Imported: 10 Mar '17 | Published: 27 Nov '08
USPTO - Utility Patents
The present invention concerns inventive polypeptides. The present invention also concerns compositions and vaccines comprising the inventive polypeptides. In other embodiments of the invention, the inventive polypeptides are provided to a subject, used to vaccinate, or used to induce immunity. Other embodiments include methods for making the inventive polypeptides and nucleic acids used to encode the inventive polypeptides.
This application claims benefit to U.S. Provisional Application No. 60/893,318, filed Mar. 6, 2007, which is incorporated by reference in its entirety. This application is also a continuation-in-part of co-pending U.S. application Ser. No. 10/096,521, filed Mar. 11, 2002, which is incorporated by reference in its entirety, which claims benefit to U.S. Provisional Application No. 60/274,392, filed Mar. 9, 2001, which is incorporated by reference in its entirety.
This invention was made with government support under 1R21A1058003, awarded by NIH/NIAID and under RG 2978-A-201 awarded by National Multiple Sclerosis Society. The U.S. Government may have certain rights in the invention.
The present invention relates generally to the field of immunology.
Induction of immunity to pathogens, toxins, and peptides expressed by tumor cells, requires the coordinated participation of the innate and adaptive immune systems. An early step is Ag internalization by APCs of the innate immune system, notably by dendritic cells (DCs), the most potent APC type, and the one best able to present Ag to nave T cells (Trombetta and Mellman, 2005). Internalized Ag is processed through the endosomal/lysosomal path. Processed peptides, bound to MHC molecules, are then delivered to the cell surface. Those T cells with appropriate receptors respond to such peptides provided co-stimulatory molecules are expressed by the DC. A second signal is often required to drive DC maturation and efficient co-stimulatory molecule expression. Ag activates B cells bearing appropriate surface immunoglobulin directly to produce IgM. CD4+ T cells, having responded to processed Ag, induce immunoglobulin class-switching from IgM to IgG.
Limited uptake of soluble antigenic peptide by DCs constrains subsequent Ag processing and presentation. Immune responses increase when Ag uptake is facilitated. IgG-immune complexes (ICs) bind to FcRs expressed on DCs and this is followed by internalization of ICs with their captured Ags. Thus, stronger Ab responses may occur when soluble Ag is complexed to IgG, than when Ag alone is administered (Wemersson et al., 1999). ICs in antibody excess can be more effective at Ag presentation than ICs at equivalence or in Ag excess (Manca et al., 1991). IC driven, FcR-mediated, Ag internalization favors DC maturation and hence expression by them of costimulatory molecules (Regnault et al., 1999). Other means to target Ag to Fc receptors on APCs have been employed in order to elicit strong immune responses against otherwise weak immunogens. Early studies, that documented the potential of this approach, employed Ag-containing anti-FcR monoclonal antibodies as a means to facilitate delivery of Ag to APCs and hence increase Ag-specific T cell responses and Ag-specific humoral responses (Snider et al., 1990; Heijnen et al., 1996; Gosselin et al., 1992; Keler et al., 2000). Modification of Ig by introduction of epitopes within the CDR region (i.e., antigenized Ig) may also enhance immune responses compared to Ag alone (Zaghouani et al., 1993, Brummeanu et al., 1996).
Immune complexes (IC) exhibit diverse biological activities; some that contribute to disease whereas others ameliorate disease. Deposition of IgG containing IC on tissue surfaces, as for example in glomeruli, can contribute to the pathogenesis of antibody-mediated autoimmune diseases. On the other hand, IC can favorably modulate T- and B-cell activation pathways via binding to Fc receptors expressed on immunocytes. Aggregated IgG (AIG) shares some features and biological activities with IC. Both modulate T-cell suppressor function (Antel et al., 1981; Durandy et al., 1981), cytokine synthesis, IgG secretion, and lymphocyte proliferation (Berger et al., 1997; Wiesenhutter et al., 1984; Ptak et al., 2000).
Monomeric IgG, or the Fc fragment thereof, can ameliorate disease progression in animal models of autoimmune disease (Miyagi et al., 1997; Gomez-Guerrero et al., 2000). Monomeric IgG can be used therapeutically, usually in massive doses, to treat antibody-mediated diseases in man. The protective effect in antibody-mediated diseases may be achieved in part through blockade of FcRs such that binding of IC to them is impeded (Clynes et al., 1998). IgG administration also favorably affects the course of T-cell mediated autoimmune diseases such as multiple sclerosis (Fazekas et al. 1997; Sorensen et al., 1998; Achiron et al., 1998). Here the basis for benefit is poorly understood though it is postulated to involve the increased production of anti-inflammatory cytokines initiated by binding of IV IgG, or complexes derived therefrom, to FcR. In both antibody and T-cell mediated processes the mechanisms and consequences of FcR engagement are fundamental to the understanding and treatment of autoimmune diseases.
Aggregated IgG has been proposed as a treatment for autoimmune diseases of humans. The use of aggregated IgG has been studied as a treatment for multiple sclerosis and other autoimmune diseases. However, aggregated IgG has major limitations. IgG is commonly aggregated by exposure to heat; the resultant aggregates are bound together in a random fashion limiting reproducibility from one preparation to the next. Preparations contain a heterogeneous collection of aggregates of varying size in diverse conformations.
U.S. Pat. Nos. 5,714,147 and 5,455,165 disclose some hybrid immunoglobulin molecules and the expression vectors encoding them. These chimeric molecules can improve the circulating plasma half-life of ligand binding molecules, and can comprise a lymphocyte homing receptor fused to an immunoglobulin constant region. Homo or hetero-dimers or tetramer hybrid immunoglobulins containing predominantly the heavy and light constant regions of immunoglobulin have been used. U.S. Pat. No. 6,046,310 discloses FAS ligand fusion proteins comprising a polypeptide capable of specifically binding an antigen or cell surface marker for use in treatment of autoimmune disorders. The fusion protein preferably comprises IgG2 or IgG4 isotype, and may comprise antibodies with one or more domains, such as the CH2, CH1 or hinge deleted. Majeau et al. (1994) discusses Ig fusion proteins used for the inhibition of T cell responses. These fusion proteins comprise IgG1 and LFA-3. Eilat et al. (1992) disclose a soluble chimeric Ig heterodimer produced by fusing TCR chains to the hinge region, CH2, and CH3 domains of human IgG1.
Immunoglobulin fusion proteins can be employed to express proteins in mammalian and insect cells (Ashkenazi, et al., 1997). Fusion protein platforms can permit the introduction of additional functions, for example, inclusion of the amino-terminal CD8 domain may result in the co-ligation of FcR on lymphocytes to MHC I on antigen presenting cells (Alcover, et al., 1993; Meyerson, et al., 1996).
Other Ig proteins and variants have also been studied for their therapeutic effect on autoimmune diseases, including a recombinant polymeric IgG that mimics the complement activity of IgM (Smith and Morrison, 1994) where the polymeric IgG is formed by the polymerization of H2L2 subunits. Greenwood et al. (1993) discusses therapeutic potency relative to the structural motifs involving the human IgG antibodies, IgG1, IgG3, and IgG4. U.S. Pat. No. 5,998,166 discloses human FcR-III variants, which can be used in the therapy or diagnosis of autoimmune diseases. U.S. Pat. No. 5,830,731 discloses novel expression vectors in which cell surface antigens cloned according to that invention appear to have diagnostic and therapeutic utility in immune-mediated infections. Cell surface antigens that are used to regulate lymphocyte activation, appear to achieve antigen aggregation in vitro by incubating lymphocytes with immobilized ligands or antibodies or their fragments (WO9942077). However, the aggregated IgG and Fc aggregates have limited reproducibility, containing a random and heterogeneous mixture of protein thereby limiting their effectiveness as therapeutic agents. Other problems include a lack of an ability to target a number of cell types with a single agent and size limitations.
In some embodiments, the inventive polypeptide has an immunoglobulin framework and consists of an Fc region linked to two arms. The Fc region consists of two Fc amino acid chains and each Fc amino acid chain is linked to one of the two arms. Each arm consists of an HCH2 polymer linked to an antigen portion, the HCH2 polymer consists of two to six linear copies of an HCH2 monomer, and the HCH2 monomer consists of at least a fragment of an HCH2 region. At least a fragment of an HCH2 region includes a hinge region; and at least one hinge region cysteine of the HCH2 monomer is mutated to serine.
In exemplary embodiments, the inventive polypeptide can consist of two amino acid chains where each amino acid chain consists of (a) an Fc portion which includes the C-terminus of the amino acid chain; (b) a polymer portion consisting of two to six linear copies of an HCH2 monomer; and (c) an antigen portion which includes the N-terminus of the amino acid chain. The N-terminus of the Fc portion is linked to the C-terminus of the polymer portion, and the N-terminus of the polymer portion is linked to the C-terminus of the antigen portion. The two amino acid chains are linked using one or more disulfide bonds located in the Fc portion of each amino acid chain. The HCH2 monomer can consist of at least a fragment of an HCH2 region, wherein the at least a fragment of an HCH2 region includes a hinge region.
In some embodiments, the inventive polypeptide has Fc amino acid chains selected from the group consisting of: an amino acid chain of the IgG1 Fc region, an amino acid chain of the IgG3 Fc region, an amino acid chain of the IgG2a Fc region, SEQ ID NO: 47, SEQ ID NO: 48, and fragments thereof.
In some embodiments, the inventive polypeptide is capable of binding to FcR or of targeting cells expressing FcR.
In some embodiments, the inventive polypeptide has the HCH2 region selected from the group consisting of: a human IgG1 HCH2 region, a human IgG2 HCH2 region, a human IgG3 HCH2 region, a human IgG4 HCH2 region, a mouse IgG2a, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 50, SEQ ID NO: 51, and fragments thereof.
In some embodiments, the inventive polypeptide has three hinge region cysteines of the HCH2 monomer mutated to serine.
In some embodiments, the antigen portion is an antigen or an epitope, which can be a protein or protein fragment, a Botulinum neurotoxin protein or fragment thereof, BoNT/A Hc, BoNT/A HcN, BoNT/A HcC, HSA1, CD8a, FABP7, PLP, MBP, PLP-MBP, PLP-PLP, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 43, or fragments thereof.
In some embodiments, a composition or vaccine comprises the inventive polypeptide.
In other embodiments, the inventive polypeptide is provided to a subject, used in a vaccine, or used to induce immunity.
Other embodiments include methods for making the inventive polypeptides or the nucleic acids used to encode the inventive polypeptides.
The present invention concerns inventive polypeptides. The present invention also concerns compositions and vaccines comprising the inventive polypeptides. This invention describes vaccines and methods of inducing immunity against an antigen using inventive polypeptides described herein. The present invention provides for improved vaccine efficacy by targeting Ag to Fc receptors using multiple copies of HCH2 of a human IgG. The vaccines of the present invention can be applied, for example, in the induction of immunity to pathogens, toxins, and peptides expressed by tumor cells. In other embodiments of the invention, the inventive polypeptides are provided to a subject. Still other embodiments include methods for making the inventive polypeptides and nucleic acids used to encode the inventive polypeptides.
Antibodies comprise a large family of glycoproteins with common structural features. An antibody is comprised of four polypeptides that form a three dimensional structure which resembles the letter Y. Typically, an antibody is comprised of two different polypeptides, termed the heavy chain and the light chain.
An antibody molecule typically consists of three functional regions: the Fc, Fab, and antigen-binding site. The Fc region is located at the base of the Y. The arms of the Y comprise the Fab region. The antigen-binding site is located at the end of each arm of the Y. The area at the fulcrum of the arms of the Y is the hinge region.
There are five different types of heavy chain polypeptides designated as , , , , and . There are two different types of light chain polypeptides designated and . An antibody typically contains only one type of heavy chain and only one type of light chain, although any light chain can associate with any heavy chain.
Antibody molecules are categorized into five classes, IgG, IgM, IgA, IgE, and IgD. The IgG class is further divided into subclasses including IgG1, IgG2, IgG3, and IgG4 for human IgG. An antibody molecule is comprised of one or more Y-units, each Y comprising two heavy chains and two light chains. For example IgG consists of a single Y-unit IgM is comprised of 5 Y-like units.
Amino acids toward the carboxyl terminal of each heavy chain polypeptide make up a constant region. Amino acids toward the amino terminal of each heavy and light chain polypeptide make up a variable (V) region. Within the variable region are hypervariable regions known as complementarity determining regions (CDRs). One heavy chain and one light chain associate to form an antigen-binding site. Each heavy chain and each light chain includes three CDRs. The six CDRs of an antigen-binding site define the amino acid residues that form the actual binding site for the antigen. CDR variability accounts for the diversity of antigen recognition.
The mature human IgG1 heavy (H) chain can span 447 amino acid residues. The Fc region of the H chain is essentially the same for all IgG1 heavy chain molecules. The Fc region is the portion of the IgG1 polypeptide that interacts with Fc receptors. The Fc region can be further subdivided into three consecutive parts, the hinge region, the CH2 domain, and the CH3 domain. For human IgG1, the binding site for Fc receptors is found within the hinge and CH2 (HCH2) region. The HCH2 region encompasses amino acid residues 216 to 340 of the human IgG1 H chain (Eu numbering). The hinge region spans residues 216 to 237 whereas the CH2 domain encompasses residues 238 to 340.
Capon, et al., 1989, Traunecker, et al., 1989, Chamow, et al., 1996, and Ashkenazi, et al., 1997 disclose some examples of fusion proteins related to immunology. A recombinant immunoglobulin fusion protein can have an amino-terminus composed of a ligand-binding domain fused to a carboxyl-terminus composed of the hinge, CH2, and CH3 regions of Ig. The Ig class sometimes used is IgG1. The hinge, CH2, and CH3 regions of IgG are collectively referred to as the Fc region of IgG. The hinge region can provide a flexible linker between the Fc region and the ligand binding domain. It also is the site of inter-chain disulphide bond formation, i.e., the covalent linking of one antibody amino acid chain to another to make the familiar dimeric structure. The hinge region (e.g., the part nearest to the CH2 domain, known as the hinge proximal region) is associated with molecular recognition and binding to Fc receptors and complement components. Thus, some recombinant immunoglobulin fusion proteins are similar to Ig but lack the variable regions and the CH1 domain, which have been replaced by the ligand-binding domain. Sometimes, the recombinant molecule is generated at the cDNA level using recombinant DNA techniques and expressed in cell culture. In some instances, the recombinant immunoglobulin fusion protein is a disulfide-linked homodimer. There have been some variations on the above described fusion proteins. For example, in addition to ligand-binding domains, other fusion partners have been placed at the amino-terminus, such as ligands, enzymes, and peptide epitopes.
The term amino acid chain includes a linear chain of amino acids. The amino acid chain can be chemically or biochemically modified (such as, but not limited to, glycosylation or phosphorylation) or derivatized amino acids, and can have a modified peptide backbone.
The term polypeptide refers to a polymeric form of amino acids of any length, and includes chemically or biochemically modified or derivatized amino acids, as well as amino acid chains having modified peptide backbones. The term includes amino acid chains that are linked, for example, by one or more disulfide bonds, proteins, amino acid chains, saccharides, or polysaccharides.
A fragment of a polypeptide or protein refers to a polypeptide that is shorter than the reference polypeptide or protein, but that can retain a biological function or activity that is recognized to be the same as the reference polypeptide or protein. Such an activity may include, for example, the ability to stimulate an immune response. A fragment may retain at least one epitope of the reference polypeptide or protein. The shorter polypeptide may retain all or part of a modification (e.g., by glycosylation or phosphorylation) of the reference polypeptide or protein.
Immunological framework refers to a molecule that comprises two arms attached to an Fc region. The Fc region has the primary structural components of an antibody Fc region, but the arms can be comprised of any molecule and thus are not limited to the Fab-antigen structures of an antibody.
In exemplary embodiments, the inventive polypeptide can consist of two amino acid chains where each amino acid chain consists of (a) an Fc portion which includes the C-terminus of the amino acid chain; (b) a polymer portion consisting of two to six linear copies of an HCH2 monomer; and (c) an antigen portion which includes the N-terminus of the amino acid chain. The N-terminus of the Fc portion is linked to the C-terminus of the polymer portion, and the N-terminus of the polymer portion is linked to the C-terminus of the antigen portion. The two amino acid chains are linked using one or more disulfide bonds located in the Fc portion of each amino acid chain. The HCH2 monomer can consist of at least a fragment of an HCH2 region, wherein the at least fragment of an HCH2 region includes a hinge region. In some embodiments, at least one hinge region cysteine of the HCH2 monomer is mutated to serine. The Fc portion can comprise, for example, SEQ ID NO: 47 or SEQ ID NO: 48.
In other exemplary embodiments, the inventive polypeptide has an immunoglobulin framework consisting of an Fc region consisting of two amino acid chains wherein each amino acid chain is linked to an arm. Each arm can consist of an HCH2 polymer linked to an antigen portion. The HCH2 polymer can consist of two to six linear copies of an HCH2 monomer, which consists of at least a fragment of an HCH2 region. In some embodiments at least one hinge region cysteine of the HCH2 monomer is mutated to serine, or another non-cysteine amino acid. Sometimes all the hinge region cysteines are mutated. In some embodiments, the Fc region comprises the linked Fc portions.
The inventive polypeptide can, for example, bind to FcR, target cells expressing FcR, or complement components.
The Fc region can be selected or derived from any animal, mammalian, mouse, or human antibody. For example, the Fc region can combine polypeptides of Fc regions from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, IgE, IgG2a, or fragments thereof. In some embodiments, the combined polypeptides are identical. Some embodiments of the fragments include fragments comprising a hinge region, a CH2 domain, and a CH3 domain. Exemplary embodiments of sequences that can be used to form the amino acid chain of the Fc region can include, but are not limited to SEQ ID NO: 47 and SEQ ID NO: 48.
Linkers can include, but are not limited to, amino acid chains, disulfide bonds, saccharides, polysaccarides, or any known linkers. For example, amino acid chains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 20 amino acids can be used as linkers.
In some embodiments, the HCH2 monomer can be selected or derived from an Fc region of any animal, mammalian, mouse, or human antibody. It can be a polypeptide from the Fc regions of, for example, but not limited to, IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM, IgE, IgG2a, or fragments thereof. For example, the HCH2 region can be selected from the group consisting of: a human IgG1 HCH2 region, a human IgG2 HCH2 region, a human IgG3 HCH2 region, a human IgG4 HCH2 region, a mouse IgG2a region, and fragments thereof. Exemplary embodiments of sequences that can be used in an HCH2 monomer include, but are not limited to, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 51, and SEQ ID NO: 52.
The HCH2 polymer can be made from, for example, 1, 2, 3, 4, 5, 6, 7, or 8 linear copies of an HCH2 monomer.
