokine production and immune cell activation but without inducing the full range of downstream effects that might lead to excessive inflammation or toxicity. The IgG4 Fc portion of the partial agonist limits FcR binding and clustering and off-target effect. A partial agonist antibody by its binding to CD40 is in competition with the CD40 ligand expressed by surrounding cells and non specific activation and cytokine release. As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or
treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]). As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and/or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. As used herein, the term “vaccination” or “vaccinating” means, but is not limited to, a process to elicit an immune response in a subject against a particular antigen. As used herein, the term "vaccine composition" is intended to mean a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in the activation of certain cells, in particular APCs, T lymphocytes and B lymphocytes. As used herein, the term “antigenic polypeptide” has its general meaning in the art and generally refers to a polypeptide that is capable of being specifically bound by an antibody or by a T cell receptor (TCR) if processed and presented by MHC molecules. An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. An antigen can have one or more epitopes or antigenic sites (B- and T- epitopes). As used herein, the term “epitope” has its general meaning in the art and a fragment of at least 8 amino acids that is recognized by an immune response component.
As used herein, the term “adjuvant” refers to a compound that can induce and/or enhance the immune response against an antigen when administered to a subject or an animal. It is also intended to mean a substance that acts generally to accelerate, prolong, or enhance the quality of specific immune responses to a specific antigen. In the context of the present invention, the term "adjuvant" means a compound, which enhances both innate immune response by affecting the transient reaction of the innate immune response and the more long-lived effects of the adaptive immune response by activation and maturation of the antigen-presenting cells (APCs) especially Dendritic cells (DCs). As used herein, the expression "therapeutically effective amount" is meant a sufficient amount of the active ingredient of the present invention to induce an immune response at a reasonable benefit/risk ratio applicable to the medical treatment. Antibodies of the present invention: The first object of the present invention relates to an antibody that is specific for CD40 wherein the heavy chain and/or the light chain is fused to a one or more antigenic polypeptide(s) that derive(s) from the protein C, protein S and/or protein P of HBV. In some embodiments, the antibody specific for CD40 is a agonist antibody and more particularly a partial agonist antibody. In some embodiments, the antibody is an IgG antibody, preferably of an IgG1 or IgG4 antibody, or even more preferably of an IgG4 antibody. In some embodiments, the antibody is a chimeric antibody, in particular a chimeric mouse/human antibody. In some embodiments, the antibody is humanized antibody. Chimeric or humanized antibodies can be prepared based on the sequence of a murine monoclonal antibody prepared as described above. DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, the murine variable regions can be linked to human constant regions using methods known in the art (see e.g., U.S. Patent No. 4,816,567 to Cabilly et al.). To create a humanized antibody, the murine CDR regions can be
inserted into a human framework using methods known in the art. See e.g., U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al. In some embodiments, the antibody is a human antibody. In some embodiments, human antibodies can be identified using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice." The HuMAb mouse® (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode un-rearranged human heavy (µ and γ) and ĸ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous µ and ĸ chain loci (see e.g., Lonberg, et al., 1994 Nature 368(6474): 856-859). In another embodiment, human antibodies can be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM mice", are described in detail in PCT Publication WO 02/43478 to Ishida et al. In