Patent Grant
9885017
The present invention relates in general to the field of immunology, and more particularly, to hepatitis C virus (HCV) immunization, vaccines, and targeting of the HCV peptides to human dendritic cells. The application also describes a bi-functional antibody fused to a HCV target antigen(s) that is directed against a dendritic cell (DC)-specific receptor.
None.
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 15, 2012, is named BHCS1118.txt and is 388,419 bytes in size.
Without limiting the scope of the application, its background is described in connection with immunostimulatory methods and compositions, including vaccines, and increased effectiveness in antigen presentation of HCV peptides in relation to HCV immunization and vaccines.
U.S. Patent Application Publication No. 2009/0238822 (Rajan et al. 2009) relates to chimeric antigens for targeting and activating antigen presenting cells to elicit cellular and humoral immune responses. The Rajan invention describes compositions and methods that contain or use one or more chimeric antigens that contain one or more pre-selected HCV antigen(s), and an immunoglobulin fragment. The invention further discloses chimeric antigens, comprising an HCV antigen and a Fc fragment of an immunoglobulin for eliciting an immune response against said antigen. The immune response is said to be enhanced upon presenting the host immune system with an immune response domain (HCV antigen from HCV core, envelope, or non-structural protein fragments) and a target-binding domain (an Fc fragment).
U.S. Patent Application Publication No. 2008/0241170 (Zurawski et al. 2008) discloses compositions and methods for making and using vaccine that specifically target (deliver) antigens to antigen-presenting cells for the purpose of eliciting potent and broad immune responses directed against the antigen. The purpose is primarily to evoke protective or therapeutic immune responses against the agent (pathogen or cancer) from which the antigen was derived.
U.S. Patent Application Publication 2010/0239575 (Banchereau et al. 2010) refers to compositions and methods for the expression, secretion, and use of novel compositions for use as, e.g., vaccines and antigen delivery vectors, to delivery antigens to antigen presenting cells. In one embodiment, the vector is an anti-CD40 antibody, or fragments thereof, and one or more antigenic peptides linked to the anti-CD40 antibody or fragments thereof, including humanized antibodies.
The present invention describes immunostimulatory compositions, vaccines, HCV vaccines, HCV antigen presenting dendritic cells, methods for increasing effectiveness of HCV antigen presentation by an antigen presenting cell, methods for increasing effectiveness of HCV antigen presentation by an antigen presenting cell, methods for increasing effectiveness of antigen presentation by an antigen presenting cell, methods for a treatment, a prophylaxis or a combination thereof against hepatitis C in a human subject, methods of providing immunostimulation by activation of one or more dendritic cells, methods to treat or prevent hepatitis C in a subject, and methods for generating a HCV presenting dendritic cell. The present invention further describes virus antigens, e.g., proteins and peptides corresponding to HCV proteins or fragments thereof, fused to heavy and/or light chain of antibodies, or fragments thereof specific for dendritic cells (DCs). The vaccine composition as described herein delivers HCV antigen specifically to DCs for the purpose of invoking an immune response. The vaccine composition may also promote efficient recall memory in hepatitis C patients.
In one embodiment the instant invention discloses an immunostimulatory composition for generating an immune response for a prophylaxis, a therapy, or any combination thereof against a Hepatitis C infection in a human or animal subject comprising: one or more antibodies or fragments thereof specific for a dendritic cell (DC) and one or more HCV antigens attached to the one or more antibodies or fragments thereof. In one aspect the composition disclosed hereinabove further comprises at least one Toll-Like Receptor (TLR) agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR8 agonists. In another aspect the composition further comprises an optional pharmaceutically acceptable carrier that is effective, in combination, to produce the immune response for prophylaxis, for therapy, or any combination thereof in the human or animal subject in need of immunostimulation. In yet another aspect the DC-specific antibody or fragment is specific for a DC specific receptor, wherein the DC-specific antibody or fragment is selected from an antibody that specifically binds to MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fcγ receptor, LOX-1, and ASPGR.
In the composition of the instant invention the HCV antigens comprises a peptide sequence derived from a HCV 1a genotype protein or a fragment thereof and the HCV antigens are selected from the group consisting of protein E1, envelope protein E2, non-structural protein NS3, non-structural protein NS4b, non-structural protein NS5b, and a fragment thereof. The one or more HCV antigens are selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and a fragment thereof and from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, E1b, and a fragment thereof. In one aspect of the composition of the instant invention the composition comprises a recombinant antibody that comprises a fusion protein and the one or more HCV antigen are at a C-terminal position relative to the one or more antibody or fragment thereof within a fusion protein. In another aspect the composition comprises a recombinant antibody, and the one or more HCV antigens are fused to a C-terminus of a heavy chain of the antibody. In yet another aspect the composition comprises a recombinant antibody, and the one or more HCV antigens are fused to a C-terminus of a light chain of the one or more antibody or fragment thereof specific for a DC.
The one or more HCV antigens are selected from the group consisting of SEQ ID NO: 12-linker A-SEQ ID NO: 13, SEQ ID NO: 12-linker A-SEQ ID NO: 11, SEQ ID NO: 12-linker B-SEQ ID NO: 14, SEQ ID NO: 14-linker B-SEQ ID NO: 12, SEQ ID NO: 12-linker B-SEQ ID NO: 10, SEQ ID NO: 10-linker B-SEQ ID NO: 12, SEQ ID NO: 9-linker
B-SEQ ID NO: 10, SEQ ID NO: 10-linker B-SEQ ID NO: 9, SEQ ID NO: 10-linker B-SEQ ID NO: 14, SEQ ID NO: 14-linker B-SEQ ID NO: 10, SEQ ID NO: 9-linker B-SEQ ID NO: 12, SEQ ID NO: 12-linker B-SEQ ID NO: 9, SEQ ID NO: 8-linker B-E1b. SEQ ID NO: 12-linker B-SEQ ID NO: 10-linker C-SEQ ID NO: 14, SEQ ID NO: 12-linker B-SEQ ID NO: 14-linker C-SEQ ID NO: 10, SEQ ID NO: 10-linker B-SEQ ID NO: 12-linker C-SEQ ID NO: 14, SEQ ID NO: 10-linker B-SEQ ID NO: 14-linker C-SEQ ID NO: 12, SEQ ID NO: 14-linker B-SEQ ID NO: 12-linker C-SEQ ID NO: 10, SEQ ID NO: 14-linker B-SEQ ID NO: 10-linker C-SEQ ID NO: 12, and SEQ ID NO: 12-linker B-SEQ ID NO: 10-linker C-SEQ ID NO: 14-linker D-SEQ ID NO: 8. In another aspect the one or more HCV antigens are attached to a C-terminus of a light chain of the recombinant antibody and selected from a group consisting of: SEQ ID NO: 9; SEQ ID NO: 11, and E1b. In yet another aspect the one or more HCV antigens are selected from the group consisting of SEQ ID NO: 9 fused to the C-terminus of a light chain and SEQ ID NO: 10-linker B-SEQ ID NO: 12-linker C-SEQ ID NO: 14 fused to the C-terminus of the heavy chain of the antibody. In a related aspect the one or more HCV antigen are chemically coupled to the one or more antibodies or fragments thereof or are attached to the one or more antibodies or fragments thereof via an affinity association. In a specific aspect the DC-specific antibody is humanized. In another aspect the composition is optimized to be administered to the human or animal subject by an oral route, a nasal route, topically, or as an injection.