The antigen portion includes, but is not limited to, antigens, polypeptides, proteins protein fragments, or any combination thereof. For example, the antigen portion can include proteins or protein fragments linked together in a serial fashion, such as PLP linked to PLP, a fragment of PLP linked to PLP, a fragment of PLP linked to another fragment of PLP, a fragment of PLP linked to a fragment of MBP, MBP linked to a fragment of PLP. In some embodiments, the antigen portion is a Botulinum neurotoxin protein, including for example, BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/A Hc, BoNT/A HcN, BoNT/A HcC, or portions, fragments, or variants thereof. In some embodiments, the antigen portion can contain at least one antigenic domain or epitope of an infectious agent, microorganism, tumor antigen, or self protein, including for example, cancer antigens (such as, sarcoma, lymphoma, leukemia, melanoma, carcinoma of the breast, colon carcinoma, carcinoma of the lung, glioblastoma, astrocytoma, carcinoma of the cervix, uterine carcinoma, carcinoma of the prostate, ovarian carcinoma, or portions, fragments, or variants thereof), antigenic domains of infectious agents, antigenic domains of viruses (such as, papilloma virus, Epstein Barr virus, herpes virus, retrovirus, hepatitis virus, influenza virus, herpes zoster virus, herpes simplex virus, human immunodeficiency virus 1, human immunodeficiency virus 2, adenovirus, cytomegalovirus, respiratory syncytial virus, rhinovirus, or portions or variants thereof), antigenic domains of a bacteria (from bacteria such as, Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, Mycoplasma pneumoniae, or portions or variants thereof), toxin polypeptides (such as, abrin, a conotoxin, diacetoxyscirpenol, ricin, saxitoxin, a Shiga-like ribosome inactivating protein, flexal, guanarito, junin, machupo, sabia, tetrodotoxin, a Botulinum neurotoxin, Clostridium perfringens epsilon toxin, a Shigatoxin, Staphylococcal enterotoxin, T-2 toxin, Bovine spongiform encephalopathy agent, epsilon toxin, ricin toxin, Staphylococcal enterotoxin B, influenza virus hemagglutinin, toxoids, or portions, fragments, or variants thereof), tumor antigens (such as, KS pan-carcinoma antigen, ovarian carcinoma antigen (CA125), prostatic acid phosphate, prostate specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigens such as: CEA, TAG-72, CO17-1A; GICA 19-9, CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19, human B-lymphoma antigen-CD20, CD33, melanoma specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, tumor-specific transplantation type of cell-surface antigen (TSTA), bladder tumor oncofetal antigen, differentiation antigen such as human lung carcinoma antigen L6, L20, an antigen of fibrosarcoma, human leukemia T cell antigen-Gp37, neoglycoprotein, a sphingolipid, EGFR, EGFRvIII, FABP7, doublecortin, brevican, HER2 antigen, polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen-APO-1, an I antigen, M18, M39, SSEA-1, VEP8, VEP9, Myl, VIM-D5, D156-22, TRA-1-85, C14, F3, AH6, Y hapten, Ley, TL5, EGF receptor, FC10.2, gastric adenocarcinoma antigen, CO-514, NS-10, CO-43, G49, MH2, a gastric cancer mucin, T5A7, R24, 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, M1:22:25:8, SSEA-3, SSEA-4, or portions, fragment, or variants thereof), autoantigens from a mammal (such as, myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), collagens, insulin, proinsulin, glutamic acid decarboxylase 65 (GAD65), an islet cell antigen, portions, fragments, or variants thereof). Other examples of antigen portions include, for example, HSA, HSA1 (HSA domain 1), HSA2 (HSA domain 2), HSA3 (HSA domain 3), Fatty Acid Binding Proteins (FABP) such as FABP1, FABP2, FABP3, FABP4, FABP5, FABP6, FABP7, FABP8, FABP9 including FABP5-like 1-7; other examples of antigen portions can be found throughout the application.
While not being by any particular theory, the immunoglobulin Fc region appears to provide some features of the IgG fusion proteins such as stability, covalent dimerization, single-step purification, and ease of detection. The intervening HCH2 polymer appears to confer increased effector function, including, for example, targeting to subsets of cells expressing FcR, increased capacity to ligate FcR, and to bind complement components. The amino-terminal domain can deliver a second signal. Thus, multiple molecular signals can be integrated into a single molecule with the potential for synergistic interaction between the domains.
The inventive polypeptide comprises multiple HCH2 regions. The polymers were developed using a cloning system that can result in the rapid addition of HCH2 units into a human IgG1 Fc region expression vector. Each HCH2 region can be composed of the hinge and CH2 domain from an Ig such as IgG1, which encompasses the region that can bind FcR and complement. In some embodiments, to prevent inter-chain disulfide bond formation between the HCH2 region of the polymer, hinge region cysteines of the HCH2 monomer unit were mutated to serines. These mutations can leave intact those hinge residues that interact with FcR and complement. The hinge within the Fc vector was not mutated thus retaining the dimeric structure of IgG. Several unique restriction sites on the 5 end can allow for the directional cloning of amino-terminal domains into the polymer expression constructs.
In some embodiments of the invention, it is not necessary for the entirety of the HCH2 region to be employed in making the HCH2 monomer. As described above, the entire human IgG1 HCH2 encompasses amino acid residues 216 to 340 of the human IgG1 H chain (Eu numbering), with the hinge region spaning residues 216 to 237 and the CH2 domain encompassing residues 238 to 340. The interactions between IgG and Fc receptors have been analyzed in biochemical and structural studies using wild type and mutated Fc. One consensus indicates that some regions for binding to Fc receptors are located in the part of the hinge region closest to the CH2 domain and in the amino-terminus of the CH2 domain that is adjacent to the hinge, including for example residues 233-239 (Glu-Leu-Leu-Gly-Gly-Pro-Ser). Mutations within this region can result in altered binding to Fc receptors. This region appears to be responsible for some of the direct interactions with Fc receptors. Further into the CH2 domain, and away from the hinge, are other residues that may, at least in some contexts, contribute to Fc receptor binding, including for example, Pro-329 which appears involved in direct contact with the Fc receptor and Asn-297 which appears to be the sole site for N-linked glycosylation within the Fc region. The presence of carbohydrate at this residue may contribute to the binding to Fc receptors. Peptides spanning residues 233-239 of IgG1 Fc may bind to FcRIII poorly.
In the examples presented below the HCH2 polymers were constructed using the human IgG1 HCH2 region that encompasses amino acid residues 216 to 340 of the human IgG1 H chain. This region contains the sequences that may contribute to Fc receptor binding as well as additional flanking residues. The flanking residues provide structural stability and spacing between the HCH2 regions. In some embodiments it can be advantageous to construct HCH2 polymers comprised of fragments within the HCH2 region instead of the entire HCH2 region. This may be done for example to reduce the size of the HCH2 monomer and hence the HCH2 polymer. One way that this could be achieved is through the deletion of flanking residues on either side of the region that has been identified with Fc receptor binding. For instance the hinge could be truncated to span residues 233 to 237 instead of residues 216 to 237 as used in the examples presented herein. Similar considerations apply to the CH2 region that spans residues 238-340 and to the hinge and CH2 regions of other Ig's including IgA, IgD, IgG2, IgG3, IgG4, and IgE. Other embodiments include different configurations of portions of HCH2 regions.
The HCH2 polymers can bind to low affinity FcR. In some instances the HCH2 polymers can bind the high affinity FcR receptors, for instance the FcRI receptor. This is a natural consequence of the high binding affinity of the high affinity FcR receptors for the HCH2 region.
In some instances it can be advantageous to construct HCH2 polymers that bind all forms of the low affinity FcR receptors such as, for example, FcRIIa, FcRIIb, FcRIIc, FcRIIIa, and FcRIIIb. In other embodiments the number and spacing of HCH2 monomers comprising the polymer are varied to increase the binding to one type of FcR receptor or conversely to decrease binding to another type of FcR receptor. In yet other embodiments alterations to the HCH2 monomer can be made to increase specificity of the polymer for one type of FcR receptor or to decrease specific binding to another type of FcR receptor. Such alterations are achieved by mutating certain amino acid residues within the HCH2 sequence to other amino acid residues. The choice of residues to mutate within the HCH2 unit can be informed by choice of target receptor specificity. In other embodiments the specific binding of the HCH2 polymers to different FcR receptors can be enhanced by the presence of and type of glycosylation of the HCH2 polymer. Choice of expression system in which to produce the HCH2 polymers in part determines the extent and type of glycosylation.
In the examples presented herein the HCH2 polymers were constructed using DNA sequences from human IgG1. In some instances it can be advantageous to construct HCH2 polymers comprised solely of human sequences to use as immunotherapeutic agents in humans. However in some embodiments the polymers are assembled from sequences of other Ig's including IgA, IgD, IgG, IgM, and IgE. In other embodiments the HCH2 polymers are assembled from sequences of more than one type of Ig, for example a polymer containing HCH2 monomers derived from IgG sequences are linked to HCH2 monomers derived from IgE sequences. In other embodiments the HCH2 polymers are comprised of non-human sequences. The choice of sequences can be determined by the target receptor and host identity (human or non-human). In yet other embodiments the hinge region cysteines are mutated to amino acid residues other than serine. In some embodiments the HCH2 monomer may be altered or mutated to bind complement components and not to bind to FcR. In other embodiments the HCH2 monomer may be altered or mutated to bind FcR and to not bind complement.
In some examples presented herein the HCH2 polymers were constructed using DNA sequences from human IgG1. The expressed proteins have been evaluated for their interactions with low affinity FcR receptors. However in some embodiments the HCH2 polymers are assembled from sequences of other Ig's including IgA, IgD, IgG, IgM, and IgE and these polymers may bind to and interact with the FcR for other Ig's including FcR, FcR, FcR, FcR, and FcRn. In other embodiments the polymers are assembled from sequences of more than one type of Ig, for example a polymer protein containing HCH2 units derived from IgG sequences and IgE sequences will interact and bind with the FcR for more than one type of Ig.
In the examples presented herein the HCH2 polymers are constructed from HCH2 monomers consisting of full length HCH2 regions. In some embodiments it may be advantageous to construct HCH2 polymers that contain HCH2 monomers that are smaller than full length HCH2 regions. HCH2 polymers derived from a smaller HCH2 monomer would have a smaller size and mass and yet still retain the ability to effectively bind to and activate FcR or complement. The reduction in the size of the HCH2 monomer is achieved by the removal of sequences that have diminished involvement in the binding to FcR or complement. In some embodiments, the identities of these sequences are known, as are the methods for their removal from the HCH2 monomer unit. The removal of these sequences could maintain the desired binding but yield a polymer of smaller mass.
Recombinant HCH2 polymer constructs can mimic the biological activity and functions of immune complexes (ICs), of aggregated IgG (AIG), and of aggregated Fc. The use of recombinant HCH2 polymer construct can offer several advantages over AIG or Fc aggregates. The number and construction of HCH2 monomers can be altered to hone interaction with FcR's. Aggregates are by nature heterogeneous with considerable variation between batches whereas inventive polypeptides are precisely defined.
The receptors can be specifically activated with constructs containing different numbers of HCH2 monomers. As shown herein, the number of repeating HCH2 monomers available to bind receptor can influence cell function. Cell function can be changed with insertion of additional HCH2 monomers. The constructs of the present invention allow for the measurement of change in receptor function based on IC size. The number of repeating HCH2 monomers included within the polymer construct is variable and can be selected to optimize biological activity. In one embodiment the HCH2 polymers are assembled as disulfide-linked homodimers. In some embodiments the HCH2 polymers are assembled as monomers (e.g., a single chain polypeptide of HCH2 monomers), or hetero- or homo-multimers, and particularly as dimers, tetramers, and pentamers.
A nonlimiting list of proteins or protein fragments are found throughout the application and include, for example, ligand-binding domains, extracellular domains of receptors, enzymes, adhesion molecules, cytokines, peptide hormones, immunoglobulin fragments (Fab), ligands, antigens, and fragments thereof. The site of the fusion of the protein or protein fragment connecting it to the linker or HCH2 polymer may be selected to optimize biological activity, stability, secretion, avidity, and binding specificity.
HCH2 polymers using IgG1 were designed using sequence data from the human IgG1 constant region gene as a guide (accession # Z17370). Several amino terminal domains have been expressed fused to the HCH2 polymers: the extracellular domain of human CD8 (accession # M12824), domain I of human serum albumin (accession # V00494), murine fatty acid binding protein 7 (accession # BC057090), human fatty acid binding protein (accession # BC012299), the Hc, HcN, HcC fragments of Botulinum neurotoxin subtype A as well as antigen portions and combinations of PLP and MBP. For some uses it may be advantageous to construct HCH2 polymers composed of the HCH2 polymer region unfused to additional protein domains or Fc or framework sequences.
In certain embodiments, the inventive polypeptides are produced by the insertion of the HCH2 polymeric region into an existing antibody sequence or the sequence of a recombinant protein. This process can be advantageous because of its simplicity. The HCH2 polymeric region is a discrete, modular DNA element designed for easy transfer from one cDNA construct to another. A modular DNA element is sometimes referred to as a cloning cassette. The HCH2 polymeric region can be used as a cloning cassette and simply spliced into the existing cDNA for any protein, thus removing several steps from the formation process. In certain circumstances the precise site of insertion within a protein sequence can be determined by experimentation. Using the approach presented in this application, existing monoclonal antibodies and recombinant immunoglobulin fusion proteins can be modified through the addition of the HCH2 polymer region.
Of course, any of the mentioned polypeptides (e.g., those specifically or generally described herein) that are used to construct the inventive polypeptide or parts or portions thereof, can be made from polypeptides that are substantially similar (as defined herein) to the mentioned polypeptides.
The inventive polypeptide may be an ingredient or component of a composition, including, for example, a vaccine, emulsion, solution, pill, or any other liquid or solid composition that may be administered to any organism including, for example, plant, animal, mammal, mouse or human. The inventive polypeptide may be glycosylated or free from glycosylation.
In some embodiments the invention includes methods for producing an inventive polypeptide comprising (1) preparing a vector comprising the nucleic acid sequence encoding the polypeptide; (2) transfecting a host cell with the vector; (3) culturing the host cell to provide expression; and (4) recovering the polypeptide.
The vector can be prepared by any known method, including but not limited to cDNA obtained from reverse transcription, de novo gene synthesis, or obtaining a cDNA template from government or commercial sources.
For example, one can isolate RNA from a cell that expresses the immunoglobulin heavy chain. The RNA can then be used to produce a cDNA using reverse transcription. An example of an expressing cell would be a cell line expressing an antibody of the Ig class of interest. Many monoclonal antibodies are expressed in SPO mouse myeloma cells. Another example is a myeloma cell line of any species that has aberrant expression of Ig heavy chains. ARH-77 (ATCC #: CRL-162) is an example of a human myeloma cell line that produces IgG1 heavy chains.
In de novo gene synthesis, cDNA sequences can be built up from smaller DNA sequences, such as oligonucleotides. The advantage of de novo synthesis is that it can provide complete control over the design of the sequences employed to construct the cDNA. This strategy can permit the removal of unwanted restriction sites while introducing others that are more desirable. The codons used in the wild-type gene can be altered to remove a codon bias and thereby improve yields of the expressed protein from the cell of choice.
A government source of cDNA template includes obtaining a cDNA clone for the proper Ig type from the IMAGE clone consortium (http://image.llnl.gov/). The IMAGE consortium or Integrated Molecular Analysis of Genomes and their Expression Consortium, serves as a repository for mammalian cDNAs for expressed genes. The IMAGE consortium has a full-length cDNA clone for nearly every human and mouse gene. In addition to these government sources, commercial sources such as OpenBiosystems are available.
Once the vector is prepared, it can be amplified by any known method including, for example, PCR.
Suitable cells for transfecting and culturing include, but are not limited to insect cells (such as, SF9 cells), mammalian cells (such as, human embryonic kidney cells, HEK 293 cells).
There are numerous resources that provide details and alternative means for the procedures that can be incorporated or used to make the inventive polypeptide. These include, for example (1) Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Vol. 3, p. 16.66, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; (2) Methods in Enzymology, Vol 216, pp 3-689, 1992, Recombinant DNA Part G; (3) Methods in Enzymology, Vol 218, pp 3-806, 1993, Recombinant DNA Part I; and (4) Current Protocols in Molecular Biology found at http://mrw.interscience.wiley.com/emrw/0471-142727/home/archive.htm#Core
Certain embodiments of the present invention involve the use of polypeptides disclosed herein to immunize subjects or as vaccines. As used herein, immunization or vaccination means increasing or activating an immune response against an antigen. It does not require elimination or eradication of a condition but rather contemplates the clinically favorable enhancement of an immune response toward an antigen. The vaccine may be a prophylactic vaccine or a therapeutic vaccine. A prophylactic vaccine comprises one or more epitopes associated with a disorder for which the individual may be at risk (e.g., Botulinum Neurotoxin antigens as a vaccine for prevention of Botulinum intoxication). Therapeutic vaccines comprise one or more epitopes associated with a particular disorder affecting the individual, such as tumor associated antigens in cancer patients.
As used herein, vaccine means an organism or material that contains an antigen in an innocuous form. The vaccine is designed to trigger an immunoprotective response. The vaccine may be recombinant or non-recombinant. When inoculated into a non-immune host, the vaccine will provoke active immunity to the organism or material, but will not cause disease. Vaccines may take the form, for example, of a toxoid, which is defined as a toxin that has been detoxified but that still retains its major immunogenic determinants; or a killed organism, such as typhoid, cholera and poliomyelitis; or attenuated organisms, that are the live, but non-virulent, forms of pathogens, or it may be antigen encoded by such organism, or it may be a live tumor cell or an antigen present on a tumor cell.
Epitope refers to an antigenic determinant of a peptide, polypeptide, or protein; an epitope comprises three or more amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids and more usually consists of at least 8 to 10 amino acids. Methods of determining spatial conformation of amino acids include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.
Certain embodiments of the present invention pertain to methods of inducing an immune response to an antigen in a subject. The term antigen means a substance that is recognized and bound specifically by an antibody or by a T cell antigen receptor. Antigens can include polypeptides, peptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids and phospholipids, fragments thereof, portions thereof and combinations thereof. The antigens can be those found in nature or can be synthetic. Antigens can elicit an antibody response specific for the antigen. Haptens are included within the scope of antigen. A hapten is a low molecular weight compound that is not immunogenic by itself but is rendered immunogenic when conjugated with an immunogenic molecule containing antigenic determinants. Small molecules may need to be haptenized in order to be rendered antigenic. In some embodiments, antigens of the present invention include peptides and polypeptides. In this regard, the immunogenic polypeptides set forth herein include an antigen polypeptide. Antigen polypeptides that may be used in the immunogenic polypeptides of the present methods include antigens from an animal, a plant, a bacteria, a protozoan, a parasite, a virus, fragments thereof or a combination thereof.
An antigen polypeptide is an amino acid sequence that under appropriate conditions results in an immune response in a subject. The immune response may be an antibody response. For example, the antibody response can be measured as an increase in antibody production, as measured by any number of techniques (e.g., ELISA). The immune response may also be a T cell response, such as increased antigen presentation to T cells, or increased proliferation of T cells.