some embodiments, the anti-CD40 antibody derives from the 12E12 antibody and comprises: - a heavy chain comprising the complementarity determining regions CDR1H, CDR2H and CDR3H, the CDR1H having the amino acid sequence GFTFSDYYMY (SEQ ID NO:4), the CDR2H having the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO:5), and the CDR3H having the amino acid sequence RGLPFHAMDY (SEQ ID NO:6), - and a light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L, the CDR1L having the amino acid sequence SASQGISNYLN (SEQ ID NO:7) the CDR2L having the amino acid sequence YTSILHS (SEQ ID NO:8) and the CDR3L having the amino acid sequence QQFNKLPPT (SEQ ID NO:9). In some embodiments, the anti-CD40 antibody derives from the 11B6 antibody and comprises: - a heavy chain comprising the complementarity determining regions CDR1H, CDR2H and CDR3H, the CDR1H having the amino acid sequence GYSFTGYYMH (SEQ ID NO:10), the CDR2H having the amino acid sequence RINPYNGATSYNQNFKD
(SEQ ID NO:11), and the CDR3H having the amino acid sequence EDYVY (SEQ ID NO:12), and - a light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L, the CDR1L having the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO:13) the CDR2L having the amino acid sequence KVSNRFS (SEQ ID NO:14) and the CDR3L having the amino acid sequence SQSTHVPWT (SEQ ID NO:15). In some embodiments, the anti-CD40 antibody derives from the 12B4 antibody and comprises: - a heavy chain comprising the complementarity determining regions CDR1H, CDR2H and CDR3H, the CDR1H having the amino acid sequence GYTFTDYVLH (SEQ ID NO:16), the CDR2H having the amino acid sequence YINPYNDGTKYNEKFKG (SEQ ID NO:17), and the CDR3H having the amino acid sequence GYPAYSGYAMDY (SEQ ID NO:18), and - a light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L, the CDR1L having the amino acid sequence RASQDISNYLN (SEQ ID NO:19) the CDR2L having the amino acid sequence YTSRLHS (SEQ ID NO:20) and the CDR3L having the amino acid sequence HHGNTLPWT (SEQ ID NO:21). In some embodiments, the agonist or partial agonist anti-CD40 antibody derives from the 12E12 antibody, 11B6 antibody or 12B4 antibody as described above. In some embodiments, the anti-CD40 antibody is selected from the group consisting of selected mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6 as described in Table A. In some embodiments, the agonist or partial agonist anti-CD40 antibody is selected from the group consisting of selected mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6 as described in Table A. mAb1 [11B6 SEQ ID NO:22 SEQ ID NO:23 VH/VkV2] mAb2 SEQ ID NO:24 SEQ ID NO:23
[11B6 VHV3/VkV2] mAb3 SEQ ID NO:25 SEQ ID NO:26 [12B4] mAb4 SEQ ID NO:27 SEQ ID NO:28 [24A3] mAb5 SEQ ID NO:29 SEQ ID NO:30 [CP870,893] mAb 6 SEQ ID NO:31 SEQ ID NO:32 [12E12] Table A: CD40 antibodies SEQ ID NO:22 (Amino acid sequence of variable heavy chain region (VH) (v2) of Humanized 11B6) EVQLVQSGAEVKKPGASVKISCKASGYSFTGYYMHWVKQAHGQGLEWIGRINPYNGATSYNQNFKDRAT LTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVTVSSAS SEQ ID NO:23 (Amino acid sequence of variable light chain (VL) Vk (v2) of humanized 11B6 VL) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSG SGSGTDFTLKISRVEAEDVGVYFCSQSTHVPWTFGGGTK SEQ ID NO:24 (Amino acid sequence of variable heavy chain region VH (v3) of humanized 11B6) EVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYMHWVRQAPGQGLEWIGRINPYNGATSYNQNFKDRVT LTVDKSTSTAYMELSSLRSEDTAVYYCAREDYVYWGQGTTVTVSSAS SEQ ID NO:25 (VH amino acid sequence of mAb3 (12B4)) EVQLQQSGPELVKPGASVKMSCKASGYTFTDYVLHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKAT LTSDKSSSTAYMELSSLTSEDSAVYYCARGYPAYSGYAMDYWGQGTSVTVSSAS SEQ ID NO:26 (VL amino acid sequence of mAb3 (12B4)) DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGT DYSLTISNLEQEDIATYFCHHGNTLPWTFGGGTK SEQ ID NO:27 (VH amino acid sequence of mAb4 (24A3 HC)) DVQLQESGPDLVKPSQSLSLTCTVTGYSITSDYSWHWIRQFPGNKLEWMGYIYYSGSTNYNPSLKSRIS ITRDTSKNQFFLQLNSVTTEDSATYFCARFYYGYSFFDYWGQGTTLTVSSAS SEQ ID NO:28 (VL amino acid sequence of mAb4 (24A3 KC)) QIVLTQSPAFMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTS YSLTISSMEAEDAATYYCQQWSSNPLTFGAGTK SEQ ID NO:29 (VH amino acid sequence of mAb5) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVT MTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSAS SEQ ID NO:30 (VL amino acid sequence of mAb5) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQANIFPLTFGGGTK
SEQ ID NO:31 (VH amino acid sequence of mAb6 (12E12 H3 Humanized HC)) EVQLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFT ISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSAS SEQ ID NO:32 (VL amino acid sequence of mAb6 (Humanized K212E12)) DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTK In some embodiments, the antibody of the present invention comprises the heavy chain having the amino acid sequence as set forth in SEQ ID NO:33 and the light chain having the amino acid sequence as set forth in SEQ ID NO:34. SEQ ID NO:33 > hAnti-CD40VH3-LV-hIgG4H EVQLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFT ISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:34 > hAnti-CD40VK2-LV-hIgGK DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC The one or more antigenic polypeptide(s) that derive(s) from the protein C, protein S and/or protein P of HBV can be derived from any genotype of HBV, such as genotype A, B, C, D, E, F, G or H. Preferably, the one or more antigenic polypeptide(s) are derived from genotype D of HBV, which is the most prevalent and virulent genotype worldwide. The antigenic polypeptide(s) can comprise one or more epitopes that are recognized by T cells and/or B cells, and can induce a humoral and/or cellular immune response against HBV. In some embodiments, the one or more antigenic polypeptide(s) that derive(s) from the protein C of HBV can be selected from two regions of the protein that are highly immunogenic and conserved among different genotypes. These regions correspond to amino acid residues 426- 498 and 512-589 of the protein C, as shown in SEQ ID NO:1. The antigenic polypeptide(s) can comprise the entire region or a fragment thereof, as long as they retain the ability to elicit an immune response against HBV. In some embodiments, the antigenic polypeptide comprises an amino acid sequence (“C3”) having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 426 to the amino acid residue at position 498 in
SEQ ID NO:1. In some embodiments, the antigenic polypeptide comprises an amino acid sequence (“C4”) having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 512 to the amino acid residue at position 589 in SEQ ID NO:1. In some embodiments, the one or more antigenic polypeptide(s) that derive(s) from the protein S of HBV can be selected from two regions of the protein that are highly immunogenic and conserved among different genotypes. These regions correspond to amino acid residues 1-35 and 288-340 of the protein S, as shown in SEQ ID NO:2. The antigenic polypeptide(s) can comprise the entire region or a fragment thereof, as long as they retain the ability to elicit an immune response against HBV. In some embodiments, the antigenic polypeptide comprises an amino acid sequence (“S1”) having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 35 in SEQ ID NO:2. In some embodiments, the antigenic polypeptide comprises an amino acid sequence (“S2”) having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 288 to the amino acid residue at position 340 in SEQ ID NO:2. In some embodiments, the one or more antigenic polypeptide(s) that derive(s) from the protein P of HBV can be selected from two regions of the protein that are highly immunogenic and conserved among different genotypes. These regions correspond to amino acid residues 1-42 and 65-124 of the protein P, as shown in SEQ ID NO:3. The antigenic polypeptide(s) can comprise the entire region or a fragment thereof, as long as they retain the ability to elicit an immune response against HBV. In some embodiments, the antigenic polypeptide comprises an amino acid sequence (“P5”) having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 42 in SEQ ID NO:3. In some embodiments, the antigenic polypeptide comprises an amino acid sequence (“P6”) having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 65 to the amino acid residue at position 124 in SEQ ID NO:3. In some embodiments, the antigenic polypeptide(s) can be fused or linked to each other, or to the heavy chain and/or light chain of the antibody, by a linker sequence. The linker sequence can be any suitable amino acid sequence that does not interfere with the function of the antibody or the antigenic polypeptide(s). In some embodiments, the linker is selected from the group consisting of FlexV1, f1, f2, f3, or f4 as described below.