Another embodiment of the present invention discloses a vaccine comprising: one or more antibodies or fragments thereof specific for a dendritic cell (DC); and one or more HCV antigens attached to the one or more antibodies or fragments thereof. The vaccine described herein further comprises at least one Toll-Like Receptor (TLR) agonist selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, and TLR8 agonists and an optional pharmaceutically acceptable carrier or an adjuvant that is effective, in combination, to produce an immune response for prophylaxis, for therapy, or any combination thereof in the human or animal subject in need of immunostimulation. In one aspect of the vaccine the DC-specific antibody or fragment is specific for a dendritic cell specific receptor. In another aspect the HCV antigen comprises a peptide sequence derived from a HCV 1a genotype protein or a fragment thereof, wherein the HCV antigen is selected from the group consisting of protein E1, envelope protein E2, non-structural protein NS3, non-structural protein NS4b, non-structural protein NS5b, and a fragment thereof. In other related aspects the DC-specific antibody is humanized and the composition is optimized to be administered to the human or animal subject by an oral route, a nasal route, topically, or as an injection.
In yet another embodiment the instant invention discloses a Hepatitis C vaccine (HCV) comprising a fusion protein comprising: (i) one or more antibodies or fragments thereof specific for a dendritic cell (DC), (ii) one or more HCV antigens located C-terminal of the antibodies or fragments thereof, (iii) 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, and (iv) one or more optional pharmaceutically acceptable carriers and adjuvants, wherein the vaccine is effective to produce an immune response, for a prophylaxis, a therapy, or any combination thereof against hepatitis C in a human or an animal subject in need thereof. In one aspect the vaccine comprises one or more optional agents selected from the group consisting of an agonistic anti-CD40 antibody, an agonistic anti-CD40 antibody fragment, a CD40 ligand (CD40L) polypeptide, a CD40L polypeptide fragment, anti-4-1BB antibody, an anti-4-1BB antibody fragment, 4-1BB ligand polypeptide, a 4-1BB ligand polypeptide fragment, IFN-γ, TNF-α, type 1 cytokines, type 2 cytokines or combinations and modifications thereof.
The instant invention in one embodiment discloses a method for increasing effectiveness of Hepatitis C virus (HCV) antigen presentation by an antigen presenting cell (APC) comprising the steps of: (i) providing an antibody conjugate comprising a dendritic cell (DC) specific antibody or a fragment thereof and one or more native or engineered HCV antigenic peptides, (ii) providing one or more APCs; and (iii) contacting the APC with the conjugate, wherein the antibody-antigen complex is processed and presented for T cell recognition. In a specific aspect of the method the antigen presenting cell comprises a dendritic cell (DC).
In another embodiment the instant invention provides a method for increasing effectiveness of antigen presentation by an antigen presenting cell (APC) comprising the steps of: i) isolating and purifying one or more dendritic cell (DC)-specific antibody or a fragment thereof, ii) providing one or more HCV antigens or antigenic peptides, iii) loading or chemically coupling the one or more HCV antigens or antigenic peptides to the DC-specific antibody to form an antibody-antigen conjugate, and iv) contacting the antigen presenting cell with the conjugate, wherein the antibody-antigen complex is processed and presented for T cell recognition.
The method as described hereinabove further comprises adding 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 and one or more optional steps comprising i) adding one or more optional agents selected from the group consisting of an agonistic anti-CD40 antibody, an agonistic anti-CD40 antibody fragment, a CD40 ligand (CD40L) polypeptide, a CD40L polypeptide fragment, anti-4-1BB antibody, an anti-4-1BB antibody fragment, 4-1BB ligand polypeptide, a 4-1BB ligand polypeptide fragment, IFN-γ, TNF-α, type 1 cytokines, type 2 cytokines or combinations and modifications thereof to the antibody-antigen conjugate and the TLR agonist prior to contacting the antigen presenting cells, ii) measuring a level of one or more agents selected from the group consisting of IFN-γ, TNF-α, IL-12p40, IL-4, IL-5, and IL-13, wherein a change in the level of the one or more agents is indicative of the increase in the effectiveness antigen presentation by the antigen presenting cell, and iii) adding one or more optional agents selected from the group consisting of an agonistic anti-CD40 antibody, an agonistic anti-CD40 antibody fragment, a CD40 ligand (CD40L) polypeptide, a CD40L polypeptide fragment, anti-4-1BB antibody, an anti-4-1BB antibody fragment, 4-1BB ligand polypeptide, a 4-1BB ligand polypeptide fragment, IFN-γ, TNF-α, type 1 cytokines, type 2 cytokines or combinations and modifications thereof.
In yet another embodiment the instant invention provides method for a treatment, a prophylaxis or a combination thereof against hepatitis C in a human subject comprising the steps of: identifying the human subject in need of the treatment, the prophylaxis, or a combination thereof against the hepatisti and administering a vaccine composition comprising one or more antibodies or fragments thereof specific for a dendritic cell (DC) and one or more HCV antigens attached to the one or more antibodies or fragments thereof. In one aspect of the method the vaccine composition further 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, and one or more optional pharmaceutically acceptable carriers and adjuvants, wherein the conjugate and agonist are each comprised in an amount such that, in combination with the other, are effective to produce an immune response, for the prophylaxis, the therapy or any combination thereof against the influenza in the human subject. In another aspect the vaccine composition further comprises one or more optional agents selected from the group consisting of an agonistic anti-CD40 antibody, an agonistic anti-CD40 antibody fragment, a CD40 ligand (CD40L) polypeptide, a CD40L polypeptide fragment, anti-4-1BB antibody, an anti-4-1BB antibody fragment, 4-1BB ligand polypeptide, a 4-1BB ligand polypeptide fragment, IFN-γ, TNF-α, type 1 cytokines, type 2 cytokines or combinations and modifications thereof. In yet another aspect the vaccine is administered to the human subject by an oral route, a nasal route, topically or as an injection.