The antigen polypeptide may be any polypeptide derived from a virus. For example, the polypeptide may be derived from adenoviridiae (e.g., mastadenovirus and aviadenovirus), herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2, Epstein-Barr virus, herpes simplex virus 5, and herpes simplex virus 6), leviviridae (e.g., levivirus, enterobacteria phase MS2, allolevirus), poxyiridae (e.g., chordopoxyirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, molluscipoxvirus, and entomopoxyirinae), papovaviridae (e.g., polyomavirus and papillomavirus), paramyxoviridae (e.g., paramyxovirus, parainfluenza virus 1, mobillivirus (e.g., measles virus), rubulavirus (e.g., mumps virus), pneumonoviridae (e.g., pneumovirus, human respiratory syncytial virus), and metapneumovirus (e.g., avian pneumovirus and human metapneumovirus), picornaviridae (e.g., enterovirus, rhinovirus, hepatovirus (e.g., human hepatitis A virus), cardiovirus, and apthovirus, reoviridae (e.g., orthoreovirus, orbivirus, rotavirus, cypovirus, fijivirus, phytoreovirus, and oryzavirus), retroviridae (e.g., mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, type D retrovirus group, BLV-HTLV retroviruses, lentivirus (e.g. human immunodeficiency virus 1 and human immunodeficiency virus 2), spumavirus), flaviviridae (e.g., hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus), togaviridae (e.g., alphavirus, e.g., sindbis virus) and rubivirus (e.g., rubella virus), rhabdoviridae (e.g., vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus, and necleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, lassa virus), coronaviridae (e.g., coronavirus and torovirus), influenza virus hemagglutinin (Genbank Accession No. J02132), human respiratory syncytial virus G glycoprotein (Genbank Accession No. Z33429), core protein, matrix protein or any other protein of Dengue virus (Genbank Accession No. M19197), measles virus hemagglutinin (Genbank Accession No. M81899), herpes simplex virus type 2 glycoprotein gB (Genbank Accession No. M14923), poliovirus I VP1, envelope glycoproteins of HIV I, hepatitis B surface antigen, diptheria toxin, streptococcus 24M epitope, gonococcal pilin, pseudorabies virus g50 (gpD), pseudorabies virus II (gpB), pseudorabies virus gIII (gpC), pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastroenteritis glycoprotein 195, transmissible gastroenteritis matrix protein, swine rotavirus glycoprotein 38, swine parvovirus capsid protein, Serpulina hydrodysenteriae protective antigen, bovine viral diarrhea glycoprotein 55, Newcastle disease virus hemagglutinin-neuraminidase, swine flu hemagglutinin, swine flu neuraminidase, foot and mouth disease virus, hog cholera virus, swine influenza virus, African swine fever virus, Mycoplasma hyopneumoniae, infectious bovine rhinotracheitis virus (e.g., infectious bovine rhinotracheitis virus glycoprotein E or glycoprotein G), infectious laryngotracheitis virus (e.g., infectious laryngotracheitis virus glycoprotein G or glycoprotein I), a glycoprotein of La Crosse virus, neonatal calf diarrhea virus, Venezuelan equine encephalomyelitis virus, punta toro virus, murine leukemia virus, mouse mammary tumor virus, hepatitis B virus core protein or hepatitis B virus surface antigen or a fragment or derivative thereof, antigen of equine influenza virus or equine herpesvirus (e.g., equine influenza virus type A/Alaska 91 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidase equine herpesvirus type 1 glycoprotein B, and equine herpesvirus type 1 glycoprotein D, antigen of bovine respiratory syncytial virus or bovine parainfluenza virus (e.g., bovine respiratory syncytial virus attachment protein (BRSV G), bovine respiratory syncytial virus fusion protein (BRSV F), bovine respiratory syncytial virus nucleocapsid protein (BRSV N), bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase), bovine viral diarrhea virus glycoprotein 48 or glycoprotein 53, Cercopithecine herpesvirus (Herpes B virus), Coccidioides posadasii, Crimean-Congo haemorrhagic fever virus, Ebola Viruses, Lassa fever virus, Marburg virus, Monkeypox virus, a reconstructed replication competent forms of the 1918 pandemic influenza virus containing any portion of the coding regions of all eight gene segments (Reconstructed 1918 Influenza virus), Rickettsia prowazekii, Rickettsia rickettsii, a South American haemorrhagic fever virus, tick-borne encephalitis complex (flavi) viruses, Central European tick-borne encephalitis, Far Eastern tick-borne encephalitis, Variola major virus (Smallpox virus), Variola minor virus (Alastrim), Yersinia pestis, Bacilius anthracis, a Botulinum neurotoxin producing species of Clostridium, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei (formerly Pseudomonas mallei), Burkholderia mallei (formerly Pseudomonas mallei), Burkholderia pseudomallei (formerly Pseudomonas pseudomallei), Coccidiodes immitis, Coxiella burnetii, Eastern Equine Encephalitis virus, Francisella tularensis, Hendra virus, Nipah Virus, Rift Valley fever virus, Venezuelan Equine Encephalitis virus, African horse sickness virus, African swine fever virus, Akabane virus, Avian influenza virus, Bluetongue virus, Camel pox virus, Classical swine fever virus, Foot-and-mouth disease virus, Goat pox virus, Japanese encephalitis virus, Lumpy skin disease virus, Malignant catarrhal fever virus, Menangle virus, Mycoplasma capricolum, Mycoplasma mycoides, Newcastle disease virus, Peste des petits ruminants virus, Rinderpest virus, Sheep pox virus, Swine vesicular disease virus, Vesicular stomatitis virus, bacillus anthracis, arenavirus, Brucella sp., Burkholderia mallei, Burkholeria pseuomalei, chlamydia psittaci, vibrio cholerae, Coxiella burnetii, ebola virus, E. coli, clostridium perfringens, Salmonella sp., Shigella sp., or a variant thereof.
Antigen polypeptides useful in the present invention may also be a cancer antigen or a tumor antigen. Any cancer or tumor antigen may be used in accordance with the immunogenic compositions of the invention including, but not limited to, KS pan-carcinoma antigen, ovarian carcinoma antigen (CA125), prostatic acid phosphate, prostate specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigens such as: CEA, TAG-72, CO17-1A; GICA 19-9, CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19, human B-lymphoma antigen-CD20, CD33, melanoma specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside GM3, tumor-specific transplantation type of cell-surface antigen (TSTA) such as virally-induced tumor antigens including T-antigen DNA tumor viruses and Envelope antigens of RNA tumor viruses, oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumor oncofetal antigen, differentiation antigen such as human lung carcinoma antigen L6, L20, antigens of fibrosarcoma, human leukemia T cell antigen-Gp37, neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR, EGFRvIII, FABP7, doublecortin, brevican, HER2 antigen, polymorphic epithelial mucin (PEM), malignant human lymphocyte antigen-APO-1, differentiation antigen such as I antigen found in fetal erythrocytes, primary endoderm, I antigen found in adult erythrocytes, preimplantation embryos, I (Ma) found in gastric adenocarcinomas, M18, M39 found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, D156-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Ley found in embryonal carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, E1 series (blood group B) found in pancreatic cancer, FC10.2 found in embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 found in Adenocarcinoma, NS-10 found in adenocarcinomas, CO-43, G49 found in EGF receptor of A431 cells, MH2 found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, T5A7 found in myeloid cells, R24 found in melanoma, 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, and M1:22:25:8 found in embryonal carcinoma cells, SSEA-3 and SSEA-4 found in 4 to 8-cell stage embryos, a T cell receptor derived peptide from a Cutaneous T cell Lymphoma, and variants thereof.
Antigen polypeptides useful in the present invention may also be an autoantigen. Autoantigens known to be associated with autoimmune disease have been described. Included are myelin proteins associated with demyelinating diseases, e.g. multiple sclerosis and experimental autoimmune encephalomyelitis; collagens and rheumatoid arthritis; acetylcholine receptor with myasthenia gravis; insulin, proinsulin, glutamic acid decarboxylase 65 (GAD65), islet cell antigen (ICA512; ICA12) with insulin dependent diabetes. Disease associated myelin proteins include myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG).
In some embodiments, the antigen polypeptide can be, but is not limited to, abrin, a conotoxin, diacetoxyscirpenol, ricin, saxitoxin, a Shiga-like ribosome inactivating protein, flexal, guanarito, junin, machupo, sabia, tetrodotoxin, a Botulinum neurotoxin, Clostridium perfringens epsilon toxin, a Shigatoxin, Staphylococcal enterotoxin, T-2 toxin, Bovine spongiform encephalopathy agent, epsilon toxin, ricin toxin, Staphylococcal enterotoxin B, or a variant thereof.
In some embodiments, the antigen polypeptide is administered with the intent of inducing an immune response. Depending on the intended mode of administration, the compounds of the present invention can be in various pharmaceutical compositions. The compositions will include a unit dose of the selected polypeptide in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and excipients. Pharmaceutically acceptable means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the fusion protein or other composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
Any method of preparation of vaccines and immunizing agents can be used, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines.
Examples of physiologically acceptable carriers include saline solutions such as normal saline, Ringer's solution, PBS (phosphate-buffered saline), and generally mixtures of various salts including potassium and phosphate salts with or without sugar additives such as glucose. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added to the composition. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. In one embodiment of the invention, adjuvants are not required for immunization.
Parenteral administration, if used, is generally characterized by injection. Sterile injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
The vaccine compositions set forth herein may comprise an adjuvant or a carrier. Adjuvants are any substance whose admixture into the vaccine composition increases or otherwise modifies the immune response to an antigen.
Adjuvants can include but are not limited to AlK(SO4)2, AlNa(SO4)2, AlNH(SO4)4, silica, alum, AI(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(12-dipalmitoyl-s-n-glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80 emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see U.S. Pat. Nos. 58,767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51 and QS-21.
Additional adjuvants or compounds that may be used to modify or stimulate the immune response include ligands for Toll-like receptors (TLRs). In mammals, TLRs are a family of receptors expressed on DCs that recognize and respond to molecular patterns associated with microbial pathogens. Several TLR ligands have been intensively investigated as vaccine adjuvants. Bacterial lipopolysaccharide (LPS) is the TLR4 ligand and its detoxified variant mono-phosphoryl lipid A (MPL) is an approved adjuvant for use in humans. TLR5 is expressed on monocytes and DCs and responds to flagellin whereas TLR9 recognizes bacterial DNA containing CpG motifs. Oligonucleotides (OLGs) containing CpG motifs are potent ligands for, and agonists of, TLR9 and have been intensively investigated for their adjuvant properties.
Other agents that stimulate the immune response can also be administered to the subject. For example, other cytokines are also useful in vaccination protocols as a result of their lymphocyte regulatory properties. Many other cytokines useful for such purposes are known, including interleukin-12 (IL-12) that has been shown to enhance the protective effects of vaccines, GM-CSF and IL-18. Thus cytokines can be administered in conjunction with antigens and adjuvants to increase the immune response to the antigens.
A vaccine composition according to the present invention may comprise more than one different adjuvant. Furthermore, the invention encompasses a therapeutic composition further comprising any adjuvant substance including any of the above or combinations thereof. It is also contemplated that ML-IAP, or one or more fragments thereof, and the adjuvant can be administered separately in any appropriate sequence.
In certain embodiments, the vaccine composition includes a carrier. The carrier may be any suitable carrier, for example a protein or an antigen presenting cell. Examples include serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. For immunization of humans, the carrier should be a physiologically acceptable carrier acceptable to humans and safe. Tetanus toxoid or diptheria toxoid are suitable carriers in one embodiment of the invention. Alternatively, the carrier may be dextrans for example sepharose.
The timing of administration of the vaccine and the number of doses required for immunization can be determined from standard vaccine administration protocols. In some instances, a vaccine composition will be administered in two doses. The first dose will be administered at the elected date and a second dose will follow at one month from the first dose. A third dose may be administered if necessary, and desired time intervals for delivery of multiple doses of a particular antigen containing HCH2 polymer can be determined. In another embodiment, the antigen containing HCH2 polymer may be given as a single dose.
For each recipient, the total vaccine amount necessary can be deduced from protocols for immunization with other vaccines. The exact amount of antigen-HCH2 polymer required can vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular fusion protein used, its mode of administration, and the like. Generally, dosage will approximate that which is typical for the administration of other vaccines, and may be in the range of about 10 ng/kg to 1 mg/kg.
Any known methods for the preparation of mixtures or emulsions of polypeptides disclosed herein and adjuvant can be used (see, e.g. Plotkin and Orenstein, eds, Vaccines, 4th Ed., 2004).
Immunizations against toxins and viral infection can be tested using in vitro assays and standard animal models. For example a mouse can be immunized with a viral antigen polypeptide expressed as a fusion protein with HCH2 polymers and delivered by the methods detailed herein. After the appropriate period of time to allow immunity to develop against the antigen, for example two weeks, a blood sample is tested to determine the level of antibodies, termed the antibody titer, using ELISA. In some instances the mouse is immunized and, after the appropriate period of time, challenged with the virus to determine if protective immunity against the virus has been achieved. Using these techniques the proper combination of antigen, adjuvant, and other vaccine components can be optimized to boost the immune response. Testing in humans can be contemplated after efficacy is demonstrated in animal models. Any known methods for immunization, including formulation of a vaccine composition and selection of doses, route of administration and the schedule of administration (e.g. primary and one or more booster doses) can be used (e.g. see Vaccines: From concept to clinic, Paoletti and McInnes, eds, CRC Press, 1999).
Generally accepted animal models can be used for testing of immunization against cancer using a tumor and cancer antigen polypeptides. For example, cancer cells (human or murine) can be introduced into a mouse to create a tumor, and one or more cancer associated antigens can be delivered by the methods described herein. The effect on the cancer cells (e.g., reduction of tumor size) can be assessed as a measure of the effectiveness of the immunization. Of course, immunization can include one or more adjuvants or cytokines to boost the immune response. The tests also can be performed in humans, where the end point is to test for the presence of enhanced levels of circulating cytotoxic T lymphocytes against cells bearing the antigen, to test for levels of circulating antibodies against the antigen, to test for the presence of cells expressing the antigen and so forth.
In some embodiments, the vaccine composition includes antigen presenting cells. The antigen presenting cell can be a dendritic cell (DC). DC may be cultivated ex vivo or derived in culture from peripheral blood progenitor cells (PBPC) and peripheral blood stem cells (PBSC). The dendritic cells may be prepared and used in therapeutic procedures according to any suitable protocol. Different protocols may be adopted to use with patients with different HLA types and different diseases. Incubation of cultured dendritic cells with HCH2 polymers of the invention is envisaged as one means of loading dendritic cells with antigen for subsequent transfer into hosts.
For any of the ex vivo methods of the invention, peripheral blood progenitor cells (PBPC) and peripheral blood stem cells (PBSC) are collected using apheresis procedures known. Briefly, PBPC and PBSC can be collected using conventional devices, for example, a Haemonetics Model V50 apheresis device (Haemonetics, Braintree, Mass.). Four-hour collections can be performed typically no more than five times weekly until, for example, approximately 6.5108 mononuclear cells (MNC)/kg patient are collected. The cells are suspended in standard media and then centrifuged to remove red blood cells and neutrophils. Cells located at the interface between the two phases (also known in the art as the buffy coat) are withdrawn and resuspended in HBSS. The suspended cells are predominantly mononuclear and a substantial portion of the cell mixture are early stem cells. The stem cells obtained in this manner can be frozen, then stored in the vapor phase of liquid nitrogen. Ten percent dimethylsulfoxide can be used as a cryoprotectant. After all collections from the donor have been made, the stem cells are thawed and pooled. Aliquots containing stem cells, growth medium, such as McCoy's 5A medium, 0.3% agar, and expansion factors (e.g. GM-CSF, IL-3, IL-4, flt3-ligand), are cultured and expanded at 37 C. in 5% CO2 in fully humidified air for 14 days.
There are three classes of Fc receptor (Gessner et al., 1998; Raghavan et al., 1996). FcRI (CD64) binds monomeric IgG with high affinity whereas AIG and IC bind preferentially to FcRII (CD32) and FcRIII (CD 16), the low affinity receptors for Fc. FcRII and FcRIII are closely related in the structure of their ligand-binding domains. In humans three separate genes, FcRIIA, FcRIIB, and FcRIIC, two of which give rise to alternatively spliced variants, code for FcRII. FcRIIa delivers activating signals whereas FcRIIb delivers inhibitory signals. The functional basis for the divergent signals arises from signaling motifs located within the cytoplasmic tails of the receptors. An immunoreceptor tyrosine-based inhibitor motif (ITIM) located in the cytoplasmic tail of the FcRIIb is involved in negative receptor signaling. The ITIM motif is a unique feature of the FcRIIb receptor as it is not apparently present in any other Fc receptor class. In contrast, an activatory immunoreceptor tyrosine-based activation motif or ITAM is located in the cytoplasmic tail of FcRIIa. ITAM motifs transduce activating signals. They are also found in the FcR -chains, which are identical to the -chains of the high affinity IgE receptor (FcRI). While FcRIIa and FcRIIb are widely expressed on myeloid cells and some T-cell subsets they are notably absent from NK cells.
Human FcRIII is also present in multiple isoforms derived from two distinct genes (FcRIIIA and FcRIIIB). FcRIIIb is unique in its attachment to the cell membrane via a glycosylphosphatidyl anchor. FcRIIIb expression is restricted to neutrophils while FcRIIIa is expressed by macrophages, and NK cells. FcRIIIa is also expressed by some y T-cell subsets and certain monocytes. FcRIIIa requires the presence of the FcR -chain or the CD3-chain for cell surface expression and signal transduction. The FcR -chain and the CD3-chain are dimeric and possess ITAM motifs. FcRIIIa forms a multimeric complex with these subunits and signaling is transduced through them. Thus, there is considerable FcR receptor heterogeneity and diverse expression profiles.
AIG and IC have been used to target FcRIIIa on immune cells, but as noted earlier production of defined AIG and IC was seen to be problematic. Assembly of complexes by physical or chemical methods is difficult to control with precision resulting in heterogeneity within complexes of similar molecular weight in addition to variations between preparations and changes in composition upon storage. Molecular cloning has been used in the present invention to create molecules that can mimic or approximate AIG and IC function with respect to their interactions with FcR and which allow for the inclusion and targeting of a second protein domain to cells expressing FcR.
The binding sites for FcRII and FcRIII map to the hinge and proximal region of the CH2 domain of IgG, the same region originally identified for FcRI (Duncan et al., 1988; Morgan et al., 1995; Lund et al., 1991). White et al. (2001) describe the cloning and expression of linear polymers of the hinge and CH2 (HCH2) fused to the Fc region of IgG1 and demonstrate their biological activity. Legge et al. (2000) have recently shown that an aggregated PLP1 immunoadhesin, unlike the monomeric form, moderates disease severity in experimental autoimmune encephalomyelitis, the rodent model for multiple sclerosis. This change is due to the dual functionality of the aggregated Fc and PLP moieties within the complex.
In the later phase of a primary immune response or in chronic responses, large ICS form. These complexes signal through the low affinity IgG receptors that recognize ICS or IgG aggregates preferentially. The low affinity receptors are of two classes FcRII (CD32) and FcRIII (CD16). FcRIIb provides an inhibitory signal for secretion of cytokines that augment immunoglobulin secretion including IgG secretion. FcRIIIa (found on NK cells, monocytes and yy6 T cells) preferentially recognizes IgG1. One thrust of this invention is directed towards activation of FcRIIIa.