QTPTNTISVTPTNNSTPTNNSNPKPNP (flexV1, SEQ ID NO:35) SSVSPTTSVHPTPTSVPPTPTKSSP (f1, SEQ ID NO:36) PTSTPADSSTITPTATPTATPTIKG (f2, SEQ ID NO:37) TVTPTATATPSAIVTTITPTATTKP (f3, SEQ ID NO:38) TNGSITVAATAPTVTPTVNATPSAA (f4, SEQ ID NO:39) In some embodiments, the antibody of the present invention comprises a light chain that is fused to one or more antigenic polypeptides that derive(s) from the protein C of HBV. In some embodiments, the antibody of the present invention comprises a light chain that is fused to a first antigenic polypeptide (“C3”) comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 426 to the amino acid residue at position 498 in SEQ ID NO:1 and/or to a second antigenic polypeptide (“C4”) comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 512 to the amino acid residue at position 589 in SEQ ID NO:1. In some embodiments, the antibody of the present invention comprises a light chain that is used to a polypeptide having the formula of [C3-f2- C4] wherein: - C3 denotes the antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 426 to the amino acid residue at position 498 in SEQ ID NO:1, - f2 denotes the linker having the amino acid sequence as set forth in SEQ ID NO:37, and - C4 denotes the antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 512 to the amino acid residue at position 589 in SEQ ID NO:1. In some embodiments, the antibody of the present invention comprises a light chain having the general formula of [hAnti-CD40VK2-LV-hIgGK-C3-f2-C4] wherein: - hAnti-CD40VK2-LV-hIgGK denotes the light chain having the amino acid sequence as set forth in SEQ ID NO:34 - C3 denotes the antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 426 to the amino acid residue at position 498 in SEQ ID NO:1,
- f2 denotes the linker having the amino acid sequence as set forth in SEQ ID NO:37, and - C4 denotes the antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 512 to the amino acid residue at position 589 in SEQ ID NO:1. In some embodiments, the antibody of the present comprises a light chain having the amino acid sequence as set forth in SEQ ID NO:40. SEQ ID NO:40> L-chain: vc116 - rAB-cetHS-puro[hAnti-CD40VK2-LV-hIgGK- C-HBV_Cpep3-f2-HBV_Cpep4] DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECARMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALTSPTSTPADSSTITPTAT PTATPTIKGLVTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVAFGV In some embodiments, the antibody of the present invention comprises a heavy chain that is fused to a first antigenic polypeptide (“S1”) comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 35 in SEQ ID NO:2 and/or to a second antigenic polypeptide (“S2”) comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 288 to the amino acid residue at position 340 in SEQ ID NO:2. In some embodiments, the antibody of the present invention comprises a heavy chain that is fused to a polypeptide having the formula of [S1-f1-S2] wherein: - S1 denotes the antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 35 in SEQ ID NO:2, - f1 denotes the linker having the amino acid sequence as set forth in SEQ ID NO:36, and, - S2 denotes the antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 288 to the amino acid residue at position 340 in SEQ ID NO:2.
In some embodiments, the antibody of the present invention comprises a heavy chain having the formula of [hAnti-CD40VH3-LV-hIgG4H-Flexv1-S1-f1-S2] wherein: - hAnti-CD40VH3-LV-hIgG4H denotes the heavy chain having the amino acid sequence as set forth in SEQ ID NO:33 - FlexV1 denotes the linker having the amino acid sequence as set forth in SEQ ID NO:35, - S1 denotes the antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 35 in SEQ ID NO:2, - f1 denotes the linker having the amino acid sequence as set forth in SEQ ID NO:36, and, - S2 denotes the antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 288 to the amino acid residue at position 340 in SEQ ID NO:2. In some embodiments, the antibody of the present invention comprises a heavy chain having the amino acid sequence as set forth in SEQ ID NO:41. SEQ ID NO:41 [hAnti-CD40VH3-LV-hIgG4H-C-Flex-v1-HBV_Spep1-f1-HBVSpep2] EVQLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFT ISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPARMGQNLS TSNPLGFFPDHQLDPASRANTANPDWDFNTSSSVSPTTSVHPTPTSVPPTPTKSSPTTPAQGNSMFPSC CCTKTSDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLV In some embodiments, the antibody of the present invention comprises a light chain that is fused to a first antigenic polypeptide (“P5”) comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 42 in SEQ ID NO:3 and/or to a second antigenic polypeptide (“P6”) comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 65 to the amino acid residue at position 124 in SEQ ID NO:3.