In another aspect the one or more antibodies or fragments thereof specific for a dendritic cell comprises antibodies specifically binds to MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fey receptor, LOX-1, or ASPGR. In yet another aspect the HCV antigen is selected from the group consisting of protein E1, envelope protein E2, non-structural protein NS3, non-structural protein NS4b, non-structural protein NS5b, and a fragment thereof, from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and a fragment thereof, or from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, E1b, and a fragment thereof.
A method of providing immunostimulation by activation of one or more dendritic cells (DCs) to a human subject for a prophylaxis, a therapy or a combination thereof against HCV is described in one embodiment of the present invention. The method comprises the steps of: a) identifying the human subject in need of immunostimulation for the prophylaxis, the therapy or a combination thereof against HCV, b) isolating one or more DCs from the human subject, c) exposing the isolated DCs to activating amounts of a composition or a vaccine comprising an anti-dendritic cell immunoreceptor (DCIR) monoclonal antibody or fragments thereof attached to one or more HCV antigens, and d) reintroducing the activated DC complex into the human subject.
The method described above further comprises the steps of contacting the one or more DCs with 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 and a pharmaceutically acceptable carrier to form an activated DC complex and the step of adding one or more optional agents selected from the group consisting of an agonistic anti-CD40 antibody, an agonistic anti-CD40 antibody fragment, a CD40 ligand (CD40L) polypeptide, a CD40L polypeptide fragment, anti-4-1BB antibody, an anti-4-1BB antibody fragment, 4-1BB ligand polypeptide, a 4-1BB ligand polypeptide fragment, IFN-γ, TNF-α, type 1 cytokines, type 2 cytokines or combinations and modifications thereof to the conjugate and the TLR agonist prior to exposing the DCs. The method disclosed hereinabove further comprises the optional step of measuring a level of one or more agents selected from the group consisting of IFN-γ, TNF-α, IL-12p40, IL-4, IL-5, and IL-13, wherein a change in the level of the one or more agents is indicative of the immunostimulation.
The present invention also discloses a method to treat or prevent Hepatitis C in a subject comprising the step of administering to the subject a fusion protein comprising an antibody or fragment thereof specific for a dendritic cell (DC) and a Hepatitis C virus antigen or antigenic peptide fused to the antibody or fragment thereof. A Hepatitis C virus antigen presenting dendritic cell (DC) is also disclosed in one embodiment of the present invention. The HCV antigen presenting DC further comprises one or more isolated dendritic cells (DCs) in contact with a fusion protein comprising an antibody or fragment thereof specific for the DC, the fusion protein further comprising a HCV peptide.
The present invention describes one or more vaccines against HCV comprising one or more antibodies or fragments thereof specific for a dendritic cell (DC) and one or more HCV antigens or antigenic domains attached to the one or more antibodies or fragments thereof. The vaccine has a general structure given by: H-w, H-w-x, H-w-x-y, or H-w-x-y-z, wherein H represents a heavy chain of an antibody or a fragment thereof specific for a DC, w, x, y, and z represent one or more HCV antigens or domains selected from the group consisting of protein E1, envelope protein E2, non-structural protein NS3, non-structural protein NS4b, non-structural protein NS5b, or any combinations thereof. In one aspect w comprises the HCV antigenic domains selected from the group consisting of ProtA, ProtB, HelB, Palm, E1b, and E2. In another aspect x comprises the HCV antigenic domains selected from the group consisting of Hel C, Hel A, Palm, ProtA, ProtB, and E1b. In yet another aspect comprises the HCV antigenic domains selected from the group consisting of Palm, ProtB, and Protb. In another aspect z comprises HCV antigenic domains selected from E2, ProtA, and HelB. In a related aspect the one or more HCV antigens or antigenic domains are linked or attached to one another by one or more flexible linkers.
Another embodiment disclosed herein relates to a vaccine comprising one or more antibodies or fragments thereof specific for a dendritic cell (DC) and one or more HCV antigens or antigenic domains attached to the one or more antibodies or fragments thereof, wherein the vaccine has a general structure given by L-w-x-y-z, wherein L represents a light chain of an antibody or a fragment thereof specific for a DC, w, x, y, and z represent one or more HCV antigens or domains selected from the group consisting of protein E1, envelope protein E2, non-structural protein NS3, non-structural protein NS4b, non-structural protein NS5b, or any combinations thereof.
In yet another embodiment the present invention discloses a vaccine comprising one or more antibodies or fragments thereof specific for a dendritic cell (DC) and one or more HCV antigens or antigenic domains attached to the one or more antibodies or fragments thereof, wherein the vaccine has a general structure given by:
Wherein H represents a heavy chain of an antibody or a fragment thereof specific for a DC, L represents a light chain of an antibody or a fragment thereof specific for the DC, w, x, y, and z represent one or more HCV antigens or domains selected from the group consisting of protein E1, envelope protein E2, non-structural protein NS3, non-structural protein NS4b, non-structural protein NS5b, or any combinations thereof.
Finally, the present invention discloses a method for generating a Hepatitis C virus (HCV) presenting dendritic cells (DCs) in a human subject comprising the steps of: providing one or more DCs and incubating the dendritic cells with a fusion protein, wherein the fusion protein comprises an antibody or fragment thereof specific for a dendritic cell and a HCV antigen fused to the antibody or fragment thereof. The method disclosed herein further comprises the step of administering to the subject an effective amount of IFNA, Ribavirin, or a combination thereof.
For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
The invention includes also variants and other modification of an antibody (or “Ab”) of fragments thereof, e.g., anti-CD40 fusion protein (antibody is used interchangeably with the term “immunoglobulin”). As used herein, the term “antibodies or fragments thereof,” includes whole antibodies or fragments of an antibody, e.g., Fv, Fab, Fab′, F(ab′)2, Fc, and single chain Fv fragments (ScFv) or any biologically effective fragments of an immunoglobulins that binds specifically to, e.g., CD40. Antibodies from human origin or humanized antibodies have lowered or no immunogenicity in humans and have a lower number or no immunogenic epitopes compared to non-human antibodies. Antibodies and their fragments will generally be selected to have a reduced level or no antigenicity in humans.