The ability of FcR to bind IgG and transmit a signal into the cell depends upon the FcRs alleles expressed, upon glycosylation, and how the receptor is associated with the signaling subunit. In addition, glycosylation patterns differ between cell types and this too can affect ligand binding to FcRIIIa. FcRIIIa on NK cells is glycosylated with high mannose oligosaccharides, whereas monocyte/macrophage FcRIIIa is not. Perhaps this imparts lower receptor affinity to monocyte/macrophage FcRIIIa relative to NK cell FcRIIIa, adding yet another level of modification to receptor function (Galon et al., 1997; Edberg et al., 1997). Thus, FcR function is regulated at several levels, which can have an impact on ligand binding and receptor signaling.
Recently, the inventors have initiated studies into the potential immunomodulatory role of immune complexes (IC) in human autoimmune syndromes. Central to these studies are the interactions between IC and FcR. The inventors have used molecular cloning to create molecules that can mimic or approximate IC function with respect to their interactions with FcR and which allow for the inclusion and targeting of a second protein domain to cells expressing FcR. The strategy pursued is to express multiple linear copies of the region of the IgG framework that binds FcR. Expressing only those determinants necessary for FcR engagement and presenting them in a particularly favorable configuration results in novel proteins that are considerably more potent than IC. Thus recombinant IC mimetic proteins described herein will provide both a valuable tool for the examination of IC deposition and in the therapeutic targeting of FcR in autoimmune disorders.
In another embodiment of the invention, the polypeptides include linked antigens. In one embodiment, the polypeptides of the invention are used to target an antigen to the cell to enhance the process of internalization and presentation of the antigen by these cells, and ultimately to stimulate an immune response. In another embodiment, the polypeptides of the invention specifically bind the antigen directly or bind to epitopes attached to the antigen, e.g., a cloned Fab fragment covalently attached to the polymer by genetic or chemical means which recognizes the antigen or epitopes attached to the antigen, and targets the bound antigen to antigen presenting cells (APC) for internalization, processing, and presentation. In another embodiment, the antigen is linked to the polymers of the invention and at the same time binds a surface receptor of an antigen-presenting cell. In another embodiment the antigen is covalently attached to the polymers of the invention by genetic or chemical means.
More broadly, the polypeptides of this invention can be linked to a cell surface marker. A cell surface marker is a protein, carbohydrate, glycolipid, etc. but most commonly comprises a protein localized to the plasma membrane of a cell having a portion exposed to the extracellular region (e.g. an integral membrane protein or a transmembrane glycoprotein), such that the extracellular portion can be specifically bound by an antibody or other ligand. The term cell surface marker also refers to a polynucleotide sequence encoding such a cell surface protein. Numerous cell surface proteins can be used as cell surface markers, such as, for example, a CD (cluster of differentiation) antigen present on cells of hematopoietic lineage (CD2, CD4, CD8, CD21), Gamma-glutamyltranspeptidase, an adhesion protein (ICAM-1, ICAM-2, ELAM-1, VCAM-1), a hormone, a growth factor, a cytokine receptor, ion channels, and the membrane-bound form of an immunoglobulin chain.
1. Polypeptides that Contain HCH2 Polymers for use in Vaccines.
Traditional vaccines consist of killed or attenuated pathogenic organisms or their products administered to develop an immune response. Drawbacks to the traditional approach include unwanted harmful immune responses, inoculation with potentially infectious pathogens, and poor immune responses. Typically these vaccines require co-administration of potent adjuvants to elicit effective antibody responses. Vaccines can be made more effective by delivering those antigenic determinants that are most likely to confer protective immunity. Early attempts to develop peptide based vaccines resulted in poor immune responses due in part to an inefficient presentation of antigen by APCs.
APCs capture, internalize and present antigen. In addition they provide important costimulatory signals to T-cells. T-cells, thus activated, are capable of stimulating the production of antibody-forming B cells. Monocytes, especially macrophages and dendritic cells, function as APC. Macrophages express all three classes of FcR constitutively whereas dendritic cells express FcRI and FcRII.
Dendritic cells (DCs) are highly specialized and are potent APCs for T-cells. As a result of this capacity DCs are often referred to as professional APCs. DCs present antigen efficiently on both MHC I and MHC II resulting in the initiation of CD8+ and CD4+ responses respectively. DCs can prime naive T-cells. Subsequent to activation by DCs, T-cells can interact with other APC. DCs have a proliferative immature stage followed by terminal differentiation into a non-proliferative mature stage. Immature DCs express FcRI and FcRII, are capable of internalizing and presenting antigen, and synthesize large amounts of MHC II. In contrast mature DCs no longer express FcRs, become fully active APCs, activate T-cells, and secrete large amounts of IL-12 (which spurs differentiation of T-cells). DCs are a more potent APC than macrophages though much less numerous.
There has been great interest in the enhancement of antigen presentation by targeting antigen to FcR expressed on APCs. Some peptide vaccines have antigenic determinants grafted into the variable region of IgG. These antigenized-antibodies increased the half-life of antigen and facilitated uptake of antigen by APCs via the FcRI receptor (Zaghouani et al., 1993; Zanetti et al., 1992). Use of antigenized-antibodies have been shown to be more effective at priming antigen specific T-cell responses than peptide alone. Antigenized-antibodies have several limiting features: Since they are directed towards FcRI alone, they can be effectively competed against by monomeric serum IgG. Secondly, the design of the molecule limits the size of the antigenizing determinant to a small peptide fragment.
More recently antigen has been expressed as a fusion protein with or chemically conjugated to monoclonal antibodies and Fab fragments directed against FcRI and FcRII (Liu et al., 1996b; Guyre et al., 1997). Using tetanus toxoid epitopes conjugated to anti-FcRI monoclonal antibody, one group reported that peptides directed to FcRI were 100 to 1000 fold more efficient than peptide alone in T-cell stimulation (Liu et al., 1996a). However, use of anti-FcRI Fab required chemical cross-linking to achieve maximal responses to antigen, thus implicating the low affinity IgG receptors (Keler et al., 2000). Disadvantages of this approach include the promiscuous binding of antigen-linked monoclonal antibody to FcRI expressed on non-APCs. Monoclonal antibodies trigger effector functions poorly. Fab fragments have the additional disadvantage of a short half-life in the circulation.
Attachment of antigen to the HCH2 polymers in the inventive polypeptides described herein for the purpose of targeting APCs has distinct advantages over existing strategies. The inventive polypeptides present antigen to low affinity receptors (FcRII and FcRIII), thus bypassing competition from monomeric serum IgG for binding to FcRI. Additionally there is no need for chemical cross-linking as is necessary when using anti-FcRI Fab. The inventive polypeptides imitate immune complexes. Antigen presented in the context of an immune complex may be a particularly appropriate substrate for APCs. Antigen-linked HCH2 polymers efficiently trigger effector functions that augment the immune response.
2. The Inventive Polypeptide in Tolerance Induction.
Immunologic tolerance can be characterized by the selective lack of an immune response, including lack of a pathogenic immune response to a specific antigen even while leaving other responses of the immune system intact. Induction and maintenance of T cell unresponsiveness to a specific antigen may be achieved by several mechanisms that can be broadly summarized as: 1) clonal deletion; 2) anergy; and 3) suppression. Clonal deletion is a process of negative selection whereby T cells with high affinity for self-antigens are deleted in the thymus. Deletion is achieved by programmed cell death (apoptosis). This process of negative selection in the thymus is known as central tolerance. Anergy represents a state of immune inactivation characterized by abolished proliferative and cytokine responses. It is induced in cells that previously responded to a given antigen and results in an unresponsive state upon re-stimulation with antigen. Since this mechanism acts upon mature T cells that have exited the thymus and reside in the peripheral compartments, this form of tolerance is termed peripheral tolerance. Anergy is induced by any of a number of molecular events and need not be permanent: it can be reversed by certain cytokines. Three common anergy-inducive mechanisms are T cell receptor (TCR) stimulation without co-stimulatory signals, sub-optimal TCR stimulation even in the presence of co-stimulation, and the autocrine inhibitory actions of IL-10. Suppression of T cell function is a third mechanism by which T cell tolerance can be achieved. Suppression ensues when regulatory T cells are induced to exert non-specific suppressive effects on antigen-specific T cells in their vicinity. This microenvironmental effect is also referred to as bystander suppression.
B cell tolerance involves concepts and mechanisms similar but not identical to those encountered in T cell tolerance. In mature B cells tolerance can be induced through a block in Ig-receptor signaling which results in impaired expression of the B7 costimulatory molecules.
Induction of tolerance to either self- or foreign-antigens provides an important therapeutic approach to the treatment of allergies, autoimmune disease and host vs. graft disease (transplant rejection). In addition, the therapeutic potential of many foreign biologically active agents is limited by their immunogenicity. Tolerance induction represents one approach for the control of immune responses directed against biologically active foreign agents, thus improving their therapeutic potential. In some instances the antigen to be tolerized is presented orally, intradermally, or intravenously. The source of antigen can be in the form of a peptide, a protein, or nucleic acid which can express a peptide or protein. The antigen is then internalized by antigen presenting cells (APC) and presented on the surface of the cell, most typically as a MHC I-antigen complex or as a MHC II-antigen complex.
The inventive polypeptides, which comprise the HCH2 polymers, have several advantageous aspects for use as vehicles for tolerance induction. Antigen(s) linked to the HCH2 polymers by chemical or genetic means are targeted to Fc receptors expressed on APC such as macrophages, B cells, and dendritic cells (DC). Fc-receptor-mediated internalization results in processing and presentation of antigen at the cell surfacethe key first step in tolerance induction.
Macrophages and DC express Fc receptors for both IgG and IgE. HCH2 polymers are expressible which bind both classes of Fc receptor simultaneouslycoaggregation of different Fc receptor classes may have advantages over targeting a single class of receptor. Ligation of FcRs induces secretion of IL10 from certain immune cells and, as already noted, IL10 induces anergy in T cells. As is observed for immune complexes, binding of inventive polypeptides to FcRs may induce a pattern of cytokine secretion that deviates T cell immune responses from a THI type response to a TH2 type response. TH2 type T cells favor the establishment and maintenance of immune tolerance. Therefore, antigens linked to the HCH2 polymers can promote tolerance induction by both the efficient presentation of antigen to APC and the simultaneous induction of mechanisms that favor establishment of immune tolerance.
a. Inventive Polypeptide ExampleModification of Recombinant Monoclonal Antibodies by the Introduction of an HCH2 Polymer
It is an embodiment of the current invention that recombinant monoclonal antibodies (mAb) can be modified by the introduction of one or more HCH2 units into the Fc region to create an HCH2 polymer of appropriate length within a monoclonal antibody. Monoclonal antibodies modified in this manner will retain their target specificity while acquiring improved or more selective effector function. HCH2 polymers greatly enhance FcFcR receptor interactions. More specifically HCH2 polymers of the current invention have greatly improved binding to and enhanced activation of FcRIIIa receptors over that seen with the Fc portion of mAb in current therapeutic use. As enhanced interaction of mAB with FcRIIIa has been documented to have therapeutic benefit in the treatment of malignancies. The inventors envisage modifying existing mAb with the introduction of an HCH2 polymer into the Fc region of the mAb. Monoclonal antibodies with this modification will have enhanced interaction with FcRIIIa receptors.
Functional IgG genes, those that direct expression of a mAb, are composed of heavy and light chain genes segments. Light chain (L) genes consist of three exons, containing the hydrophobic leader sequence, the variable regions and the L constant region (CL). Separating the exons are the intervening sequences or introns. Similarly, the variable region of a functional Ig heavy chain (H) gene has a separate exon for each of the leader sequence, the variable region, and H chain constant region (CH1). The H gene also contains the Fc region that is composed of separate exons for the hinge, the CH2 region and CH3 regions. Once again the exons are separated by introns. The expression of mAb in mammalian cells typically involves cloning both the H and L gene segments from functional Ig genes into either a single expression vector or separate expression vectors (one for L, one for H genes) that posses the Ig promoter region. Once subcloned the expression vectors possessing the L and H genes are transfected into an appropriate cell line for expression. The use of gene segments insures the presence of intronic sequences, which contain enhancer and other elements that collectively allow for high levels of Ig expression in B cells and myeloma cells. Ig expression systems utilizing the Ig promoter and intronic genetic elements limit protein expression to cells of lymphoid derivation however.
More recently, Ig expression systems have been developed that use viral promoters and enhancer combinations, such as CMV. The use of viral promoter/enhancer combinations permits strong expression in both lymphoid and non-lymphoid cells lines such as CHO and COS (Norderhaug, et al., 1997). Inclusion of the intronic enhancer from the Ig H gene also directs high level expression in lymphoid cells. Additionally, H and L gene segments are no longer necessary for efficient expression and can be replaced by their corresponding cDNA's (McLean, et al., 2000).
The introduction of HCH2 polymers into mAb can be achieved by any of several approaches. In one method, using known molecular cloning techniques, H chain gene segments within expression vectors are modified by the insertion of the HCH2 polymer cloning cassette into the 5 end of the hinge exon. The modified hinge exon now consists of the HCH2 polymer fused in frame to the hinge sequences. The vector containing the modified H gene is introduced in conjunction with an L gene into an appropriate cell line for mAb expression. Another method is to replace the Fc gene segment with a cDNA segment comprising a splice acceptor signal, the HCH2 polymer fused to an Ig Fc cDNA and a polyA signal. The modified H gene is then transferred into an Ig expression vector capable of directing Ig expression without Ig gene intronic sequences. The vector containing the modified H gene is introduced in conjunction with an L gene into an appropriate cell line for expression.
The insertion of HCH2 polymers into mAb expressed from cloned cDNA within expression vectors can also be achieved using similar techniques. For instance, the cDNA encoding the Fc region can be removed from the H chain cDNA and replaced with a DNA segment encoding the HCH2 polymer fused to an Fc cDNA. Conversely, the cDNA encoding the H chain leader, variable and CH1 region can be excised and transferred to vectors containing the HCH2 polymer region genetically fused to an Fc cDNA. Alternatively, the HCH2 polymer cassette can be introduced into the H chain cDNA at the appropriate site. This site would sometimes be the junction between the CH1 region and the hinge. Subsequently, the modified H chain cDNA is then transferred into an Ig expression vector capable of directing Ig expression without Ig gene intronic sequences. The vector containing the modified H chain cDNA is introduced in conjunction with an L chain expression vector into an appropriate cell line for expression.
While interaction with FcRIIIa receptors is important for the efficacy of several mAb in clinical use, the methods of modification described above are general. In other embodiments of the inventive polypeptides, HCH2 polymers can be introduced into mAb to enhance specificity for other individual FcR receptors, classes of FcR receptors, as blocking reagents for FcR receptors, or for binding to complement factors.
Autoimmune diseases are processes in which the immune system mounts an attack against body tissue components. This attack may be mediated by anti-tissue component antibodies produced by B lymphocytes or by cell-mediated tissue destructive processes mediated by T cells, by NK cells, and by monocytes/macrophages. In some autoimmune diseases several tissue damaging mechanisms may operate either concurrently or sequentially. The inventive polypeptides of the current invention can be used in the treatment of autoimmune diseases. They can be used to alter immunity and to deliver therapeutic agents to a delivery site in a patient where the therapeutic agent is effective.
The number of autoimmune diseases is considerable and some persons may have more than one autoimmune disease. Similarly, signs and symptoms may cover a wide spectrum and severity may also vary widely between afflicted individuals and over time. The reasons why some persons develop autoimmunity while others do not are imperfectly understood but certain recurring themes can be signaled. In many autoimmune processes there is a genetically determined propensity to develop disease. Among the genes that have been linked to propensity to develop autoimmunity are those of the major histocompatibility complex. In addition, environmental factors are thought to play a role. During embryonic development many of those immune system cells that are capable of reacting against self-components are eliminated but some remain so that essentially everyone is at least theoretically capable of mounting an autoimmune response. This observation implies that under normal circumstances potentially auto-aggressive cells are held in check by physiologic restraint mechanisms and that a contributor to the pathogenesis of autoimmunity is a failure of normal restraint mechanisms.
Examples of commonly encountered autoimmune disorders include but are not limited to: systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, Guillain-Bare syndrome, other immune mediated neuropathies including chronic inflammatory demyelinating polyneuropathy, multiple sclerosis and other immune-mediated central nervous system demyelinating diseases, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myasthenia gravis, scleroderma/systemic sclerosis, and dermatomyositis/polymyositis to name some of the more commonly encountered entities. Additional autoimmune diseases include acute glomerulonephritis, nephrotic syndrome, and idiopathic IgA nephropathy among autoimmune processes that affect the kidneys.
Examples of autoimmune processes that affect the formed elements of the blood are autoimmune aplastic anemia, autoimmune hemolytic anemia, and idiopathic thrombocytopenic purpura.
Autoimmune diseases that affect the endocrine organs include Addison's disease, idiopathic hypoparathyroidism, Grave's disease, Hashimoto's thyroiditis, lymphocytic hypophysitis, autoimmune oophoritis, and immunologic infertility in the male.
The liver may also be the target of autoimmune processes. Examples include autoimmune hepatitis, hepatitis C virus-associated autoimmunity, immunoallergic reaction drug-induced hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis.
Autoimmune processes of the intestinal tract include pernicious anemia, autoimmune gastritis, celiac disease, Crohn's disease, and ulcerative colitis.
Cutaneous autoimmune diseases include dermatitis herpetiformis, epidermolysis bullosa acquisita, alopecia totalis, alopecia greata, vitiligo, linear IgA dermatosis, pemphigus, pemphigoid, psoriasis, herpes gestationis, and cutaneous lupus including neonatal lupus erythematosus.
Additional autoimmune diseases with rheumatological features include CREST syndrome, ankylosing spondylitis, Behet's disease, juvenile rheumatoid arthritis, Sjgren's syndrome, and eosinophilia-myalgia syndrome.
Autoimmune diseases can affect the heart. Examples include myocarditis and idiopathic dilated cardiomyopathy, rheumatic fever, Chaga's disease and possibly some components of atherosclerosis.
There can be an autoimmune component to inflammatory diseases of the blood vessels. Examples include giant cell arteritis, Kawasaki's disease, Henoch-Schonlein purpura, polyarteritis nodosa, Goodpasture's syndrome, immune complex vasculitis, Wegener's granulomatosis, Churg-Strauss syndrome, Takayasu arteritis, necrotizing vasculitis, and anti-phospholipid antibody syndrome.
Autoimmune diseases of the central and peripheral nervous systems can occur as a remote effect of malignant tumors. Rarely these same entities occur in the absence of a tumor. Examples include the Lambert-Eaton syndrome, paraneoplastic myelopathy, paraneoplastic cerebellar degeneration, limbic encephalitis, opsoclonus myoclonus, stiff man syndrome, paraneoplastic sensory neuropathy, the POEMS syndrome, dorsal root ganglionitis, and acute panautonomic neuropathy.