In some embodiments, the antibody of the present invention comprises a light chain that is fused to a polypeptide having the general formula of [P5-f3-P6] wherein: - P5 denotes the antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 42 in SEQ ID NO:3, - f3 denotes the linker having the amino as set forth in SEQ ID NO:38, and - P6 denotes the second antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 65 to the amino acid residue at position 124 in SEQ ID NO:3. In some embodiments, the antibody of the present invention comprises a light chain having the general formula of [hAnti-CD40VK2-LV-hIgGK-P5-f3-P6] wherein: - hAnti-CD40VK2-LV-hIgGK denotes the light chain having the amino acid sequence as set forth in SEQ ID NO:34, - P5 denotes the antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 1 to the amino acid residue at position 42 in SEQ ID NO:3, - f3 denotes the linker having the amino as set forth in SEQ ID NO:38, and - P6 denotes the second antigenic polypeptide comprising an amino acid sequence having at least 90% of identity with the amino acid sequence that ranges from the amino acid residue at position 65 to the amino acid residue at position 124 in SEQ ID NO:3. In some embodiments, the antibody of the present invention comprises a light chain having the amino acid sequence as set forth in SEQ ID NO:42. SEQ ID NO:42 > L-chain: vc118 - rAB-cetHS-puro[hAnti-CD40VK2-LV-hIgGK- C-HBV_Pol_Pep5-f3-HBV_Pol_Pep6] DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECARPFLLAQFTSAICSVVRRAFPHCLAFSYMDDVVLGAKSVQHLESLFTAVTNFLLSLGIHLN PNKTKRWGYSLHFMGYVITSTVTPTATATPSAIVTTITPTATTKPLPLHPAAMPHLLVGSSGLSRYVAR LSSNSRNNNNQYGTMQNLHDSCSRQLYVSLMLLYKTFGWKLHLYSHPIV
In some embodiments, the antibody of the present invention comprises a heavy chain having the general formula of [hAnti-CD40VH3-LV-hIgG4H-Flexv1] wherein: - hAnti-CD40VH3-LV-hIgG4H denotes the heavy chain as set forth in SEQ ID NO:33, and - Flexv1 denotes the linker having the amino acid sequence as set forth in SEQ ID NO:35. In some embodiments, the antibody of the present invention comprises a heavy chain having the amino acid sequences as set forth in SEQ ID NO:43. SEQ ID NO: 43> [hAnti-CD40VH3-LV-hIgG4H-C-Flex-v1] EVQLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFT ISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK PSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNP In some embodiments, the antibody of the present invention consists of the CD40.HBV(S/C) antibody that comprises: - a heavy chain having the general formula of [hAnti-CD40VH3-LV-hIgG4H- Flexv1-S1-f1-S2] as defined above and, - a light chain having he general formula of [hAnti-CD40VK2-LV-hIgGK-C3-f2- C4] as defined above. In some embodiments, the antibody of the present invention consists of the CD40.HBV(S/C) antibody that comprises - a heavy chain having the amino acid sequence as set forth in SEQ ID NO:41 and, - a light chain having the amino acid sequence as set forth in SEQ ID NO: 40. In some embodiments, the antibody of the present invention consists of the CD40.HBV(P) antibody that comprises: - a heavy chain having the amino acid sequence as set forth in SEQ ID NO:33 and, - a light chain having the general formula of [hAnti-CD40VK2-LV-hIgGK-P5-f3- P6] as defined above.
In some embodiments, the antibody of the present invention consists of the CD40.HBV(P) antibody that comprises: - a heavy chain having the amino acid sequence as set forth in SEQ ID NO:33 and, - a light chain having the amino acid sequence as set forth in SEQ ID NO:42. In some embodiments, the antibody of the present invention consists of the CD40.HBV(P) antibody that comprises: - a heavy chain having the amino acid sequence as set forth in SEQ ID NO:43 and, - a light chain having the amino acid sequence as set forth in SEQ ID NO:42. In some embodiments, the amino acid sequence herein described comprise one or more sequences originating from the restriction cloning site(s) present in the polynucleotide encoding for said amino acid sequence. Typically, said sequences may consist of 2 amino acid residues and typically include AP, AS, AR, PR, SA, TR, and TS sequences. The antibodies of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides, by standard techniques for production of polypeptides. For instance, the antibodies of the invention can be synthesized by recombinant DNA techniques as is now well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly) peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques. Polynucleotides, vectors and host cells of the present invention: A further object of the invention relates to a polynucleotide that encodes for a heavy chain and/or light chain of the antibody of the present invention. Typically, said polynucleotide is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.