As used herein, the terms “Ag” or “antigen” refer to a substance capable of either binding to an antigen binding region of an immunoglobulin molecule or of eliciting an immune response, e.g., a T cell-mediated immune response by the presentation of the antigen on Major Histocompatibility Antigen (MHC) cellular proteins. As used herein, “antigen” includes, but is not limited to, antigenic determinants, haptens, and immunogens, which may be peptides, small molecules, carbohydrates, lipids, nucleic acids or combinations thereof. The skilled immunologist will recognize that when discussing antigens that are processed for presentation to T cells, the term “antigen” refers to those portions of the antigen (e.g., a peptide fragment) that is a T cell epitope presented by MHC to the T cell receptor. When used in the context of a B cell mediated immune response in the form of an antibody that is specific for an “antigen”, the portion of the antigen that binds to the complementarity determining regions of the variable domains of the antibody (light and heavy) the bound portion may be a linear or three-dimensional epitope. In the context of the present invention, the term antigen is used on both contexts, that is, the antibody is specific for a protein antigen (CD40), but also carries one or more peptide epitopes for presentation by MHC to T cells. In certain cases, the antigens delivered by the vaccine or fusion protein of the present invention are internalized and processed by antigen presenting cells prior to presentation, e.g., by cleavage of one or more portions of the antibody or fusion protein.
As used herein, the term “conjugate” refers to a protein having one or more targeting domains, e.g., an antibody, and at least one antigen, e.g., a small peptide or a protein. These conjugates include those produced by chemical methods, such as by chemical coupling, for example, coupling to sulfhydryl groups, and those produced by any other method whereby one or more antibody targeting domains and at least one antigen, are linked, directly or indirectly via linker(s) to a targeting agent. An example of a linker is a cohesin-dockerin (coh-doc) pair, a biotin-avidin pair, histidine tags bound by Zn, and the like.
As used herein, the term “Antigen Presenting Cells” (APC) refers to cells that are capable of activating T cells, and include, but are not limited to, certain macrophages, B cells and dendritic cells. “Dendritic cells” (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). These cells can be isolated from a number of tissue sources, and conveniently, from peripheral blood, as described herein. Dendritic cell binding proteins refers to any protein for which receptors are expressed on a dendritic cell. Examples include GMCSF, IL-1, TNF, IL-4, CD40L, CTLA4, CD28, and FLT-3 ligand.
For the purpose of the present invention, the term “vaccine composition” is intended to mean a composition that can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in a production of antibodies or simply in the activation of certain cells, in particular antigen-presenting cells, T lymphocytes and B lymphocytes. The vaccine composition can be a composition for prophylactic purposes or for therapeutic purposes, or both. As used herein, the term “antigen” refers to any antigen which can be used in a vaccine, whether it involves a whole microorganism or a subunit, and whatever its nature: peptide, protein, glycoprotein, polysaccharide, glycolipid, lipopeptide, etc. They may be viral antigens, bacterial antigens, or the like; the term “antigen” also comprises the polynucleotides, the sequences of which are chosen so as to encode the antigens whose expression by the individuals to which the polynucleotides are administered is desired, in the case of the immunization technique referred to as DNA immunization. They may also be a set of antigens, in particular in the case of a multivalent vaccine composition which comprises antigens capable of protecting against several diseases, and which is then generally referred to as a vaccine combination, or in the case of a composition which comprises several different antigens in order to protect against a single disease, as is the case for certain vaccines against whooping cough or the flu, for example. The term “antibodies” refers to immunoglobulins, whether natural or partially or wholly produced artificially, e.g. recombinant. An antibody may be monoclonal or polyclonal. The antibody may, in some cases, be a member of one, or a combination immunoglobulin classes, including: IgG, IgM, IgA, IgD, and IgE.
The term “adjuvant” refers to a substance that enhances, augments or potentiates the host's immune response to a vaccine antigen.
The term “gene” is used to refer to a functional protein, polypeptide or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences, and fragments or combinations thereof, as well as gene products, including those that may have been altered by the hand of man. Purified genes, nucleic acids, protein and the like are used to refer to these entities when identified and separated from at least one contaminating nucleic acid or protein with which it is ordinarily associated.
As used herein, the term “nucleic acid” or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., α-enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
As used herein, “polynucleotide” or “nucleic acid” refers to a strand of deoxyribonucleotides or ribonucleotides in either a single- or a double-stranded form (including known analogs of natural nucleotides). A double-stranded nucleic acid sequence will include the complementary sequence. The polynucleotide sequence may encode variable and/or constant region domains of immunoglobulin that are formed into a fusion protein with one or more linkers. For use with the present invention, multiple cloning sites (MCS) may be engineered into the locations at the carboxy-terminal end of the heavy and/or light chains of the antibodies to allow for in-frame insertion of peptide for expression between the linkers. As used herein, the term “isolated polynucleotide” refers to a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof. By virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotides” are found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence. The skilled artisan will recognize that to design and implement a vector can be manipulated at the nucleic acid level by using techniques known in the art, such as those taught in Current Protocols in Molecular Biology, 2007 by John Wiley and Sons, relevant portions incorporated herein by reference. Briefly, the encoding nucleic acid sequences can be inserted using polymerase chain reaction, enzymatic insertion of oligonucleotides or polymerase chain reaction fragments in a vector, which may be an expression vector. To facilitate the insertion of inserts at the carboxy terminus of the antibody light chain, the heavy chain, or both, a multiple cloning site (MCS) may be engineered in sequence with the antibody sequences.
As used herein, the term “polypeptide” refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. The term “domain,” or “polypeptide domain” refers to that sequence of a polypeptide that folds into a single globular region in its native conformation, and that may exhibit discrete binding or functional properties.
As used in this application, the term “amino acid” means one of the naturally occurring amino carboxylic acids of which proteins are comprised. The term “polypeptide” as described herein refers to a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides.” A “protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
A polypeptide or amino acid sequence “derived from” a designated nucleic acid sequence refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 3-5 amino acids, preferably at least 4-7 amino acids, more preferably at least 8-10 amino acids, and even more preferably at least 11-15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence. This terminology also includes a polypeptide expressed from a designated nucleic acid sequence.
As used herein, the terms “stable,” “soluble,” or “unstable” when referring to proteins is used to describe a peptide or protein that maintains its three-dimensional structure and/or activity (stable) or that loses immediately or over time its three-dimensional structure and/or activity (unstable). As used herein, the term “insoluble” refers to those proteins that when produced in a cell (e.g., a recombinant protein expressed in a eukaryotic or prokaryotic cell or in vitro) are not soluble in solution absent the use of denaturing conditions or agents (e.g., heat or chemical denaturants, respectively). The antibody or fragment thereof and the linkers taught herein have been found to convert antibody fusion proteins with the peptides from insoluble and/or unstable into proteins that are stable and/or soluble. Another example of stability versus instability is when the domain of the protein with a stable conformation has a higher melting temperature (Tm) than the unstable domain of the protein when measured in the same solution. A domain is stable compared to another domain when the difference in the Tm is at least about 2° C., more preferably about 4° C., still more preferably about 7° C., yet more preferably about 10° C., even more preferably about 15° C., still more preferably about 20° C., even still more preferably about 25° C., and most preferably about 30° C., when measured in the same solution.