Autoimmune diseases may affect the visual system. Examples include Mooren's ulcer, uveitis, and Vogt-Koyanagi-Harada syndrome.
Other autoimmune processes, or ones in which autoimmunity may contribute to disability, include interstitial cystitis, diabetes insipidus, relapsing polychondritis, urticaria, reflex sympathetic dystrophy, and cochleolabyrinthitis.
The list of autoimmune processes given above, while extensive, is not intended to be exhaustive. Rather it is intended to document that autoimmunity is a wide-ranging clinical phenomenon.
1. Multiple Sclerosis
This disease is characterized by destruction of CNS myelin and of the axons which myelin ensheathes. The illness can begin with focal attacks of tissue destruction in the white matter of the CNS which cause loss of neuronal function and as one attack follows another progressively accumulating disability. After a time most multiple sclerosis patients experience a decline in the frequency of their attacks but this decline is accompanied by a shift in the natural history of the illness to a slow but inexorable worsening of their neurological disabilities. The switch from a relapsing-remitting course to a progressive one ultimately occurs in better than 80% of multiple sclerosis victims.
Multiple sclerosis is an inflammatory disease. Lymphocytes and macrophages move from the blood into the CNS and attack and destroy myelin and ultimately the myelin forming cells known as oligodendrocytes. The process is one of autoimmunity but the precise target within the CNS against which the immune response is directed remains unknown. There is a genetically determined predisposition to develop multiple sclerosis but there is compelling evidence that environmental factors have a role as well, though the nature of the environmental factors in cause remains unknown.
There have been advances in the treatment of multiple sclerosis in recent years. Five agents are approved for the treatment of MS. These are interferon beta1a, interferon betalb, glatiramer acetate, natalizumab, and novantrone. All five modulate immune responses in a manner that favorably alters the hitherto bleak natural history of MS. Unfortunately all five are only modestly effective and each has side effects that are often troublesome. The present invention offers the prospect of a more efficient and effective therapy for MS.
Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for MS and serves as a useful model for the study of autoimmune diseases. EAE is a disease of the central nervous system and may be induced in susceptible animals by immunization with neuroantigens. EAE may also be adoptively transferred from one animal to the next by the serial transfer of T cells reactive against encephalitogenic determinants of myelin proteins or by the injection of T cell clones reactive against encephalitogenic determinants of myelin proteins. Myelin proteins that may be targets of the autoreactive response include proteolipid apoprotein (PLP), myelin basic protein (MBP), and myelin oligodendrocyte protein (MOG). Depending on the type and strain of animal used, the mode of induction, and the neuroantigen administered, the disease may be acute and monophasic in nature, or alternatively chronic, or relapsing-remitting.
Affected animals develop flaccid tails, paralysis of the hindlimbs, and incontinence. In severe disease, movement of the forelimbs may also become impaired and animals may become moribund. Histological analysis of the CNS reveals an inflammatory cell infiltrate during the acute stages of disease that may be accompanied by demyelination of the neurons during chronic phases of the disease. EAE is widely used for the study of autoimmune disease and serves as a model for testing potential efficacy of experimental drugs for the treatment of MS and for the treatment of autoimmune diseases in general.
The proteins of the current invention were tested for their effect on disease activity in a mouse model of EAE to gain insight into their potential use as therapeutics for the treatment of MS and other autoimmune diseases. Products of the current invention inhibited EAE in the SJL/J mouse. Administration of construct HSA1Fc and in particular of HSA1R4 decreased clinical disease activity during the early acute stages of disease and decreased the frequency of and severity of relapses at later time points as compared to saline-treated controls. Decreased inflammatory cell infiltrates were observed in the CNS of construct-treated animals compared to saline treated-controls.
As modifications or changes may be made in the structure of the polynucleotides and or proteins of the present invention, while obtaining molecules having similar or improved characteristics, such biologically functional equivalents are also encompassed within the present invention.
1. Modified Polynucleotides and Polypeptides
The biological functional equivalent may comprise a polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to encode the wild-type or standard protein. This can be accomplished owing to the degeneracy of the genetic code, i.e., the presence of multiple codons, which encode for the same amino acids. In one example, one of skill in the art may wish to introduce a restriction enzyme recognition sequence into a polynucleotide while not disturbing the ability of that polynucleotide to encode a protein.
In another example, a polynucleotide can be engineered to contain certain sequences that result in (and encode) a biological functional equivalent with more significant changes. Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, and such like. So-called conservative changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges on the protein's ability to carry out its designated function. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.
In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a biologically functional equivalent protein or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity, such as binding to FcRs. Biologically functional equivalents are thus defined herein as those proteins (and polynucleotides) in which selected amino acids (or codons) may be substituted.
In general, the shorter the length of the molecule, the fewer the changes that can be made within the molecule while retaining function. Longer domains may have an intermediate number of changes. The full-length protein will have the most tolerance for a larger number of changes. However, it must be appreciated that certain molecules or domains that are highly dependent upon their structure may tolerate little or no modification.
Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, or the like. An analysis of the size, shape or type of the amino acid side-chain substituents reveals that arginine, lysine or histidine are all positively charged residues; that alanine, glycine or serine are all of similar size; or that phenylalanine, tryptophan or tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine or histidine; alanine, glycine or serine; or phenylalanine, tryptophan or tyrosine; are defined herein as biologically functional equivalents.
To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity or charge characteristics, these are: isoleucine (+4.5); valine (+4.2); Leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8); tryptophan (0.9); tyrosine (1.3); proline (1.6); histidine (3.2); glutamate (3.5); glutamine (3.5); aspartate (3.5); asparagine (3.5); lysine (3.9); or arginine (4.5).
Hydropathic amino acid index can be used to confer interactive biological function on a protein (Kyte Doolittle, 1982). In some instances, certain amino acids may be substituted for other amino acids having a similar hydropathic index or score or still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids with hydropathic indices can be within 2 or within 1, or within 0.5.
The substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments, as in certain embodiments of the present invention. U.S. Pat. No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity or antigenicity, i.e., with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.01); glutamate (+3.01); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (0.4); proline (0.51); alanine (0.5); histidine (0.5); cysteine (1.0); methionine (1.3); valine (1.5); leucine (1.8); isoleucine (1.8); tyrosine (2.3); phenylalanine (2.5); tryptophan (3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids with hydrophilicity values can be within 2, or within 1, or within 0.5.
The term substantially similar means a variant amino acid sequence that is at least 80% identical to a native amino acid sequence, or at least 90% identical. The percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). Some default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353 358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are known. Naturally occurring variants are also encompassed by the invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the native protein, wherein the native biological property is retained.
While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration of the encoding DNA; taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid. A table of amino acids and their codons is presented below for use in such embodiments, as well as for other uses, such as in the design of probes and primers and the like.
The term functionally equivalent codon is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (see Codon Table, above).
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5 or 3 sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5 or 3 portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
3. Altered Amino Acids
The present invention, in many aspects, relies on the synthesis of peptides and polypeptides in cyto, via transcription and translation of appropriate polynucleotides. These peptides and polypeptides will include the twenty natural amino acids, and post-translational modifications thereof. However, in vitro peptide synthesis permits the use of modified or unusual amino acids. A table of exemplary, but not limiting, modified or unusual amino acids is provided herein below.
In addition to the biological functional equivalents discussed above, the present inventors also contemplate that structurally similar compounds may be formulated to mimic the key portions of peptide or polypeptides of the present invention. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and, hence, also are functional equivalents.
Certain mimetics that mimic elements of protein secondary and tertiary structure are described in Johnson et al. (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody or antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
Some successful applications of the peptide mimetic concept have focused on mimetics of -turns within proteins, which are known to be highly antigenic. Likely -turn structure within a polypeptide can be predicted by computer-based algorithms. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.
Other approaches have focused on the use of small, multidisulfide-containing proteins as attractive structural templates for producing biologically active conformations that mimic the binding sites of large proteins (Vita et al., 1998). A structural motif that appears to be evolutionarily conserved in certain toxins is small (30-40 amino acids), stable, and highly permissive for mutation. This motif is composed of a beta sheet and an alpha helix bridged in the interior core by three disulfides.
Beta II turns have been mimicked successfully using cyclic L-pentapeptides and those with D-amino acids. (Weisshoff et al., 1999). Also, Johannesson et al. (1999) report on bicyclic tripeptides with reverse turn-inducing properties.
Methods for generating specific structures have been disclosed in the art. For example, alpha-helix mimetics are disclosed in U.S. Pat. Nos. 5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structures render the peptide or protein more thermally stable, also increase resistance to proteolytic degradation. Six, seven, eleven, twelve, thirteen and fourteen membered ring structures are disclosed.
Methods for generating conformationally restricted beta turns and beta bulges are described, for example, in U.S. Pat. Nos. 5,440,013; 5,618,914; and 5,670,155. Beta-turns permit changed side substituents without having changes in corresponding backbone conformation, and have appropriate termini for incorporation into peptides by standard synthesis procedures. Other types of mimetic turns include reverse and gamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos. 5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S. Pat. Nos. 5,672,681 and 5,674,976.
In certain embodiments, the present invention concerns novel compositions comprising at least one proteinaceous molecule, such as a polypeptide with multiple HCH2 regions. As used herein, a proteinaceous molecule, proteinaceous composition, proteinaceous compound, proteinaceous chain or proteinaceous material generally refers to, but is not limited to, a protein of greater than about 200 amino acids or the full length endogenous sequence translated from a gene; a polypeptide of greater than about 100 amino acids; or a peptide of from about 3 to about 100 amino acids. All the proteinaceous terms described above may be used interchangeably herein.
In certain embodiments the size of at least one proteinaceous molecule may comprise, but is not limited to, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater amino molecule residues, and any range derivable therein.
As used herein, an amino molecule refers to any amino acid, amino acid derivative, or amino acid mimic as would be known to one of ordinary skill in the art. In certain embodiments, the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues. In other embodiments, the sequence may comprise one or more non-amino molecule moieties. In particular embodiments, the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.
Accordingly, the term proteinaceous composition encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not limited to those shown in Table 3.
In certain embodiments the proteinaceous composition comprises at least one protein, polypeptide or peptide. In further embodiments the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide. As used herein, the term biocompatible refers to a substance that produces no significant untoward effects when applied to, or administered to, a given organism according to the methods and amounts described herein. Organisms include, but are not limited to, a bovine, a reptilian, an amphibian, a piscine, a rodent, an avian, a canine, a feline, a fungus, a plant, an archebacteria, or a prokaryotic organism, with a selected animal or human subject being sometimes preferred. Such untoward or undesirable effects are those such as significant toxicity or adverse immunological reactions. In some embodiments, biocompatible protein, polypeptide or peptide containing compositions will generally be mammalian proteins or peptides, or synthetic proteins or peptides, each essentially free from toxins, pathogens and harmful immunogens.
Proteinaceous compositions may be made by any technique known, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials. The nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art. One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/). The coding regions for these known genes may be amplified or expressed using the techniques disclosed herein or otherwise known. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known.
In certain embodiments a proteinaceous compound may be purified. Generally, purified will refer to a specific protein, polypeptide, or peptide composition that has been subjected to fractionation to remove various other proteins, polypeptides, or peptides, and which composition substantially retains its activity, as may be assessed, for example, by the protein assays, that may be known for the specific or desired protein, polypeptide or peptide.
In certain embodiments, the proteinaceous composition may comprise at least one antibody. As used herein, the term antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD, and IgE. Generally, IgG or IgM may be preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
Polypeptide regions of proteinaceous compounds may be linked via a linker group. A linker group is able to join the compound of interest via a biologically-releasable bond, such as a selectively-cleavable linker or amino acid sequence.
The term antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab, Fab, F(ab)2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known. Means for preparing and characterizing antibodies are also known (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
It is contemplated that virtually any protein, polypeptide, or peptide containing component may be used in the compositions and methods disclosed herein. However, the proteinaceous material may be biocompatible. Proteins and peptides suitable for use in this invention may be autologous proteins or peptides, although the invention is clearly not limited to the use of such autologous proteins. As used herein, the term autologous protein, polypeptide or peptide refers to a protein, polypeptide or peptide that is derived from or obtained from an organism. Organisms that may be used include, but are not limited to, a bovine, a reptilian, an amphibian, a piscine, a rodent, an avian, a canine, a feline, a fungus, a plant, or a prokaryotic organism, with a selected animal or human subject being sometimes being preferred. The autologous protein, polypeptide or peptide may then be used as a component of a composition intended for application to the selected animal or human subject. It can be biocompatible (i.e. from mammalian origin for mammals, from human origin for humans, from canine origin for canines, etc.; it is autologous; it is non-allergenic, or it is non-immunogenic).
Autoimmune disease often involves both T-cell and B-cell mediated components that may act dependently or independently of one another, simultaneously or sequentially, resulting in a host-damaging disease often characterized by tissue or cell compromise and a loss of one or more bodily functions. Fc receptors and proteins of the complement cascade are often intimately associated with the generation of the autoimmune response, the regulation of the ongoing immune response, and the effector phase of the immune response (i.e. those mechanisms that lead to tissue or cell destruction or damage). The inventive polypeptides, through their ability to bind Fc receptors or complement, may influence disease outcome by their impact upon one or more of these areas.
The inventive polypeptides may favorably alter disease activity by multiple pathways depending on the fusion protein design and type of disease treated. Inventive polypeptides may be designed to contain; multiple units of HCH2 regions, or portions thereof, able to bind Fc receptors, multiple units of HCH2 regions able to bind complement components, or both. It is contemplated that the inventive polypeptide design can be modified to maximize potential benefits achieved from its use in treating a specific disease and its composition may vary from one disease to the next. For example, for the treatment of some diseases it may be preferable to retain the Fc receptor binding ability of the fusion proteins but exclude or diminish binding of components of the complement cascade. The obverse may be preferred for the treatment of other diseases.
The effect of the inventive polypeptides on disease outcome will depend not only on whether they contain multiple units able to bind Fc receptors, multiple units able to bind complement components, or both, but also on other protein domains that may be coexpressed in the inventive polypeptides to give them an additional function, binding capability, or other added feature. An additional modification to the inventive polypeptides includes the binding of additional proteins, protein domains, or peptides to the inventive polypeptides that give them an additional function, binding capacity, or other added feature. The flexibility in the fusion protein design enables the inventors to, depending on disease type, modify the inventive polypeptides to maximize their therapeutic potential. It is an embodiment of the current invention that in addition to the treatment of autoimmunity, modifications of the inventive polypeptides as described above are applicable to their use in the treatment of neoplasms, the treatment of infections by viruses or other pathogens, the treatment of warts, and the purposeful induction of an immune response directed against a particular antigen or antigens, as for example in a vaccine.
Inventive polypeptides able to bind Fc receptors may influence disease outcome through multiple mechanisms including but not limited to blocking Fc receptor accessibility to endogenously produced Ig and immune complexes. Such blockade would be expected to limit self-antigen presentation by antigen presenting cells and to, as a consequence, diminish autoimmune responses. Blockade of Fc receptors may also limit or diminish tissue and cell destruction. Tissue and cell destruction in autoimmune disease can be mediated by Fc receptor-expressing effector cells (monocytes, neutrophils, macrophages, microglia, NK cells, as well as other cell types) that bind self-antigen reactive Ig bound to tissue or cells. For example, in ATP, the inventive polypeptides could limit platelet destruction and clearance by the body by decreasing their uptake by Kupffer cells in the liver and spleen via Fc receptor-mediated mechanisms. Similarly inventive polypeptides might limit demyelination in the CNS in multiple sclerosis or acetylcholine receptor destruction of motor neural endplates in myasthenia gravis by decreasing macrophage accessibility to Ig bound to self Ag in target tissues. The inventive polypeptides may favorably alter numerous autoimmune diseases via similar mechanisms.
The inventive polypeptides may modify autoimmune disease by activating cells through Fc receptors and thereby altering the secretion of immunomodulators, the expression of specific cell surface markers, or the type or magnitude of specific cell functions. Modulation of protein secretion might include the decreased or increased production of interleukins including but not limited to IL-2, IL-4, IL-10, IL-12, IL-18; cytokines including but not limited to TGF, TNF, TNF; interferons , , and ; growth factors, and products of the arachidonate cascade. Cellular functions that may be altered include cellular cytotoxicity, cell division, and activation state.
The inventive polypeptides may also be used to suppress or amplify immunity to a specific antigen. Autoimmune disease may be treated by inducing tolerance to a specific antigen or by deviating the autoimmune response to a specific antigen from a harmful pathogenic one to a less harmful type. For example, in multiple sclerosis the elaboration of type 1 cytokines (IL-12, IL-2) in response to autoantigen is generally thought to be deleterious to the host while induction of a type 2 response (IL-4, IL-10) is thought to be protective. The purposeful deviation of the immune response from a Th1 type to a Th2 type would likely be beneficial in the treatment of multiple sclerosis. In contrast, a Th2 type response is thought to be harmful in other autoimmune diseases such as lupus erythematosus, and consequently the purposeful deviation of the response to autoantigen in this disease from a Th2 type response to a Th1 type response would likely be beneficial. Thus, modification of the inventive polypeptides would vary depending on the disease type and the mechanisms involved.
It is an embodiment of the current invention to coexpress one or more protein domains of a specific antigen or bind one or more specific antigens or antigenic determinants to the inventive polypeptides that would induce a protective immune response, deviate a harmful immune response to a less harmful one, or induce a state of nonresponsiveness to antigen (Lasalle et al., 1994). For example, the inventors contemplate, in the treatment of multiple sclerosis, to coexpress a neuroantigen peptide in the fusion protein that induces a protective Th2 type response or an unresponsive state. A nonlimiting list of potential neuroantigens that might be used for the treatment of multiple sclerosis include proteolipid protein, myelin basic protein and myelin oligodendrocyte glycoprotein. Similarly, a T cell receptor or Ig domain may be expressed in the fusion protein that would induce a protective anti-T cell receptor or anti-idiotype response. The inventors contemplate that varying the protein coexpressed based upon disease type should allow the inventive polypeptides to be used for the treatment of numerous autoimmune diseases.
As mentioned earlier, the adaptive immune system is often referred to as having two components, cellular immunity (or Th1 type response) and humoral immunity (or Th2 type response). Response to an antigen evokes one or both of these components. Immunomodulators such as lymphokines and monokines that promote one component often inhibit the other. Thus a strong cellular response will often occur in the presence of a blunted humoral response and vice versa. Factors important to the development of one or the other response include the presence or absence of cytokines, costimulatory factors, as well as other factors that are known to those familiar in the art (Lasalle et al., 1994). For example the presence of IL-4 has been shown to enhance a Th2 type response while the presence of interferon gamma induces a Th1 type response (Swain et al., 1988). In the treatment of autoimmune disease, neoplasms, or viral infections, or in the induction of immunity to pathogens by vaccine based therapies, selective modulation of one or both of these components may be used. The coadministration of cytokines, steroids, or other immunomodulators may be used in the treatment of varying diseases or when attempting to induce immunity to an antigen or antigens based upon the type of response desired.