So, a further object of the invention relates to a vector comprising a polynucleotide of the present invention. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40, LTR promoter and enhancer of Moloney mouse leukemia virus, promoter and enhancer of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1 beta d2-4 and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication- defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94/19478. A further object of the present invention relates to a host cell which has been transfected, infected or transformed by a polynucleotide and/or a vector according to the invention. The polynucleotides of the invention may be used to produce an antibody of the present invention in a suitable expression system. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells vectors and plants. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Kluyveromyces or Saccharomyces yeasts. Mammalian host cells include Chinese Hamster Ovary (CHO cells) including dhfr- CHO cells (described in Urlaub and Chasin, 1980) used with a DHFR selectable marker, CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells, for example GS CHO cell lines together with GS XceedTM gene expression system (Lonza), or HEK cells.
The present invention also relates to a method of producing a recombinant host cell expressing the antibody according to the invention, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant polynucleotide or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and/or secrete said antibody. Such recombinant host cells can be used for the production of antibodies of the present invention. The host cell as disclosed herein are thus particularly suitable for producing the antibody of the present invention. Indeed, when recombinant expression are introduced into mammalian host cells, the polypeptides are produced by culturing the host cells for a period of time sufficient for expression of the antibody in the host cells and, optionally, secretion of the antibody into the culture medium in which the host cells are grown. The antibodies can be recovered and purified for example from the culture medium after their secretion using standard protein purification methods. Pharmaceutical and vaccine compositions: The antibodies as described herein may be administered as part of one or more pharmaceutical compositions. Except insofar as any conventional carrier medium is incompatible with the antibodies of the present invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents,
coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The antibodies as described herein are particularly suitable for preparing vaccine composition. Thus a further object of the present invention relates to a vaccine composition comprising an antibody of the present invention. In some embodiments, the vaccine composition of the present invention comprises an adjuvant. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is Incomplete Freund’s adjuvant (IFA) or other oil based adjuvant that is present between 30-70%, preferably between 40-60%, more preferably between 45-55% proportion weight by weight (w/w). In some embodiments, the adjuvant is Polyinosinic-polycytidylic acid (poly (I:C)). In some embodiments, the vaccine composition of the present invention comprises at least one Toll- Like Receptor (TLR) agonist which is selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR8 agonists. Therapeutic methods: The antibodies as well as the pharmaceutical or vaccine compositions as herein described are particularly suitable for inducing an immune response against HBV and thus can be used for vaccine purposes. Therefore, a further object of the present invention relates to a method for vaccinating a subject in need thereof against HBV comprising administering a therapeutically effective amount of the antibody of the present invention. In some embodiments, the antibodies of the present invention are particularly suitable for the treatment of HBV infection, and more particularly for the curative treatment of patients suffering from HBV infection. Chronic HBV infection is indeed associated with an increased risk of developing hepatocellular carcinoma (HCC), the most common form of primary liver cancer and one of the leading causes of cancer-related death worldwide. The mechanism by which HBV induces HCC is not fully understood, but it involves chronic inflammation, oxidative stress, DNA damage, immune evasion, and genomic instability. Current therapies for chronic HBV infection, such as nucleoside analogues and interferons, can suppress viral replication and reduce liver inflammation, but they rarely achieve complete clearance of the
virus and do not eliminate the risk of HCC. Therefore, there is an unmet medical need for novel therapeutic strategies that can eradicate HBV infection and prevent or reverse its oncogenic effects. The vaccine of the present invention, based on antibodies that target HBV antigens, may offer several advantages over existing therapies. First, the antibodies may have a direct antiviral effect by neutralizing the virus and preventing its entry into hepatocytes. Second, the antibodies may have an immunomodulatory effect by activating the innate and adaptive immune system against HBV-infected cells, enhancing the clearance of viral particles and covalently closed circular DNA (cccDNA), the stable form of HBV genome that persists in the nucleus of infected cells. Third, the antibodies may induce long-lasting immunity against HBV, preventing viral reactivation and recurrence of infection, and thus reducing the risk of HCC development and progression. Therefore, the vaccine of the present invention may represent a promising therapeutic option for patients with chronic HBV infection, especially those who have a high risk of developing HCC or who have already developed HCC. The vaccine may provide a more effective and durable cure of HBV infection, as well as a potential prevention or treatment of HCC. In particular, the present invention relates to a method of curing a HBV infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a first antibody CD40.HBV(S/C) and/or a second antibody CD40.HBV(P) wherein the CD40.HBV(S/C) and CD40.HBV(P) are defined above. In some embodiments, the first antibody CD40.HBV(S/C) and the second antibody CD40.HBV(P) may be administered simultaneously or separately, depending on the patient's condition and the physician's discretion. For example, the patient may receive a single dose of a combination of the two antibodies, or a sequential or alternating regimen of the two antibodies, as long as the total amount of each antibody is within the therapeutically effective range. The simultaneous or separate administration of the two antibodies may have a synergistic or additive effect in curing HBV infection and preventing or treating HCC. Alternatively, the patient may receive only one of the two antibodies, if the other antibody is contraindicated or not available. The optimal mode and schedule of administration of the two antibodies may be determined by clinical trials or empirical methods known in the art.