As used herein, the term “in vivo” refers to being inside the body. The term “in vitro” used as used in the present application is to be understood as indicating an operation carried out in a non-living system.
As used herein, the term “treatment” or “treating” means any administration of a compound of the present invention and includes (1) inhibiting the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., arresting further development of the pathology and/or symptomatology), or (2) ameliorating the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., reversing the pathology and/or symptomatology).
As used herein, “pharmaceutically acceptable carrier” refers to any material that when combined with an immunoglobulin (Ig) fusion protein of the present invention allows the Ig to retain biological activity and is generally non-reactive with the subject's immune system. Examples include, but are not limited to, standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as an oil/water emulsion, and various types of wetting agents. Certain diluents may be used with the present invention, e.g., for aerosol or parenteral administration, that may be phosphate buffered saline or normal (0.85%) saline.
Substantial similarity of a peptide refers to similarity of a peptide as reflected in the amino acid sequence of the peptide. Identity of a continuous stretch of least 8 amino acids in an antigenic epitope of the peptide may be sufficient to establish substantial identity that enables cross reactivity. A first peptide and a second peptide are substantially similar in this regard if they have substantial similar antigenic epitopes so that immunization with the first peptide causes an immune response against the second peptide.
A fragment of an antibody, as used in the present application, refers to a portion of an antibody, created by protein engineering including proteolysis, or genetic engineering including recombination of nucleic acids; the fragment of an antibody retains specificity for the antigen.
A fragment of a peptide used as antigen refers to a portion of the peptide that retains its immunogenicity. A person of ordinary skill in the art will recognize that a continuous stretch of least 8 amino acids in an antigenic epitope of the peptide may be sufficient I order for a peptide to retain its immunogenicity.
Recombinant protein or antibody is generated by genetic engineering of nucleic acid encoding the protein or antibody and subsequent translation of the coding sequence by a cell or in a cell-free translation system.
The present invention describes a vaccine composition for delivering a HCV antigen specifically to DCs for the purpose of invoking an immune response In one embodiment, due to the high polymorphism of HCV, a sequence that is representative of most of circulating HCV sequence was selected. Based on sequence variation HCV can be classified into 6 genotypes that differs one to the other on the basis of sequence identity. World wide, 1 genotype is the most represented and also the most difficult to treat with the current IFNa-Ribavirin double therapy. More precisely, 1a genotype is the most represented subsequence in industrial country, and especially in US.
In one embodiment, 1a genotype was used as target sequence to derive a vaccine. It was observed that sequence alignment with all available 1a sequences found in data bases (euHCVdb and Los Alamos National Laboratory) showed less than 70% of sequence identity and the sequence of the HCV antigen would have to be adjusted accordingly.
A mosaic sequence was derived using the mosaic vaccine tools at www.hiv.lanl.gov/content/sequence/MOSAIC/ interface choosing mosaic sequence cocktail, 1 as cocktail size and 9 as epitope size. We used 249 sequences for E1 mosaic, 656 sequences for E2 mosaic, 213 sequences for NS3 mosaic and 310 sequences for NS5b mosaic. All sequences correspond to complete genes of 576, 1089, 1893, 1773 nucleotides respectively and found in euHCVdb (euhcvdb.ibcp.fr/euHCVdb/).
HCV antigen choice: HCV is an RNA enveloped virus. Virions are consisted by 4 structural proteins Core, E1, E2 and p7. As an RNA virus replication is based on viral proteins that need to be expressed after infection. Six non-structural proteins (NS2, NS3, NS4a, NS4b, NS5a, NS5b) are necessary to establish and maintain replication and virus production. HCV targets the liver and can infect barely all the liver with 90% of hepatocytes infected. However, the virus is able to replicate only in 30% of hepatocytes. Infected cells presented at their surface epitopes coming from structural proteins, while infected virus-producing cells presented all HCV antigens, structural and non structural.
Because HCV targets a vital organ such as the liver, therapeutic vaccine need to be very specific in order to avoid complete liver destruction and death of the patients. Indeed, we choose for our therapeutic vaccine antigens that are only found in infected virus producing hepatocytes, and then target antigen will be non-structural proteins. Moreover, NS3 and NS4b are highly immunogenic in chronic infected patients, as efficient as structural core or E1 E2 structural proteins. Therefore the present inventors included NS5b as an antigen too.
In one embodiment, NS3 and NS5b were chosen because of their possible expression as recombinant protein and the availability of their 3D structure.
Description of an embodiment of a vaccine: A particular embodiment of a vaccine consisted of bifunctional antibodies, which were directed against Dendritic Cells specific receptors and have target antigens fused at C terminus part of heavy chain. This allows unique targeting of DC and more precisely different DC subset that expressed different receptors, DC activation through the targeted receptor, and direct delivery of antigen to DC. In turn antigens are presented more efficiently and APC function is associated to cytokine secretion that orient T cells activation towards different functions.
Design of domains: It is not readily predictable whether any particular non-structural viral protein will be efficiently expressed as a direct antibody-antigen fusion protein. Commonly, fusion proteins may not be soluble and not be secreted. The present application describes that by using flexible linker modules, fragmenting the antigen coding sequence, and varying the fragment order, efficient secretion of recombinant antibody-antigen vaccines bearing extensive stretches of non-structural proteins can be achieved. The current application describes a first testing of constructs by expression of antibody fused to individual HCV non-structural proteins, then linking those that are expressible as soluble protein to each other to maximize the antigen load. Domains were first designed based on the 3D structure of the corresponding full-length proteins. Domains were design as the minimal structured regions in between unfolded loops. Length of the loops was varied in order to increase expression of corresponding domains. Pymol software was used to visualize 3d structures. The domains that expressed at the C-terminal of the antibody heavy chain are represented by SEQ ID NOs: 7-14.
Multiple combinations of individual domains have been made in order to provide as much HCV antigen as possible. In some embodiments, each single domain is separated from the next by flexible linkers, which can be as small as two amino acids (e.g., AS) but can also be longer, e.g., 3, 4, 5, 6, 7 8, 9, 10, 12, 15, 18, 20, 25 or 30 amino acids long.
In another embodiment, domains were also expressed at the C-terminus part of the light chain, and used in combination with heavy chain fused to multiple HCV domains. This allows the formation of a combine antibody with 3HCV domains fused to the heavy chain and one fused to the light chain.