1. Neoplastic Cell Targets
Many so-called tumor antigens have been described, any one of which could be employed as a target in connection with the combined aspects of the present invention. A large number of exemplary solid tumor-associated antigens are listed herein below. The preparation and use of antibodies against such antigens is known, and exemplary antibodies include from gynecological tumor sites: OC 125; OC 133; OMI; Mo v1; Mo v2; 3C2; 4C7; ID3; DU-PAN-2; F 36/22; 4F7/7A10; OV-TL3; B72.3; DF3; 2C8/2F7; MF 116; Mov18; CEA 1-H5; CA 19-9 (1116NS19-9); H17-E2; 791T/36; NDOG2; H317; 4D5, 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8; HMFG2; 3.14.A3; from breast tumor sites: DF3; NCRC-11; 3C6F9; MBE6; CLNH5; MAC 40/43; EMA; HMFG1 HFMG2; 3.15.C3; M3, M8, M24; M18; 67-D-11; D547Sp, D75P3, H222; Anti-EGF; LR-3; TA1; H59; 10-3D-2; HmAB1,2; MBR 1,2,3; 24171; 24-172 (3E12); F36/22.M7/105; C11, G3, H7; B6.2; B11; Cam 171; SM3; SM4; C-Mul (566); 4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, 5B8; OC 125; MO v2; DU-PAN-2; 4F7/7A10; DF3; B723; cccccCEA 11; H17-E2; 314A3; FO23C5; from colorectal tumor sites: B723; (17-1A) 1083-17-1A; CO17-1A; ZCE-025; AB2; HT-29-15; 250-30.6; 44X14; A7; GA733; 791T/36; 28A32; 28.19.8; X MMCO-791; DU-PAN-2; ID3; CEA 11-H5; 2C8/2F7; CA-19-9 (1116NS19-9); PR5C5; PR4D2; PR4D1; from melanoma sites 41; 82 M17; 96.5; 118.1, 133.2, (113.2); L1, L10, R10(R19); 112; K5; 61; R24; 51; 225.28S; 465.12S; 9227; F11; 376.96S; 465.12S; 1595; 1595; Mel-14; Mel-12; Me3-TB7; 225.28SD; 763.24TS; 705F6; 436910; M148; from gastrointestinal tumors: ID3; DU-PAN-2; OV-TL3; B72.3; CEA 11-H5; 314 A3; C COL1; CA-19-9 (1116NS19-9) and CA50; OC125; from lung tumors: 4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8; MO v2; B72.3; DU-PAN-2; CEA 11-H5; MUC8-22; MUC2-63; MUC2-39; MUC7-39; and from miscellaneous tumors: PAb 240; PAb 246; PAb 1801; ERIC1; M148; FMH25; 61; CA1; 3F8; 4F7/7A10; 2C8/2F7; CEA 11-H5.
Another means of defining a targetable tumor is in terms of the characteristics of a tumor cell itself, rather than describing the biochemical properties of an antigen expressed by the cell. Accordingly, the ATCC catalogue exemplifies human tumor cell lines that are publicly available (from ATCC Catalogue). Exemplary cell lines include J82; RT4; ScaBER; T24; TCCSUP; 5637; SK-N-MC; SK-N-SH; SW 1088; SW 1783; U-87 MG; U-118 MG; U-138 MG; U-373 MG; Y79; BT-20; BT-474; MCF7; MDA-MB-134-VI; MDA-MD-157; MDA-MB-175-VII; MDA-MB-361; SK-BR-3; C-33 A; HT-3; ME-180; MS751; SiHa; JEG-3; Caco-2; HT-29; SK-CO-1; HuTu 80; A-253; FaDu; A-498; A-704; Caki-1; Caki-2; SK-NEP-1; SW 839; SK-HEP-1; A-427; Calu-1; Calu-3; Calu-6; SK-LU-1; SK-MES-1; SW 900; EB1; EB2; P3HR-1; HT-144; Malme-3M; RPMI-7951; SK-MEL-1; SK-MEL-2; SK-MEL-3; SK-MEL-5; SK-MEL-24; SK-MEL-28; SK-MEL-31; Caov-3; Caov-4; SK-OV-3; SW 626; Capan-1; Capan-2; DU 145; A-204; Saos-2; SK-ES-1; SK-LMS-1; SW 684; SW 872; SW 982; SW 1353; U-20S; Malme-3; KATO III; Cate-1B; Tera-1; Tera-2; SW579; AN3 CA; HEC-1-A; HEC-1-B; SK-UT-1; SK-UT-1B; SW 954; SW 962; NCI-H69; NCI-H128; BT-483; BT-549; DU4475; HBL-100; Hs 578Bst; Hs 578T; MDA-MB-330; MDA-MB-415; MDA-MB-435S; MDA-MB-436; MDA-MB-453; MDA-MB-468; T-47D; Hs 766T; Hs 746T; Hs 695T; Hs 683; Hs 294T; Hs 602; JAR; Hs 445; Hs 700T; H4; Hs 696; Hs 913T; Hs 729; FHs 738Lu; FHs 173We; FHs 738B1; NIH:0VCAR-3; Hs 67; RD-ES; ChaGo K-1; WERI-Rb-1; NCI-H446; NCI-H209; NCI-H146; NCI-H441; NCI-H82; H9; NCI-H460; NCI-H596; NCI-H676B; NCI-H345; NCI-H820; NCI-H520; NCI-H661; NCI-H510A; D283 Med; Daoy; D341 Med; AML-193 and MV4-11.
One may consult the ATCC Catalogue of any subsequent year to identify other appropriate cell lines. Also, if a particular cell type is desired, the means for obtaining such cells, or their instantly available source, are known. An analysis of the scientific literature will thus readily reveal an appropriate choice of cell for any tumor cell type desired to be targeted.
Recent technological advances allow rapid and efficient comparisons of gene expression in neoplastic tissue to that of normal tissue. These technological advances include but are not limited to differential gene analysis using gene chip arrays and protein arrays. Using these technologies one is able to compare mRNA species and proteins expressed in neoplastic tissue to that found in normal tissue. Those mRNA species or proteins that are differentially expressed in neoplastic tissue compared to normal tissue may be readily discerned. Proteins found to be preferentially expressed in neoplastic tissue or in neoplastic cells using these screening technologies serve as likely candidates for the further development of cancer or tumor specific therapies. It is an embodiment of the current invention that tumor associated proteins or tumor specific proteins discovered using these technologies may be employed as targets in connection with the combined aspects of the present invention.
Combination of the inventive polypeptides with other therapeutic agents is contemplated for use in the clinical treatment of various diseases that involve altering immunity, inflammation or neoplasms.
Naturally, before wide-spread use, animal studies and clinical trials will be conducted. The various elements of conducting a clinical trial, including patient treatment and monitoring, are known, especially in light of the present disclosure.
The present invention contemplates that the inventive polypeptides may be used in combination with other therapies. Therapies for autoimmune diseases include but are not limited to interferon-, interferon-, i.v. immunoglobulins, monoclonal antibodies such as h5G1.1-mAb, polyclonal antibodies such as anti-RhoD (WinRho SDF), retinoic acid and other immunomodulatory agents such as glatiramer acetate.
Therapies for diseases that involve inflammation include, but are not limited to non-steroidal inflammatory drugs (NSAIDs) such as cyclo-oxygenase 2 (COX-2) inhibitors.
The present invention contemplates that the inventive polypeptides may be used as an adjuvant in combination with vaccines. Vaccines include, for example, mAb 105AD7 anti-idiotype vaccine, mAb 11D10 anti-idiotype vaccine, mAb 3H1 anti-idiotype vaccine, GM2, GM2-KLH, and MUC-1 antigen among many others.
Cancer therapies include a variety of combination therapies that are contemplated with the inventive polypeptides including immunological, chemical and radiation based treatments. Combination immunotherapies include, for example, interleukin-2, monoclonal or bispecific antibodies such as Rituximab, Herceptin (Trastuzumab), mAb Lym-1, mAb ml70, mAb BC8, mAb Anti-B1 (tositumomab), Campath-1H, anti-CEA mAb MN-14, mAb HuG1-M195, mAb HuM291, mAb 3F8, mAb C225 (cetuximab), anti-Tac mAb (daclizumab), and mAb hLL2 (epratuzumab).
Combination immunotherapies also include monoclonal antibodies (mAb) linked to toxins or other agents. Examples include mAb gemtuzumab ozogamicin (mylotarg), mAb Mono-dgA-RFB4, mAb ibritumomab tiuxetan (IDEC-Y2B8), and Anti-Tac(Fv)-PE38. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate or any analog or derivative variant thereof.
For precancerous conditions such as benign prostatic hyperplasia, a second therapeutic agent selected from an -1 adrenergic receptor blocker such as terazosin, doxazosin, prazosin, bunazosin, indoramin, tamsulosin, prazicin or alfuzosin; a 5--reductase enzyme blocker such as finasteride or an azasteroid derivative; a combination of an -1 adrenergic receptor blocker, and a 5--reductase enzyme blocker, a potassium channel opener such as minoxidil, and a retinoic acid derivative.
Various combinations may be employed, for instance where the inventive polypeptide is A and the radio-, chemotherapeutic or other therapeutic agent is B:
The terms contacted and exposed, when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
The therapy including inventive polypeptides may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and inventive polypeptide are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and the fusion protein would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 h of each other or within about 6-12 h of each other, with a delay time of only about 12 h being also possible. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) elapse between the respective administrations.
Pharmaceutical compositions of the present invention comprise an effective amount of one or more inventive polypeptides, therapeutic agents or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier. Aqueous compositions of the present invention comprise an effective amount of the inventive polypeptides, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrases pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
As used herein, pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). The use of such media and agents for pharmaceutical active substances is known. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologic Standards.
The biological material should be dialyzed to remove undesired small molecular weight molecules or lyophilized for more ready formulation into a desired vehicle, where appropriate. The active compounds will then generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intranasal, intralesional, or even intraperitoneal routes. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The inventive polypeptides can be formulated into a composition in a free base, in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars or sodium chloride can be included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intranasal, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, Remington's Pharmaceutical Sciences 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used, including cremes.
In certain embodiments, the use of liposomes or nanoparticles is contemplated for the formulation and administration of the fusion proteins or analogs thereof. The formation and use of liposomes is generally known and is also described below.
Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles are easily made.
Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 , containing an aqueous solution in the core.
The following information may also be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one may operate at the same time.
The therapeutic agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, by inhalation (e.g. aerosol inhalation), by injection, by infusion, by continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other methods or any combination of the foregoing (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990).
The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
In any case, the composition may comprise various antioxidants to retard oxidation of one or more component.
In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof. In some cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof.
In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants in the present invention. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in some embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.
In certain embodiments the inventive polypeptides are prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. Some carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the invention, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.
In certain embodiments an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations of the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
Additional formulations which are suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, or about 1% to about 2%.
The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
The invention also provides for the use of adjuvants as components in an immunogenic composition compatible with the purified proteins to boost the immune response resulting from vaccination. One or more adjuvants can be selected from the group comprising saponins (e.g, GP-0100), or derivatives thereof, emulsions alone or in combination with carbohydrates or saponins, and aluminum-based adjuvants (collectively, alum or alum-based adjuvants) such as aluminum hydroxide, aluminum phosphate, or a mixture thereof. Aluminum hydroxide (commercially available as Alhydrogel) was used as alum in the Examples. A saponin is any plant glycoside with soapy action that can be digested to yield a sugar and a sapogenin aglycone. Sapogenin is the nonsugar portion of a saponin. It is usually obtained by hydrolysis, and it has either a complex terpenoid or a steroid structure that forms a practicable starting point in the synthesis of steroid hormones. The saponins of the invention can be any saponin as described above or saponin-like derivative with hydrophobic regions, especially the strongly polar saponins, primarily the polar triterpensaponins such as the polar acidic bisdesmosides, e.g. saponin extract from Quillsjabark Araloside A, Chikosetsusaponin IV, Calendula-Glycoside C, chikosetsusaponin V, Achyranthes-Saponin B, Calendula-Glycoside A, Araloside B, Araloside C, Putranjia-Saponin III, Bersamasaponiside, Putrajia-Saponin IV, Trichoside A, Trichoside B, Saponaside A, Trichoside C, Gypsoside, Nutanoside, Dianthoside C, Saponaside D, aescine from Aesculus hippocastanum or sapoalbin from Gyposophilla struthium, saponin extract Quillaja saponaria Molina and Quil A. In addition, saponin may include glycosylated triterpenoid saponins derived from Quillaja Saponaria Molina of Beta Amytin type with 8-11 carbohydrate moieties as described in U.S. Pat. No. 5,679,354. Saponins as defined herein include saponins that may be combined with other materials, such as in an immune stimulating complex (ISCOM)-like structure as described in U.S. Pat. No. 5,679,354. Saponins also include saponin-like molecules derived from any of the above structures, such as GPI-0100, such as described in U.S. Pat. No. 6,262,029. The saponins of the invention can be amphiphilic natural products derived from the bark of the tree, Quillaja saponaria. They can consist of mixtures of triterpene glycosides with an average molecular weight (Mw) of 2000. In another embodiment of the invention a purified fraction of this mixture is used.
Any of the compositions described herein may be comprised in a kit. In a non-limiting example, an inventive polypeptide, a nucleic acid coding for an inventive polypeptide or additional agent, may be comprised in a kit. The kits will thus comprise, in suitable container means, an inventive polypeptide, a nucleic acid coding for an inventive polypeptide or an additional agent of the present invention. The inventors envisage other components that may be included in a kit. These include but are not limited to immunodetection agents such as peroxidase and alkaline phosphatase linked monoclonal and polyclonal antibodies, immunoprecipitation reagents such as protein A- or protein G-linked beads, immune cell purification reagents such as magnetic beads, cloning reagents for the purpose of manipulating an expression vector, protein expression reagents including prokaryotic and eukaryotic cell lines for the purpose of protein expression.
The kits may comprise a suitably aliquoted inventive polypeptide or additional agent compositions of the present invention, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, or suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the inventive polypeptide, lipid, additional agent, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
Therapeutic kits of the present invention comprise an inventive polypeptide, other polypeptide, peptide, inhibitor, gene, vector or other effectors. Such kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of an inventive polypeptide in a pharmaceutically acceptable formulation. The kit may have a single container means, or it may have distinct container means for each compound.
When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being sometimes preferred. The inventive polypeptide composition may also be formulated into a syringeable composition, in which case, the container means may itself be a syringe, pipette, or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, or even applied to or mixed with the other components of the kit.
However, the components of the kit may be provided as dried powder(s). When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the immunoglobulin fusion protein formulation is placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer or other diluent.
The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
Irrespective of the number or type of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate inventive polypeptide within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.
As used herein the specification, a or an may mean one or more. As used herein in the claim(s), when used in conjunction with the word comprising, the words a or an may mean one or more than one. As used herein another may mean at least a second or more. As used herein, or takes on its usual meaning in that it also includes the conjunctive sense of and.
Some of the abbreviations used in this application can be found in Table 4.
The following examples are included to demonstrate some embodiments of the invention. It will be appreciated that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Examples 1, 2, and 3 describe the cloning and construction of the Fc framework region and HCH2 polymer region of the ligands described in this application. This sequence in the mature polypeptide referred to as R4 is 742 amino acids long and is SEQ ID NO: 13.
The Fc region of human IgG1 corresponds to the constant region domains that include the hinge region and CH2 and CH3 domains, (HCH2-CH3). The cDNA for the Fc region was isolated to serve as template for HCH2 polymer construction. In addition the HCH2 polymers wer expressed as fusion to the Fc region. While the Fc region was derived from human IgG1 cDNA one could equally use the HCH2-CH3 domains from human IgG3 for this same purpose. To obtain the HCH2-CH3 sequence for human IgG1, total RNA was isolated from the cell line ARH-77 (ATCC #: CRL-162) using the method of Chomczynski and Sacchi (Chomczynski, 1986). cDNA was produced from the total RNA using reverse transcription. First strand cDNA synthesis was primed with 100 mol random hexamers using 200 U SuperScript II reverse transcriptase (Invitrogen) and 5 g of total RNA in a 20 L reaction mixture that was 500 M in dNTPs (Pharmacia), 1 U RNasin/L (Promega), 10 M in DTT, and 1 in first strand buffer. Reaction proceeded at 42 C. for 50 min.
The fragment containing the HCH2-CH3 region (corresponding to amino acid residues 226-457) was subcloned using RT-PCR, the primer, FRM-5p-H3, which introduced a HindIII site immediately 5 of the hinge region and a second primer, FRM-3p-Sal, which introduced a SalI site immediately 3 of the stop codon (Table 5). PCR reactions were carried out in a volume of 50 L and consisted of 1PCR buffer (10 mM Tris pH 8.3 and 50 mM KCL), 1.5 mM MgCl2, 150 M of dNTPs, 15 mol each of forward and reverse primers, 5 L of reverse transcription products and 1.25 U TAQ polymerase. Cycling parameters consisted of 30s denaturation at 94 C., 1 min annealing at 60 C. and 1 min extension at 72 C. and reactions proceeded for 30 cycles. The contents of 4 identical PCR reactions were pooled and extracted once with 1:1:0.05 mixture of phenol:CHCl3:isoamyl alcohol (PCIA) and subsequently extracted 1 with CHCl3. DNA was recovered by precipitation with sodium acetate, pH5.4, and ethanol. DNA pellets were washed 1 with 75% ethanol, and air dried. Amplified cDNAs were dually digested for two hours in a 120 L digestion buffer containing 150 U of HindIII and 150 U SalI. The digest was extracted 1 with PCIA and 1 with CHCl3 and DNA was recovered by ethanol precipitation. DNA pellets were washed 1 with 75% ethanol, air dried and resuspended in 15 L of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The HindIII and SalI digested PCR products were ligated into like-digested pBSKS+vector (Stratagene).
The ligation reaction contained 50-100 ng of vector, 20-400 ng of insert, 2 L of 10 reaction buffer (660 mM Tris-HCl, pH 0.5, 50 mM MgCl2, 50 mM DTT, 10 mM ATP), 1 L of 5 mM ATP, and 5 U T4 DNA ligase in a final volume of 20 L. The ligation reaction proceeded overnight at 16 C. 10 L of ligation product was used to transform DH5a cells (subcloning-efficiency, Invitrogen). Transformed bacteria were plated onto LB-Amp plates. 10 colonies from each transformation were grown up over-night in LB-Amp medium and mini-prep DNA was isolated and analyzed by HindIII and SalI digestion.
Plasmid preparation: To produce larger quantities of plasmid, single colonies were used to inoculate 75 mL of LB medium supplemented with the appropriate antibiotic and the cultures were grown over-night. Plasmid DNA was isolated from bacteria using the Qiagen plasmid midi purification kit following the manufacturers protocol (Qiagen). Plasmid DNA was resuspended in TE buffer and UV absorbance at 260/280 nm is used to determine concentration and purity. Plasmid concentration and purity was confirmed by electrophoresis on agarose gels and visualization of DNA by ethidium bromide staining. Two positive clones were analyzed by DNA sequencing to verify sequence integrity. The resulting clone pFRM-HS was used for further expression construct assembly as describe in the following examples. Primers for this and subsequent steps involving IgG1 cloning were designed using sequence data from the human IgG1 constant region gene as a guide (accession # Z17370).