One of the advantages of the antibodies CD40.HBV(S/C) and CD40.HBV(P) is that they can be administered to the patient at different doses, depending on the patient's response and tolerance. For example, the patient may receive a low dose of one or both antibodies initially, and then gradually increase the dose until the desired therapeutic effect is achieved. Alternatively, the patient may receive a high dose of one or both antibodies for a short period of time, and then reduce the dose to maintain the effect. The dose adjustment may also depend on the stage and severity of the HBV infection, as well as the presence of any co-infections or co-morbidities. The dose optimization may be performed by the physician based on the clinical evaluation of the patient and the pharmacokinetic and pharmacodynamic properties of the antibodies. In some embodiments, the patient may receive a personalized dose of one or both antibodies, based on the patient's genetic, epigenetic, or immunological profile. The personalized dose may be determined by using biomarkers, bioinformatics, or machine learning methods known in the art. The different doses of the antibodies CD40.HBV(S/C) and CD40.HBV(P) may provide more flexibility and efficacy in curing HBV infection and preventing or treating HCC. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The antibodies and the pharmaceutical or vaccine compositions as herein described may be administered to the subject by any route of administration and in particular by oral, nasal, rectal, topical, buccal (e.g., sub-lingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active agent which is being used.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES: Figure 1: Production and quality control of the CD40.HBV(S/C) and CD40.HBV(P) vaccines. Six peptides, including two from HBs, two from HBc, and two from HBV Pol (genotype D ayw), ranging from 35 to 97 amino acids, were selected through in silico screening. These peptides were chosen for their high content of HLA class-I and -II peptides, their haplotype coverage of 99% of the global population, their conservation among major circulating HBV strains, and their biochemical stability. These peptides contain epitopes known to stimulate HBV-specific T cells (see Table-1). (A) Diagram illustrating the CD40.HBV(S/C) vaccine. The 12E12 (VH3) anti-CD40 mAb was fused to HBs Peptides (pep) 1 and 2 at the C- terminal end of the heavy (H)-chain via a flexible linker (FlexV1), with another flexible linker (f1) connecting Pep-1 and Pep-2. HBc Pep-3 and Pep-4 were fused at the C-terminal end of the light (L)-chain, using a flexible linker (f2) between Pep-3 and Pep-4. (B) Diagram illustrating the CD40.HBV(P) vaccine. It uses the same clone as in (A) but with HBV Pol Pep-5 and Pep- 6 fused to the C-terminal end of the L-chain, using a flexible linker (f3) between Pep-5 and Pep-6. (C) SDS-PAGE with Coomassie staining of the CD40.HBV(S/C) vaccine under non- reducing (nr) and reducing (r) conditions. The molecular weights (kDa) of the ladder are indicated. Bands corresponding to the recombinant H- or L-chains are marked by arrows. The predicted sizes of the CD40.HBV(S/C) mAb are 205 kDa without glycosylation, with H-chains and L-chains predicted to be 64.7 kDa and 37.7 kDa, respectively. (D) SDS-PAGE with Coomassie staining of the CD40.HBV(P) vaccine under non-reducing (nr) and reducing (r) conditions. The predicted size of the CD40.HBV(P) mAb is 191 kDa without glycosylation, with H-chains and L-chains predicted to be 52.1 kDa and 43.3 kDa, respectively. (E) Size- exclusion chromatography (SEC) analysis of CD40.HBV(S/C) using a 10/300 column, with standard molecular weights indicated by dotted lines. A single elution peak was observed at the expected elution time. (F) Same analysis as in (E), but for CD40.HBV(P). Figure 2: in vitro stimulation of human HBV-specific T cells using DC-targeting vaccines. (A) PBMCs from 19 NUC-treated HBV-infected donors were collected and stimulated for 8 days with 1 nM of either CD40.HBV(P) or CD40.HBV(S/C) vaccines. This resulted in