Preparation of targeting constructs: Anti human DCIR and CD40 V region form H and L chain were cloned in a IgG4 backbone. Spe I cloning site was introduced at the end of the carboxy terminus to clone in frame antigen sequences. HCV antigens from NS3 and NS5b viral proteins represented as subdomains of these proteins were subcloned as a Spe-Not fragment in Nhe-Not linearized pIRES vector.
HCV-domains were designed based on the 3D-structure of the corresponding full-length proteins (PDB code IJXP for NS3protease, 1HEI for NS3Helicase and 1GX5 for NS5b). 3D-structures were visualized using PyMol software. Domains were designed as the minimal structured regions in between unfolded loops. Length of the loops was varied in order to increase expression of corresponding domains fused to the recombinant antibody. For multiple domains cloning, linkers were introduced between domains using Spe-Not/Nhe-Not strategy. Mosaic sequences, used in this study, corresponding to the maximum HCV-domains expressed as antibody-antigen recombinant fusion proteins are shown below. They included amino acids 95 to 180 from NS3Protease, amino acids 132 to 254 from NS3Helicase and a recombinant fusion of amino acids 55 to 80; 172 to 261 and 276 to 362 from NS5bPolymerase. Spe, Nhe and Not introduced cloning sites are underlined.
SEQ ID NOS: 1-6 show the amino acid sequence of the HCV proteins E1, E2, NS3, and NS5b mosaic sequences. Membrane domains are underlined. The full-length protein NS3 contains 631 amino acids and is also presented as being cut in its two enzymatic activities proteins: NS3Protease and NS3Helicase. These may also be produced as recombinant proteins N-terminal fused to either histidine tag or Cohesin tag.
SCLWMMLLISQAEA
APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCING
The nucleotide sequences are presented herein below.
ACTAGT
ACTCCTTGTACCTGCGGCTCATCCGACCTGTACCTGGTCACCCG
ACTAGT
GTGACTGTGCCCCACCCCAATATCGAAGAGGTGGCCCTTAGTAC
ACTAGT
GTGCTGGACTCTCACTACCAGGATGTCCTGAAGGAAGTAAAAGC
SEQ ID NOS: 7-14 show the HCV antigen domains E1a, E2, ProtA, Prot B, Hel A, Hel B, Hel C, and NS5 bpalm. These were expressed as antibody fusion proteins. For all constructs, amino acids TS and AS (shown in red) have been added for cloning purpose to the mosaic HCV sequence. NS5b palm has been constructed based on NS5b 3D structure (1C2P). It is based on structural domain corresponding of the palm domain of NS5b polymerase and do not correspond to the linear amino acid sequence;
TSVGQLFTFSPRRHWTTQDCNCSIYPGHITGHRMAWDMMMNWSPTTAVVA
In SEQ ID NO: 7 membrane domain and predicted unfolded regions have been removed from E1 mosaic 192 aa sequence to increase expression of the Ab fusion protein.
TSETHVTGGSAARTTAGLAGLFTPGAKQNIQLINTNGSWHINRTALNCND
In SEQ ID NO: 8 the membrane domain has been removed for E2 mosaic sequence.
NS3Protease has been cut in 2 structural domains based on its 3D structure (IJXP).
TSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCI
TSTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGP
FQVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSK
VTVPHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDE
PSGMFDSSVLCECYDAGCAWYELTPAETTVRLRAYMNTPGLPVCQDHL
VTAS
TSVLDSHYQDVLKEVKAAASKVKANALYDVVSKLPLAVMGSSYGFQYSPG
HCV sequence and HCV domains constructions: Due to the high polymorphism of HCV, a sequence that is representative of most of circulating HCV sequence was selected.
A mosaic sequence was derived using the mosaic vaccine tools at http://www.hiv.lanl.gov/content/sequence/MOSAIC/ interface choosing mosaic sequence cocktail, 1 as cocktail size and 9 as epitope size. We used 213 sequences for NS3 mosaic and 310 sequences for NS5b mosaic. All sequences correspond to complete genes of 1893, 1773 nucleotides respectively and found in euHCVdb (available on the internet at: euhcvdb.ibcp.fr/euHCVdb/).
Synthetic corresponding genes were purchased from Bio Basic Inc. (Ontario Canada). For cloning purposes, Spe cloning site was introduced at 5′ end and Nhe, EcoRI and Not I at the 3′ end. HCV domains were then constructed by PCR. NS3Protease domain B was construct using the synthetic gene cloned in pUC57 as template and the following primers: NS3Protease domain B forward: 5′-GAGCTCGGATCCACTAGTACTCCTTGTACCTGCGGCTCATCC−3′ (SEQ ID NO: 148) NS3Protease domain B reverse: 5′-GCCCGCGGCCGCGAATTCTCAGCTAGCACTCTGCGGCACTGCTGGGGG-3′ (SEQ ID NO: 149). NS3Helicase domain B was ordered directly as a synthetic gene. For NS5bPolymerase Palm domain construction, regions coding for amino acids 172 to 261 and 276 to 362 were amplified using NS5b synthetic gene and the respective following primers: Ns5b Palm (aa 172-261) forward: 5′-TCTAAAGTCAAAGCGAACGCTCTGTACGATGTCGTTTCC-3′ (SEQ ID NO: 150), Ns5b Palm (aa 172-261) reverse: 5′-ACCGGAAGCGCGACAGCGGCCAACGTACAGGCGTTCGGT-3′ (SEQ ID NO: 151), NS5b Palm (aa 276-362) forward: 5′-ACCGAACGCCTGTACGTTGGCCGCTGTCGCGCTTCCGGT-3′ (SEQ ID NO: 152), NS5b Palm (aa 276-362) reverse: 5′-GCGGCCGCGAATTCttAGCTAGCGGTGATCAGCTCCAG-3′ (SEQ ID NO: 153). Amplified products were then used as templates together with annealed primers 5′-CAAGCCCAACCCCACTAGTGTGCTGGACTCTCACTACCAGGATGTCCTGAAGGAAGTAAAAG CAGCCGCTTCTAAAGTCAAAGCGAACGCTCTGTACGAT-3′ (SEQ ID NO: 154) and 5′-ATCGTACAGAGCGTTCGCTTTGACTTTAGAAGCGGCTGCTTTTACTTCCTTCAGGACATCCTG GTAGTGAGAGTCCAGCACACTAGTGGGGTTGGGCTTG-3′ (SEQ ID NO: 155) in a final PCR using primers 5′-CAAGCCCAACCCC-3′ (SEQ ID NO: 156) and 5′-GCGGCCGCGAATTCTTAGCTAGCGGTGATCAGCTCCAG-3′ (SEQ ID NO: 157). The amplified NS5bPolymerase Palm domain was then cloned in TA vector and sub-cloned in XX vector using Nhe/Not strategy.