This example describes the isolation and construction of a cDNA coding for the hinge and CH2 region (HCH2) used for the construction of the HCH2 polymer. The region of Fc that binds to FcRI, FcRII, and FcRIII is found within the HCH2 region. The HCH2 region (corresponding to amino acid residues 226-350 of IgG1) was isolated as a separate monomer unit using PCR. The hinge region within the HCH2 monomer unit was modified using PCR mutagenesis to change the three cysteines that form inter-chain disulfide bridges between Fc units to serines. Since the mature polypeptide will contain from 2 to 6 HCH2 units in each polypeptide chain, we mutated the three cysteines in each hinge region (H) so that aberrant disulfide bonds do not form during the translation of the mRNA to a polypeptide. The polymer was constructed using three differing constructs, referred to as ENH, SNH, and SH3. These units differ one from another only at their 5 or 3 ends in that they have different flanking restriction sites as detailed below. These units allow for the construction of the HCH2 polymers as detailed in Example 3.
The first unit produced, composed of 5 EcoRI-HCH2-NruI-HindIII 3, is termed ENH to denote the sequence of restriction sites and the delta is included in front of the hinge, H, to denote that the hinge region was mutagenised. The ENH construct served as the starting unit for polymer construction. This was accomplished by amplifying the HCH2 region (corresponding to amino acid residues 226-350 of IgG1) using a 5 primer, Hingel (Table 5) which introduced single nucleotide changes in each of the three hinge cysteine codons resulting in their alteration to serine residues. The 5 primer also introduced an EcoRI site immediately 5 of the hinge region. The 3 primer, CH2NH3 (Table 5), directed the amplification of the CH2 domain and introduced an in-frame 3 NruI site separated by a 6 nucleotide spacer from a HindIII site. Clone pFRM-HS was the template for the PCR reactions. PCR reactions conditions were identical to those described in Example 1. PCR reactions were pooled, extracted with phenol: Chloroform to remove the Taq polymerase and the amplified DNA was recovered with sodium acetate precipitation as described for Example 1. Amplified cDNA was dually digested for two hours in a 120 L digestion buffer containing 150 U of EcoRI and 150 U HindIII. The digest was extracted and DNA was recovered by ethanol precipitation as described for Example 1. The EcoRI and HindIII digested PCR products were ligated into like-digested pBSKS+vector (Stratagene). Ligation reaction conditions were the same as described for Example 1. 10 L of ligation product was used to transform DH5a cells (subcloning-efficiency, Invitrogen). Transformed bacteria were plated onto LB-Amp plates. 10 colonies from each transformation were grown up over-night in LB-Amp medium and mini-prep DNA was isolated and analyzed by EcoRI and HindIII digestion.
Two clones identified as positive by restriction analysis were used to inoculate 75 mL cultures to produce larger quantities of plasmid using Qiagen midi columns as described for Example 1. The clones were analyzed by DNA sequencing to verify sequence integrity. Clone pENH18 was used in subsequent cloning steps.
Two additional constructs, an extension unit designated pSNH, and a capping unit designated pSH3, were generated. These varied from pENH18 only in their flanking restriction sites. pSNH has 5 SmaI-HCH2-NruI-HindIII 3 and was amplified using pENH18 as template and primers that introduced the flanking restriction sites (Table 5). The second construct, pSH3, contains 5 SmaI-HCH2-HindIII 3 and was amplified from pENH18 template using a 5 primer, SMA-DELH, and a 3 primer, CH2H3 (Table 5), which introduced a single HindIII site that flanks the 3 end of the CH2 domain. In both instances, the techniques and conditions for the PCR reactions, restriction digest, ligation and plasmid preparation are identical to those described for Example 1. For polymer construction, both pSNH and pSH3 plasmids were digested with SmaI and HindIII. The digestion released the 5 SmaI-HCH2-NruI-HindIII 3 and 5 SmaI-HCH2-HindIII 3 inserts from the vector. The restriction digests were extracted once with 1:1:0.05 mixture of phenol:CHCl3:isoamyl alcohol (PCIA) and subsequently extracted 1 with CHCl3. DNA was recovered by precipitation with sodium acetate, pH5.4, and ethanol. DNA pellets were washed 1 with 75% ethanol, and air dried. The pelleted digests were resuspended in 20 L of TE, mixed with 6 loading dye (0.025% xylene cyanol, 0.025% bromphenol blue, and 50% sucrose in Tris-EDTA buffer) and loaded onto 1% low-melt agarose gels in TAE running buffer. The inserts were visualized on a UV gel box, and the inserts were excised from the gel and transferred to microfuge tubes. The DNA inserts were purified from the gel using the QIAEX II Gel Extraction kit (Qiagen) following manufacturers instructions. The inserts were eluted in 50 L of TE and stored for use in polymer construction (Example 3).
Polymers composed of HCH2 units were built using the scheme presented in FIG. 1. The HCH2 polymers were constructed by the sequential addition of a single starting unit (ENH), multiple extension units (SNH), and ended by addition of a single capping unit (SH3).
Clone pENH18 was digested with NruI and HindIII resulting in a 5 blunt end and a 3 sticky end. Next a 5 SmaI-HCH2-NruI-HindIII 3 insert, isolated as described in Example 2, was ligated into the linearized vector resulting in the in-frame insertion of a HCH2 repeat unit at the 3 end of the pENH18 starting unit. The insertion also regenerated the original sequence of restriction sites (NruIspacer-Hin dIII) that were used in the next round of extension. Conditions for the ligation reaction were identical to those described for Example 1. The ligation mixture is transformed into DH5a cells. Transformed bacteria were plated onto LB-Amp plates and 10 colonies from the transformation were grown up over-night in LB-Amp medium and min-prep DNA was isolated and analyzed by EcoRI and HindIII digestion to confirm the insertion. Two colonies were expanded into 75 mL of LB-Amp broth and grown overnight. Plasmid DNA was isolated using the Qiagen midi columns as described in Example 1. Sequence integrity was confirmed with DNA sequencing. The extension process continued with NruI and HindIII digestion of the nascent polymer vector followed by ligation with the next SNH insert as described above. This cycle of digestion, ligation, transformation, and plasmid isolation was repeated twice more to generate the HCH2 polymer sequence for R4. In the final round of polymer construction a capping unit (SH3 insert) is ligated into the polymer instead of the SNH insert. This resulted in the loss of the internal cloning site but importantly it results in an identical junction between all the inserted HCH2 units of the polymer. The result was the stepwise insertion of HCH2 units into the framework expression vector. Directionality of HCH2 insertion was maintained by the use of non-compatible flanking restriction sites but HCH2 insertion was confirmed with DNA sequencing at each step.
The junction between the HCH2 units was composed of the fusion of the 5 NruI half-site to the 3 SmaI half-site, resulting in an in-frame Gly-Ser spacer between the protein domains. Choice of restriction sites determines the amino acid composition of the spacer. Guiding the choice of restriction sites was the desire to introduce spacers between the HCH2 units that were composed of small, hydrophilic amino acids such as glycine and serine. The completed polymer constructs were liberated from the pBSKS+cloning vector by digestion with EcoRI and HindIII and the inserts were purified from low-melt agarose gels as described in Example 2. The polymer inserts were ligated into EcoRI and Hind III digested pFRM-HS resulting in the in-frame joining of the HCH2 polymers to the IgG1 framework region (FIG. 2.).
Examples 1, 2, and 3, provide a step by step process that one can use to produce a set of constructs that contain the framework region of human IgG1 with 2, 3 or 4 HCH2 units. The steps given here use specific sequences from human IgG1. However, this procedure can be readily modified to create polypeptides that contain up to 6 HCH2 repeat units by increasing the number of cycles of SNH insertion used to create the linear polymers.
This example describes the preparation of fusion proteins formed between the antigen, HSA1, and the polypeptide, R4, for its use in vaccines.
Background. HSA1 was selected as an antigen since it is poorly antigenic in the mouse. For this reason, we chose to use it to show the utility of using the R4 polypeptide to increase immune responses to a weak antigen in a vaccine formulation. Although HSA1 is used as the antigen in this specific example, these same steps can be used to link other polypeptide antigens to the R4 polypeptide for use in vaccines. HSA1, which spans residues 1-197 of the mature HSA polypeptide (Minghetti et al., 1986), is 67% identical and 82% similar to its murine homolog. HSA1, although weakly antigenic for mice, contains both T and B cell epitopes, and accordingly, provides a useful study of techniques to facilitate T cell-dependent Ab responses against weak Ags (Kenney et al., 1989; Marusic-Galesic et al., 1991; Marusic-Galesic et al., 1992). HSA can be converted into a stronger Ag when presented to APCs in an IC, and is widely employed as a carrier for haptens (Marusic-Galesic et al., 1991; Marusic-Galesic et al., 1992). HSA1 was expressed in a construct with R4 (HSA1R4). HSA1 was also expressed as an Fc fusion protein (HSA1Fc), or with a 6 Histidine tag (HSA1) to be used as comparators in these examples. HSA1Fc is an IgG fusion protein where HSA1 has been fused to the framework region of IgG as described in Example 1. The experiments described below also serve to demonstrate the general utility of the expression system Method. HSA1, HSA1Fc, and HSA1R4 cloning. Total RNA was isolated from cell line Hep G2 (ATCC HB-8065) using the method of Chomczynski and Sacchi (1987). First strand cDNA synthesis was primed with 100 pmol random hexamers using 200 U SuperScript II reverse transcriptase (Invitrogen, Carlsbad, Calif.) and 5 g of total RNA in a 20 L reaction mixture that was 500 M in dNTPs (Pharmacia, Piscataway, N.J.), 1 U RNasin/L (Promega Corp., Madison, Wis.), 10 M in DTT, and 1 in first strand buffer. Reaction proceeded at 42 C. for 50 min. Domain 1 of mature HSA (HSA1) was amplified from Hep G2 cDNA using PCR, the forward primer Dom1-F (5-GGCCGCATCTCGAGATGAAGTGGGTAACCTTTATTTCC-3; SEQ ID NO:11), and the reverse primer Dom1-R (5-CCGCATGAATTCTCTCTGTTTGGCAGACGAAGCCTT-3; SEQ ID NO:12). The leader sequence and the first 197 amino acid residues of mature HSA (i.e., HSA1) (Minghetti et al., 1986) were amplified, and flanking 5 Xho 1 and 3 Eco RI sites were introduced. The PCR product was digested with Xho I and Eco RI and ligated into like-digested pBSKS+ cloning vector (Stratagene, La Jolla, Calif.) to produce clone pHSA-BS.
The HSA1 fragment was subcloned into the (White et al., 2001) Fc and HCH2 polymer expression vectors described in Examples 1 and 3 to yield pHSA1Fc, pHSAIR2, pHSA1R3 and pHSA1R4 respectively. To express HSA1 with a 6HIS-tag, a short linker that introduces a His tag and a 3 stop codon was ligated into the Eco RI and Sal I sites of pHSA-BS. Fusion protein cDNAs were transferred into the baculovirus expression vector, pFastBacl (Invitrogen), by digestion with Bam HI and Sal I and subsequent ligation of the isolated cDNA fragments into the same sites on pFastBacl to produce pHSA1-FB, pHSA1Fc-FB and pHSA1R4FB. The pFastBacl vector places fusion protein constructs under the control of a strong baculovirus-specific promoter for expression in insect cells. The vector is also used to generate virus that express the recombinant proteins. The pFastBacl expression constructs were transformed into DH10Bac competent cells (Invitrogen) following manufacturer's instructions and correctly recombined virus was identified using PCR.
Cell line SF9 (ATCC CRL-1171) was maintained in ExCell 420 serum free medium (JRH Biosciences, Lenexa, Kans.) supplemented with 100 u/ml penicillin and 100 g/ml streptomycin. For bacmid transfection, 1106 cells were plated into each well of a 6 well cluster and allowed to grow overnight. Transfection medium was replaced with 2 ml fresh ExCell 420 without antibiotics. Two hours later, Bacmid DNA (6 g) was transfected into SF9 cells using Cellfectin reagent (Invitrogen). After 9 hours, the medium was replaced with fresh medium containing antibiotics. Forty-eight hours later, medium containing virus was harvested and used in a second round of viral amplification.
For protein expression, 100 ml of medium supplemented with 1% Pluronic F-68 (Invitrogen) in shaker flasks was seeded with 4105 SF9 cells/ml and shaken at 110 RPM at 27 C. for 24 hours at which time virus was introduced. Conditioned medium was harvested 72 hours later, and the protease inhibitor PMSF (Research Organics, Cleveland, Ohio) plus pepstatin A (Peptides International, Louisville, Ky.) were added to a final concentration of 1 mM and 1 M respectively. HSA1Fc and HSA1R4 were purified using protein G-Sepharose (Pharmacia) as described previously (White et al., 2001).
The 6His tagged HSA1 protein was purified using a Ni2+ immobilized resin (Ni-NTA, Qaigen, Valencia, Calif.). Prior to application to the column, interfering ions and peptides were removed by dialyzing the conditioned medium (12,000-14,000 MWCO Spectrapor tubing) against 20 mM Tris, pH 7.9 and 0.5 M NaCl (TN) with 5 mM imadazole for 36 h (1 buffer change). Dialyzed conditioned medium was loaded onto a 2.5 mL bed column at a rate of 1 ml/min. The column was washed with buffer TN with 30 mM imadazole, and HSA1 was eluted from the column with 0.5 M imidazole in buffer TN. Eluted proteins were dialyzed extensively against endotoxin free PBS pH 7.0, tested for endotoxin content using the Kinetic-QCL limulus amebocyte assay (BioWhittaker, Walkersville, Md.), aliquoted, and stored at 70 C. for future use.
Results: The expressed polymers are stable, secreted, and soluble and are readily concentrated to useful levels. The proteins are glycosylated, as documented by the difference in predicted and observed molecular weights. Yields correlate inversely with protein size and fall in the range of 0.8 to 2.0 g/mL of conditioned medium.
To express HSA1R4 in HEK293 cells, the coding region was transferred from the pFastBac vector into the pcDNA3.0 mammalian expression vector (Invitrogen). The pcDNA3.0 vector uses the strong CMV viral promoter to drive gene expression in a wide variety of mammalian cells. The vector also expressed the geneticin/G418 resistance gene permitting the selection of stably expressing cell lines. The HSA1R4 coding regions were liberated from pFastBac vector by digestion with Bam HI and Sal I and the excised DNA fragment was purified from agarose gels using techniques described in Example 2. The fragment was ligated into Bam HI and Xho I digested pcDNA3.0 expression vector. The ligation conditions were identical to those described in Example 1. The HSA1R4-pcDNA3.0 expression construct was transfected into HEK293 cells using lipofectamine (Invitrogen). Two days post-transfection, cells were subjected to selection with culture medium supplemented with 500 ug/mL of geneticin (G418). Cells were passaged for 1 month in G418 selection medium at which time they were seeded into 100 mLs of growth medium in 500 mL Erlenmeyer flasks and grown with gentle shaking (100 rpm) for two weeks. Conditioned medium was harvested after two weeks and HSA1R4 was isolated from the conditioned medium using protein G affinity chromatography as described in Example 4.
Results: HSA1R4 is well expressed in HEK293 cells. The expressed protein has comparable polyacrylamide gel migration as HSA1R4 produced in SF9 insect cells.
For studies in mice, HCH2 polymers were produced that are composed of murine IgG2a sequences. Murine IgG2a is syntenic with human IgG1.
Murine HCH2 fragment subcloning and hinge mutagenesis: Examples 1, 2 and 3 describe the procedure for assembling linear HCH2 polymers from small cDNA fragments containing the HCH2 region. The techniques and reaction conditions used were the same as those described in Examples 1, 2, and 3. This procedure was applied to produce HCH2 polymers from mouse IgG2A HCH2 cDNA. To obtain the template sequence for mouse IgG2a, total RNA was isolated from the murine cell line F50-8A5.5 and the fragment containing the HCH2-CH3 region was subcloned using RT-PCR, the primer, Mu_FRM5p-H3, which introduced a HindIII site immediately 5 of the hinge region and a second primer, Mu_FRM3p-S, which introduced a SalI site immediately 3 of the stop codon (Table 6). The resulting clone Mu_FRM-HS was characterized by DNA sequencing and used as a sequence template for further rounds of PCR. Prior to murine HCH2 polymer construction, PCR mutagenesis was used to change the three cysteines that form inter-chain disulfide bridges between Fc units to serines. As detailed in Example 2, the hinge cysteines were mutated to serines, the HCH2 region amplified and a 5 flanking EcoRI site and 3 flanking NruI and HindIII site were introduced using primers Mu_Hinge-F, Mu_CH2NH3 in a PCR amplification wherein the IgG2A framework clone, Mu_FRM-HS, served as template. The HCH2-ENH region was subcloned and served as template for the production of two additional HCH2 region fragments that differ from HCH2_ENH only by 5- or 3-flanking restriction sites: Fragment HCH2-SNH, which differs from HCH2-ENH only by the presence of a 5 flanking Sma I site was produced using primers Mu_XS_DELH and Mu_CH2NH3 (Table 6) in a PCR amplification wherein HCH2-ENH served as template. Fragment HCH2-SH differs from HCH2-SNH only by the removal of the NruI site from the 3 flanking sequences, leaving Hind III site intact. HCH2-SH was produced using primers Mu_XS_DELH and Mu_CH2H3 in a PCR amplification wherein HCH2-SNH served as template. Three HCH2 region fragments result; HCH2-ENH, HCH2-SNH, and HCH2-SH. All possess mutations that alter hinge region cysteines to serines.
Murine HCH2 polymer construction: Polymers composed of murine HCH2 units were built using the scheme presented in FIG. 1. As a first step, clone HCH2-ENH was opened at the 3 flanking Nru I sites and Hind III sites using restriction endonucleases. Clone HCH2-SNH was digested with Sma I and Hind III and the insert thus liberated was gel purified and ligated into compatible sites in the HCH2-ENH clone. The result of the ligation was the tandem addition of one HCH2 fragment to another. The insertion also regenerated the original sequence of restriction sites (NruIspacer-HindIII) which were used in the next round of extension. Repeating this process of digestion and ligation adds HCH2 units in a stepwise manner. In the last round of polymer construction an insert derived from Sma I and Hind III digestion of HCH2-SH was used resulting in an HCH2 polymer with a flanking 3 Hind III site. The completed murine R4 polymer was digested with EcoRI and HindIII to release the polymer inserts from the cloning vector and ligated into like-digested Mu_FRM-HS resulting in the in-frame joining of the HCH2 polymers to the murine IgG2a framework region. HCH2 polymer and framework region were liberated by digestion with Eco RI and Sal I and ligated into like digested pFactBac expression vector (FIG. 2).