significant activation of memory CD4⁺ T cells specific to HBV S, C, and P peptides, as shown by intracellular cytokine staining (ICS) and elevated production of Th1 cytokines (IFN-γ, TNF, IL-2; denoted as "Cyt⁺") compared to non-stimulated controls (NS). (B) The Th1 responses elicited by the DC-targeted vaccines were significantly stronger than those induced by an equivalent dose of non-targeted overlapping peptide pools (OVLP). (C) CD4⁺ T cell responses to HBc peptides induced by CD40.HBV(S/C) were dose-dependent and exceeded those triggered by the non-targeted control vaccine IgG4.HBV(S/C) (P = 0.05). (D) When PBMCs were stimulated with 10 nM of CD40.HBV(P) or CD40.HBV(S/C), specific CD4⁺ T cell responses against each peptide were observed in 9 patients out of 10. Additionally, CD8⁺ T cell responses were detected in 60% of donors, with 20% responding to the S peptide and 40% to the C and P peptides (using a 2-fold increase over background as the response threshold). Figure 3: In vivo humoral cellular responses of DC-targeting vaccines in hCD40Tg C57BL/6 mice. Groups of hCD40 Tg mice were immunized intraperitoneally with the DC- targeting HBV vaccine candidates to evaluate both humoral (anti-HBs and HBc IgG) and cellular responses (IFNg ELISpot on splenocytes, FACS phenotyping of T and B cells). The vaccines were administered in a 3-week prime/boost homologous regimen with Poly-ICLC (clinical-grade Hiltonol, 50µg), and the animals were sacrificed one week post-boost. (A) Immunizations with CD40.HBV(S/C) alone showed a dose-dependent expansion of germinal- center (GC) B cells (left) and antibody-secreting cells (ASC; right) in the spleens. Based on these findings, a vaccine dose of 8.6µg of CD40.HBV(S/C) combined with an equivalent amount of Pol peptides (5.1µg of CD40.HBV(P)) were selected. (B) This dosing confirmed the induction of GC B cells (left) and ASC (right). (C) The GC reaction was associated with significant IgG production in the sera, specific to both HBs (left) and HBc (right), as measured by Luminex. Dotted lines: prime and boost. Non-parametric unpaired t-tests, ****, P<0.0001; **, P<0.01. Figure 4: In vivo T-cell responses of DC-targeting vaccines in hCD40Tg C57BL/6 mice. IFN-g T cell responses were measured by ELISpot. (A) A significant advantage in targeting HBs peptides to CD40 was shown in a dose-dependent manner compared to the IgG4(S/C) non- targeting vehicle. (B) When immunizing with CD40.HBV(S/C) and CD40.HBV(P) together (880ng of each peptide), significant IFNg responses were observed to both the S and P peptides. Non-parametric unpaired t-tests, ****, P<0.0001; **, P<0.01.
EXAMPLE: The results of the in vivo experiments in hCD40Tg C57BL/6 mice demonstrate that the DC- targeting HBV vaccine candidates can elicit strong and specific humoral and cellular immune responses against both the HBs and HBc antigens. The vaccines consist of fusion proteins that target the CD40 receptor on DCs and deliver HBV-derived peptides to the MHC class I and II pathways. As shown in Figure 3, the immunization with CD40.HBV(S/C) alone or in combination with CD40.HBV(P) induced a robust expansion of germinal center B cells and antibody-secreting cells in the spleen, as well as high levels of IgG antibodies in the serum. The antibodies were specific for both the HBs and HBc antigens, indicating a broad coverage of the viral envelope and core proteins. As shown in Figure 4, the immunization also triggered potent IFN-gamma T cell responses to both the S and P peptides, indicating a successful activation of CD8+ and CD4+ T cells by the DC-targeting vaccines. These results suggest that the DC- targeting HBV vaccine candidates have the potential to induce protective immunity against HBV infection and chronic liver disease.
Table-1 down selected vaccine HBV peptides:
REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.