Chimeric Recombinant Antibodies Purification: For construct selection, chimeric DC-specific antibodies were transiently expressed in HEK293 cells and purified from the supernatant using Protein A sepharose chromatography. DNA from chimeric constructs expressed in HEK293 was then sub-cloned in cetHSpuro vector as AgeI/NotI fragment for expression in CHO cells after stable transfection. Antibodies were purified from supernatants using ProteinA sepharose.
Patients were recruited at the Baylor Hospital Liver Transplant Clinic (BHLTC, Dallas, Tex.) after obtaining informed consent. The study was approved by the Institutional Review Board of the Baylor Health Care System (Dallas, Tex.). Peripheral blood (100 ml) was collected at the BHLTC from 29 chronic HCV-infected adult patients and one healthy donor in contact with chronic HCV-infected patient. Leukapheresis were collected at Baylor University Medical Center Apharesis Collection Center (Dallas, Tex.) from all the enrolled individuals within 30 days after the first visit. Patient information is summarized in Table I.
Preparation of dendritic cells and PBMCs: PBMCs were isolated from heparinized blood on Ficoll density gradients. Monocytes were enriched from the leukapheresis according to cellular density and size by elutriation (Elutra™, CaridianBCT, Lakewood, Colo.) as per the manufacturer's recommendations. Elutriation Fraction 5 consisted mainly on monocytes (85% on average). Cells were cryopreserved in 10% DMSO 50% FCS 10% culture medium before use. For dendritic cell generation, monocytes were resuspended in serum-free CellGro DC culture medium (CellGenix Technologie Transfer Gmbh, Germany) at a concentration of 1 106 cells/ml. Media were supplemented with 100 ng/ml granulocyte-macrophage colony-stimulated factor (GMCSF, Leukine, Berlex, Wayne, N.J.) and 500 UI/ml alpha-interferon (IFN-α, Intro A, IFN-α-2b, Merck/Schering-Plough, Kenilworth, N.J.). After 24 h of culture at 37 degree Celsius, 5% CO2, fresh cytokines were added. On day 3, recombinant antibody vaccines were added at various concentration (5 nM, 0.5 nM or 0.05 nM) or peptide cluster controls (2 μM each peptide) as indicated. Alternatively, TLR agonists (polyIC, 25 μg/ml; CL075 1 μg/ml; or PAM3, 200 ng/ml; all from Invivogen) were added in the culture at the same time as vaccine candidates or peptide controls. DC were pulsed for 16 h before harvest and used in PBMCs co-culture.
Expansion of Antigen-specific T cells in DC/PBMCs coculture. Frozen PBMCs from leukapharesis were thawed, washed by centrifugation and resuspended at 2×106 cells/ml in cRPMI medium. Autologous DC loaded with vaccine candidates or peptides cluster controls were co-cultured with PBMCs in a 24 well tissue plate at a ratio of 1/20 and incubated for a total of 10 days. IL2 (20 IU/ml, Aldesleukine, ProleukineR; Bayer Healthcare and Novartis, Emeryville, Calif.) was added every two days. At day 9, PBMCs from a 24-well plate were washed, distributed in 2 wells in a 96-well plates and rested for 24 h. The specificity of the T-cell response elicited by vaccine candidate loaded-DC was assessed by restimulation of PBMCs with peptide clusters (2 μM each peptide). For each condition, a negative background control was included as a restimulation without peptides.
Flow cytometry: After 1 hour of peptide clusters restimulation, BFA (Sigma) was added for the last 5-6 h to block cytokine secretion. The cells were stained for surface markers with a combination of fluorochrome antibodies (perCP-CD3, PE-CD8, APCH7-CD4), fixed, permeabilized and intracellular-stained with a mixture of APC-IFNγ, FITC-IL2 and PEcy7-TNFα antibodies. For CTL marker function analysis, FITC-CD107a antibody was added with BFA in the culture medium and the following antibodies combination was used for the surface staining: PerCP-CD3, pacific blue-CD8, APCH7-CD4 and for the intracellular staining: PE-IFNγ, APC-GranzB, APCcy7-TNFα. All antibodies were purchased from BD sciences except APC-GranzB (Invitrogen). Cells were analyzed on a FACS-Canto collecting 500,000 events, and results analyzed using FlowJo software. Most of the data were displayed as two colors plot to measure IFN-γ and TNF-α production in CD3+CD8+ or CD3+CD4+ cells.
Luminex: Supernatants of DC-PBMCs co-culture were harvested 48 h after PBMCs restimulation with peptide clusters. Cytokine multiplex assays were employed to analyzed IFN-γ, IL-10, and IL-13.
Evaluation of embodiments of vaccines: Vaccine candidate were tested in targeting experiment by co-culture of vaccine with PBMCs from chronic HCV infected patients or chronic HCV infected patients cured after IFNa-Ribavirin therapy. The data show that anti-CD40 or anti-DCIR vaccines bearing a HCV NS3HelB antigen can recall a potent memory antigen-specific anti-CD4+ T cell response in vitro using immune cells from HCV infected patients. In this in vitro culture system anti-CD40 and anti-DCIR are equally potent vaccines—these DCs express both receptors. Anti-DCIR vaccine construct bearing longer HCV antigen coverage induced multifunctional CD4+ antigen specific T cells against multiple HCV epitopes.
The data further show that anti-DCIR vaccines bearing a HCV NS3HelBC antigen can recall a potent memory antigen-specific anti-CD4+ T cell response in vitro using immune cells from HCV infected patients. This response is directed against multiples HCV epitopes. In this in vitro culture system, both concentration used for anti-DCIR HCV-NS3HelBC targeting are equally potent in contrast to anti-DCIR HCV-NS3HelB vaccine.
Longer construct are equally potent to recall multi epitopes HCV specific T cells. The data in e.g.,
The data in
Similar responses are induced in multiple different chronic HCV infected patients either cured or after therapy or in treatment failure.
The data in
The data in
CD8+ antigen specific T cells were obtained after TLR agonist introduction in the co-culture of vaccine with PBMC cells from HCV patients.
The data in
The data in
All tested HCV patients are able to recall CD4+ and CD8+ HCV specific memory after DC-targeting with HCV vaccine candidates.
It was also observed that different combinations of HCV domains on vaccine candidate are equally equivalent to recall CD4+HCV memory. Moreover, HCV antigen combination where two domains are borne on heavy chain and one on light chain is more efficient than having the 3 borne by heavy chain.
The vaccine candidates described in the present invention also showed the ability induce cross reactivity recall memory responses in patients infected with an HCV genotype different from those used to build the vaccine (
Non-limiting examples different DC-specific antibodies or fragments (both nucleotide and protein sequences) that may be used in the preparation of the HCV vaccine of the present invention are shown herein below, the nomenclature corresponding to the target (e.g., Anti_CLEC_6_9B9.2G12_Heavy Hv-V-hIgG4H-C—is an anti-CLEC-6 antibody from the mouse hybridoma clone “9B9.2G12” (which is the source of the anti-CLEC-6 antibody sequence); heavy chain “H” variable region “v” (which can be humanized) heavy and is an IgG4 constant region isotype. The same nomenclature applies to light chains (from mouse Kappa light chains), and the antigens.
Humanized anti-CD40-HCV vaccine is: hAnti-CD40VK2-LV-hIgGK-C×hAnti-CD40VH3-LV-hIgG4H-C-Flex-v1-HelB-f1-ProtB-f2-NS5BPalm, wherein the portion of HelB are underlined, the portions of ProtB are bold and the portions of NS5BPalm are italicized. The linker sequence (in bold italics) is flanked by the transition sequence “AS” that bracket the linker sequences
ASVTVPHPNI
EEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKLVAL
GINAVAYYRGLDVSVIPTSGVVVVVATDALMTGFTGDFDSVIDCNTC
VTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAP
GERAS ASTPCTCGSSDLYLVTRHADVIPVRRRGDS
RGSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVCTRGVAKAVD
FIPVENLETTMRSPVFTDNSSPPAVPQSAS ASVLDS
HYQDVLKEVKAAASKVKANALYDVVSKLPLAVMGSSYGFQYSPGQ
RVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDL
DPQAFVAIKSLTERLYVGRCRASGVLTTSCGNTLTCYIKARAACRAA
GLQDCTMLVCGDDLVVICESAGVQEDAASLRAFTEAMTRYSAPPG
DPPQPEYDLELITAS
Humanized anti-DCIR-HCV 1st generation vaccine is: [hAnti-DCIRVK4-LV-hIgGK-C]×[hAnti-DCIRVH1-LV-hIgG4H-C-Flex-v1-HelB-f1-ProtB-f2-NS5BPalm] wherein the portion of HelB are underlined, the portions of ProtB are bold and the portions of NS5BPalm are italicized. The linker sequence (in bold italics) is flanked by the transition sequence “AS” that bracket the linker sequences
ASVTV
PHPNIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKKKCDELAAKL
VALGINAVAYYRGLDVSVIPTSGVVVVVATDALMTGFTGDFDSVIDCNT
CVTQTVDFSLDPTFTIETTTLPQDAVSRTQRRGRTGRGKPGIYRFVAP
GERAS ASTPCTCGSSDLYLVTRHADVIPVRRRGDSR
GSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFI
PVENLETTMRSPVFTDNSSPPAVPQSAS SVLDSHYQ
DVLKEVKAAASKVKANALYDVVSKLPLAVMGSSYGFQYSPGQRVE
FLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTEEATYQCCDLDPQ
ARVAIKSLTERLYVGRCRASGVLTTSCGNTLTCYIKARAACRAAGLQD
CTMLVCGDDLVVICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQ
PEYDLELITAS
Humanized anti-CD40-HCV vaccine is: hAnti-CD40VK2-LV-hIgGK-C-ViralHCVhelicasefgtB×hAnti-CD40VH3-LV-hIgG4H-C-Flex-v1-ProtB-f1-NS5BPalm, wherein the portion of ViralHCVhelicasefgtB are underlined, the portions of ProtB are bold and the portions of NS5BPalm are italicized. The linker sequence (in bold italics) is flanked by the transition sequence “AS” that bracket the linker sequences.
IPLEVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTS
GVVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTL
PQDAVSRTQRRGRTGRGKPGIYRFVAPGERAS
ASTPCTCGSSDLY
LVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGIF
RAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQSAS
ASVLDSHYQDVLKEVKAAASKVKANALYDVVSKLPLAVMG
SSYGFQYSPGQRVEFLVQAWKSKKTPMGFSYDTRCFDSTVTESDIRTE
EAIYQCCDLDPQARVAIKSLTERLYVGRCRASGVLTTSCGNTLTCYIKAR
AACRAAGLQDCTMLVCGDDLVVICESAGVQEDAASLRAFTEAMTRYS
APPGDPPQPEYDLELITAS
Humanized anti-DCIR-HCV 2nd generation vaccine is: [hAnti-DCIRVK4-LV-hIgGK-C-ViralHCVhelicasefgtB]×[hAnti-DCIRVH1-LV-hIgG4H-C-Flex-v1-ProtB-f1-NS5BPalm], wherein the portion of ViralHCVhelicasefgtB are underlined, the portions of ProtB are bold and the portions of NS5BPalm are italicized. The linker sequence (in bold italics) is flanked by the transition sequence “AS” that bracket the linker sequences.
EVIKGGRHLIFCHSKKKCDELAAKLVALGINAVAYYRGLDVSVIPTSGVV
VVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTLPQDA
VSRTQRRGRTGRGKPGIYRFVAPGERAS
ASTPCTC
GSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPA
GHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVETDNSSPPAVPQ
SAS
ASVLDSHYQDVLKEVKAAASKVKANA
LYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSKKTPMGESYD
TRCFDSTVTESDIRTEEATYQCCDLDPQARVAIKSLTERLYVGRCRAS
GVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGDDLVVICESAGV
QEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITAS
Linkers can be a small as 2 amino acids, e.g., AS, but can also be longer, e.g., SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO.: 166); PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO.: 167); TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO.: 168); TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO.: 169) or QTPTNTISVTPTNNSTPTNNSNPKPNP (SEQ ID NO:170).
It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
U.S. Patent Application Publication No. 2009/0238822: Chimeric HCV Antigens for Eliciting an Immune Response.
U.S. Patent Application Publication No. 2008/0241170: Vaccines Based on Targeting Antigen to DCIR Expressed on Antigen-Presenting Cells.
U.S. Patent Application Publication No. 2010/0239575: Anti-CD-40 Antibodies and Uses Thereof.
This application is a divisional of co-pending U.S. patent application Ser. No. 13/430,206 filed Mar. 26, 2012, which claims priority to U.S. Provisional Application Ser. No. 61/467,840, filed Mar. 25, 2011, and U.S. Provisional Application Ser. No. 61/529,700, filed Aug. 31, 2011, the entire contents of each of which are incorporated herein by reference.
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| Number | Date | Country | |
|---|---|---|---|
| 20140199763 A1 | Jul 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61467840 | Mar 2011 | US | |
| 61529700 | Aug 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13430206 | Mar 2012 | US |
| Child | 14152448 | US |