Domain I of murine serum albumin (MSA1): Total RNA was isolated from mouse liver using the method of Chomczynski and Sacchi (1987). First strand cDNA synthesis was primed with 100 mol random hexamers using 200 U SuperScript II reverse transcriptase (Invitrogen, Carlsbad, Calif.) and 5 g of total RNA in a 20 L reaction mixture that was 500 M in dNTPs (Pharmacia, Piscataway, N.J.), 1 U RNasin/L (Promega Corp., Madison, Wis.), 10 M in DTT, and 1 in first strand buffer. Reaction proceeded at 42 C. for 50 min. MSA1 was amplified from murine liver cDNA using PCR, the forward primer MSA_DomI_F (SEQ ID NO: 21) (5 GGCCGCATGGATCCAAAATGAAGTGGGTAACCTTTCTC 3), and the reverse primer MSA_DomI_R (SEQ ID NO: 22) (5 CCGCATGAATTCTCTCTGACGGACAGATGAGACC 3). The resulting cDNA spans the first 221 amino acid residues, including the leader sequence, of mouse serum albumin (i.e., MSA1) and flanking 5 Bam HI and 3 Eco RI sites were introduced. The amplified cDNA was digested with Bam HI and Eco RI and ligated into like-digested pFastBac expression vector into which the mR4 polymer and associated framework region had already been transferred. The resulting expression construct directs expression of an amino-terminal MSA1 fused to a polymer of 4 HCH2 regions in tandem followed by an IgG2a framework region on the carboxyl end (MSA1mR4). MSA1mR4 was expressed in SF9 cells and purified as described in Example 5 (FIG. 4).
To examine the structural integrity and antigenic content, the recombinant proteins were resolved on SDS-PAGE gels and analyzed by Western blot. Proteins were electrophoresed on 7% SDS-PAGE gels (Laemmli et al., 1970) and transferred to nitrocellulose membranes (MSI). Membranes were blocked overnight in 5% non-fat milk in Tris-buffered saline, pH 7.4 (TBS). For analysis of Fc domains, a total of 50 ng of recombinant protein or 0.5 g of control proteins (human IgG and BSA) were loaded onto the gels. The membrane was incubated for two hours with horse radish peroxidase (HRP)-labeled goat anti-human Fc polyclonal antibody (Caltag) used at 1:10000 dilution in a binding buffer consisting of 0.1% non-fat milk and 0.1% normal goat serum in TBS. The blot was washed with TBS-tween and detection performed using the ECL-plus chemoluminescent reagent following manufacturers instructions (Amersham). For direct visualization of proteins, gels were stained with Coomassie brilliant blue.
Results: As shown in FIG. 3A the HCH2 polymers are expressed, stable, and secreted. The observed molecular weight is larger than predicted for the peptide backbone alone, which indicates that the proteins are glycosylated (see Table 7). A Western blot probed with antibodies directed against human Fc revealed binding to the HCH2 polymers in a fashion similar to the IgG control (FIG. 3B).
Insect cells are known to express proteins that can have altered carbohydrate moieties. These alterations may weaken binding of complement factor C1q to these proteins. For this reason the binding of C1q to HCH2 polymers expressed in insect cell line SF9 was investigated. An assay examining the binding of C1q to human IgG or to HCH2 polymers expressed in insect cells was undertaken. Various concentrations of human C1q were allowed to bind to monomeric human IgG, to HSA1Fc, or to HSA1R4 previously immobilized onto wells of a 96 well ELISA plate. The extent of C1q binding was detected using a goat anti-human C1q polyclonal antibody.
C1q Binding Assay. Binding of human C1q to monomeric human IgG, HSA1Fc, and HSA1R4 was determined using modifications of a previously described ELISA protocol (Hinton et al., 2006). Ligands (2-10 ug/ml) were diluted in PBS and coated onto Costar high-binding ELISA assay plates overnight at 4 C. Plates were washed with 0.05% Tween-20 in PBS (PBS-T) and overlain with 4 g/ml of C1q (Calbiochem) prepared in PBS-T with 0.1% gelatin (PTG) for four hours at room temperature. Plates were washed with PBS-T and incubated for 1 hour with goat anti-human Clq (Calbiochem, La Jolla, Calif.) diluted 1:1000 in PTG. Plates were washed with PBS-T and incubated for 1 hour with rabbit anti-goat IgG conjugated to horse radish peroxidase diluted 1:10,000 in PTG. The rabbit anti-goat IgG detecting antibody was preincubated with 2.5 ng/mL of human IgG to eliminate residual cross-reactivity to human Igs. Finally, plates were washed with PBS-T and developed with 0.5 mg/mL o-phenylenediamine (Sigma) peroxidase substrate. Absorbance was measured at 450 nm using a ThermoMax plate reader (Molecular Devices).
The results, shown graphically in FIG. 5, demonstrate that HCH2 polymers isolated from an insect cell expression system engage C1q more weakly than native IgG.
The receptor binding assay measures the binding of HCH2 polymers, Fc fusion proteins or IgG to recombinant ligand-binding domains of FcRI, FcRIIa, FcRIIb, FcRIIIa-158F and FcRIIIa-158V. FcRI has high affinity for the Fc region of IgG and binds avidly to HCH2 polymers, Fc fusion proteins, or monomeric IgG. The low affinity Fc receptors, FcRIIa, FcRIIb, FcRIIIa-158F and FcRIIIa-158V bind the Fc regions of IgG with low affinity.
Recombinant Fc receptor ligand binding domains: PCR was used to amplify ligand binding domains (LBD) and to add the 6His Tag for easy purification. The templates for PCR were the full-length cDNAs IMAGE clones for each receptor that were acquired from OpenBiosystems. The PCR products were digested with Hind III and Eco RI and ligated into like digested expression plasmid vector pcDNA3.1 (Invitrogen). Fc receptor expression vectors were transfected into HEK293 cells using lipofectamine (Invitrogen) mediated transfection. The histidine tagged Fc receptors were purified by immobilized metal affinity chromatography using a Ni2+ immobilized resin (Ni-Sepharose 6 Fast Flow, GE Biosciences). His tagged Fc receptors were extensively dialyzed against endotoxin fee PBS pH 7.0.
Fc Receptor binding assay: The recombinant FcRI, FcRIIa, FcRIIb, FcRIIIa-158F and FcRIIIa-158V receptors were coated onto 96-well ELISA plates at 4 g/mL in DPBS, pH 7.6. FcRI was coated onto plates at 2 g/mL. Receptors were incubated overnight at 4 C. Wells were washed once with DPBS+0.5% Tween-20 (PBST) and blocked by the addition of 200 L of 1% Sanalac (Conagra, Irvine, Calif.) in DPBS to prevent non-specific binding. Blocking proceeded over night at 4 C. Plates were washed 4 with PBST to remove non-adherent receptors and blocking buffer. Human IgG, HSA1Fc, or HSA1R4 were diluted in 1% Sanalac in DPBS at the indicated concentrations and 0.1 mL was added to duplicate wells and incubated for 3 hours to allow receptor binding to occur. The plates were washed 4 with PBST to remove unbound ligands. Ligands were detected by the addition of Protein-G conjugated to horseradish peroxidase (HRPO). Protein-G binds the CH3-CH2 interface on IgG and thus binds to a single site on each ligand. Detection was achieved by incubating with the OPD substrate at 1 mg/mL (o-phenylenediamine, Sigma P-1526) in citrate buffer, pH 5.4. Data was acquired on the ThermoMax plate reader using the dual wavelength endpoint (450 nm-650 nm) method and expressed as OD 450 after correction for blank absorbance.
Results and Discussion: HCH2 polymers bind better to all of the human low-affinity FcRs tested, at all concentrations tested, than native human IgG, or the Fc fusion protein control protein (FIG. 6). It should be noted that the highest concentration tested, 20 ug/mL, represents the amount of circulating immune complexes present in normal human blood. Thus the concentration range used in this study parallels physiologically relevant concentrations of FcR ligands.
HCH2 polymers bind avidly to low affinity Fc receptors. This property distinguishes HCH2 polymers from Fc fusion proteins or IgG that do not have significant binding to the low-affinity Fc receptors. Fc fusion proteins alone or IgG alone do not bind to the low affinity Fc receptors. In order for them to bind they must first be modified by incorporation into an immune complex. HCH2 polymers alone are sufficient to bind low affinity Fc receptors. The ability of HCH2 polymers to bind directly to low affinity Fc receptors distinguishes HCH2 polymers from Fc fusion proteins or IgG.
This example shows that the HCH2 polymers bind to more than one FcR type expressed on the surface of living cells and that they bind to the ligand binding site of the receptors.
Method. Binding of HSA1R4 to FcRs was determined by flow cytometry using the human monocytic cell line, U937. U937 cells constitutively express FcRI and FcRII (Liao et al., 1992). U937 cells (ATCC#: CRL-1593.2, Rockville, Md.) were maintained in RPMI 1640 supplemented with 10% FBS and 2 mM L-Glutamax. Cells were suspended in wash buffer (1% OVA in DPBS) at 1107 cells/ml. To detect binding, 5 g of HSA1R4 was added to a 0.05 ml suspension of cells. The cells were incubated at 4 C. for 20 min, washed, and resuspended in 0.05 ml of wash buffer containing affinity purified anti-HSA FITC conjugated goat IgG (1:100 dilution, Bethyl Labs, Montgomery, Tex.). To show specificity of binding, U937 cells were preincubated at 4 C. for 20 min with 5 g of monoclonal antibodies to FcRI (CD64; clone 10.1; BD Biosciences) and to FcRII (CD32; clone FLI8.26; BD Biosciences) to block the ability of HSA1R4 to bind to FcRs. Cells were analyzed using a FACScan II (BD Biosciences, San Jose, Calif.).
Results and Discussion. Increased fluorescence is uniformly observed when U937 cells are incubated with HSA1R4 followed by FITC conjugated goat anti-HSA polyclonal IgG to detect surface bound HSA1R4 (FIG. 7A). To detect binding to specific FcRs, U937 cells were pre-incubated with blocking monoclonal antibodies (mAbs) to FcRI, to FcRII, or to both. Decreased fluorescence is observed following pre-incubation with either mAb while pre-incubation with both reduces fluorescence to background levels (FIG. 7A, 7B, 7C). Thus, HSA1R4 appears to bind exclusively to FcRI and FcRII receptors on U937 cells. These data show that the HCH2 polymers bind to both FcRI and FcRII on the surface of U937 cells. In addition, the data indicate that the HCH2 polymers bind to the ligand binding site on both FcRI and FcRII.
To assess potential HCH2 polymerFcRIII interactions, the HCH2-polymers, HSA1R2, HSA1R3, and HSA1R4 were assayed for their ability to activate NK cells within PBMC isolates and compared to responses achieved using the Ig-fusion protein, HSA1Fc. NK cells express both the low affinity IL-2 receptor, and FcRIII (CD16) (Nagler et al., 1990). When primed with high levels of IL-2 (1 ng/mL), NK cells mount a proliferative response to CD16 ligation. This triggered response was used as a test of the fitness of the recombinant molecules to engage FcR.
PBMC Purification and Proliferative Assays.
Peripheral blood mononuclear cells (PBMC) from four healthy donors were isolated from heparinized blood on a Ficoll-Paque gradient (Pharmacia Biotech Inc) and suspended in AIM V defined serum free medium (Gibco BRL). Recombinant protein stocks were initially prepared in RPMI 1640 (concentration 1 mg/ml). Recombinant protein stocks were diluted in AIM V medium (Fisher Scientific) to achieve the desired final concentrations as indicated in the drawings. PBMC were plated at a final concentration of 2106 cells/ml in 96 well flat bottom plates (0.200 ml/well final volume). Cells were incubated for 72 hours in a humidified incubator at 37 C. in 5% atmospheric CO2. During the last 5 hours of culture, wells were pulsed with 1 Ci of [methyl-3H] thymidine (Amersham Corp). Cells were harvested using a PhD cell harvester (Cambridge Technologies). Radioactivity was determined using a Beckman Scintillation Counter LS 5000TD (Beckman Instruments).
Results. The HCH2 polymer constructs, expressing domain one of human serum albumin were tested for their ability to induce proliferative responses in PBMC. Use of these ligands allows us to determine the impact of decreasing HCH2 repeat number on FcRIII triggered cell activation. PBMC were incubated with decreasing concentrations of HSA1R4, HSA1R3, and HSA1R2, in the presence of IL-2 and proliferative responses were measured as above. PBMC were also incubated with decreasing doses of HSA1Fc plus IL-2 to allow comparison with the HCH2 polymers. As shown in FIG. 8, PBMC proliferative responses triggered by the HCH2 polymers correlated with the number of HCH2 repeats present within the ligand. As the number of HCH2 units increased within each polymer so did its ability to induce proliferation by PBMC. HSA1R4 was the most effective ligand for inducing proliferative responses of PBMC; greater proliferative responses were observed in PBMC in response to all doses of HSA1R4 tested than in response to HSA1R3, HSA1R2 or HSA1Fc (FIG. 8).
The purpose of this example is to demonstrate the feasibility of using R4 as an antigen delivery vehicle to increase antigen specific antibody responses. In this example we have used domain one of human serum albumin (HSA1) as an antigen fused to the amino terminus of R4 to generate the polypeptide we term, HSA1R4. In Example 4 we showed how the construct, HSA1R4, was produced. In Example 5 we showed how HSA1R4 could be expressed and purified. We have used the HSA1R4 polypeptide to immunize mice and compared the responses achieved to those obtained using the antigen alone, HSA1.
Though we have used HSA1 in this example there are numerous other antigenic epitopes that could be expressed at the amino terminus of R4 to generate a hybrid molecule. The polypeptide containing the antigenic epitope(s) could be linked to the amino terminus of R4, as is shown in this example where HSA1 is used as an antigen, or alternatively the antigenic epitopes could be linked to the carboxyl end of R4. Numerous antigens of choice may be used as detailed in this patent application. In some instances, the polypeptide sequence would be less than 500 amino acids long and soluble in aqueous solutions.
Mice. SJL/J mice and C57BL6 mice, 5 to 6 wk old, from Jackson Laboratories (Bar Harbor, Me.) or from Taconic (Germantown, N.Y.), were maintained in a Barrier facility and acclimated for one to two wks before study. Animal care and experiments were performed according to NIH guidelines, as approved by the animal use committee of the University of Chicago.
Immunization. HSA1, HSA1Fc, or HSA1R4, dissolved in 0.15 ml saline, was injected into a tail vein. For s.c. injections, HSA1, HSA1Fc, HSA1R4, or ovalbumin (OVA; Sigma Corp., St. Louis, Mo.) were suspended in Ribi adjuvant (Sigma Corp.) according to manufacturer's instructions. Ribi adjuvant contains MPL and synthetic TDM incorporated into a mix of squalene and Tween-80, and serves as an immunostimulant with little toxicity. Proteins were dissolved in 2 ml of saline at 0.25 mg/ml, transferred into vials containing 0.5 mg of MPL and 0.5 mg of TDM and vortexed for 4 min to create an oil-in-water emulsion. Mice were immunized subcutaneously at two sites, one on each flank. A total volume of 0.1 ml containing from 0.125 g to 25 g of protein was injected at each site. Mice were bled retro-orbitally.
ELISA. ELISA plates (Corning Inc., Corning, N.Y.) were overlain with 0.1 ml/well of carbonate buffer (0.1M, pH 8.4) containing 5 g of HSA or OVA and incubated at room temperature for 5 hours. Wells were treated by the addition of 0.1 ml/well of 0.25% Sanalac in DPBS to prevent non-specific binding (Conagra, Irvine, Calif.). After 2 hours at room temperature, wells were washed with 0.5% Tween-20 in DPBS (wash buffer) and 1:200, 1:250, 1:500, 1:2, 500, 1:12, 500, 1:62, 500, 1:125,000, 1:250,000, 1:500,000, 1:1,000,000, and 1:2,000,000 dilutions of serum samples in 0.25% Sanalac were added to duplicate wells. Sera from nave mice were diluted 1:200 and added to duplicate wells to provide background O.D. values. Plates were left overnight at 4 C., then washed, and overlain with a cocktail of biotinylated rat mAbs (each at 0.5 g/ml) specific for murine Ig (mAbs clones: anti-IgG1 A85-1; anti-IgG2b R12-3; anti-IgG3 R40-82; all from Invitrogen: and anti-IgG2c5.7 from BD Biosciences). IgG2, was measured since SJL and C57BL6 mice express IgG2c rather than IgG2a (Martin et al., 1998). To quantitate levels of HSA1-reactive IgG1 or IgG2c, wells were overlain with biotinylated Abs specific for those isotypes. Following incubation with biotinylated Abs, wells were washed, and overlain with 0.1 ml of affinity purified peroxidase-conjugated goat anti-biotin Ab (1:500 dilution:Zymed, South San Francisco, Calif.) for 45 min. Wells were washed, and 0.2 ml of ortho-phenylenediamine (1 mg/ml) and H2O2 (1 l/ml) in citrate buffer (0.1 M, pH 4.5) was added to each well. Absorbance was measured 15 min later using a ThermoMax Microplate Reader (Molecular Devices Corp., Sunnyvale, Calif.). Serum dilutions were considered positive when their O.D. values exceeded twice the mean O.D. values obtained from wells containing non-immune sera. As a control, absorbance values were measured from wells not coated with HSA or OVA but overlain with immune sera. Absorbance values of control wells always approximated those found in blanks.
Statistics. Ab titers were compared using Student's unpaired T test.
Results and Discussion
In this example we tested HSA1R4 in the high responder mouse strain SJL and in the low responder mouse strain C57BL6. SJL mice respond to vaccination with high antibody titers to antigen while C57BL6 mice respond to vaccination with lower antibody titers. SJL mice were injected intravenously with 50 g of HSA1, HSA1Fc, or HSA1R4, and serum was obtained 14 days later, and Ab responses were assayed by ELISA. Anti-HSA Ab titers were not detected in mice given HSA1 alone at the minimum 1:200 serum dilution used as a cutoff (FIG. 9). Mice given HSA1R4 or HSA1Fc developed substantial Ab responses to HSA (FIG. 9). HSA specific titers were fourfold higher in mice injected with HSA1R4 than in mice injected with HSA1Fc (p0.05). Equal mass weights of the proteins were injected so that the amount of HSA1 in HSA1Fc was 2.5 times that in HSA1R4 (FIG. 9). Anti-HSA IgG1 and IgG2, were increased in response to both immunizations indicating activation of both Th1 and Th2 type T cells (FIG. 10). These data show that SJL mice injected with HSA1 covalently linked to R4 generate greater antibody responses to HSA1 than in mice injected with HSA1 alone or to HSA1Fc. When Ag/Ab complexes are injected intravenously in mice, greater Ab responses are observed than with Ag alone (Wernersson et al., 1999; Wernersson et al., 2000; Getahun et al., 2004).
Vaccines are typically injected subcutaneously. Accordingly, the efficacy of HSA1R4 as an Ag delivery agent was assessed. HSA1R4 was emulsified in Ribi adjuvant. Ribi adjuvant contains monophosphoryl lipid A (MPL) which signals through Toll-like receptor 4 (TLR4) to activate APC maturation and to increase co-stimulatory molecule expression (Ismaili et al., 2002; Martin et al., 2003). Mice were immunized with HSA1 alone, HSA1Fc, or HSA1R4 (50 g/mouse), and anti-HSA Ab titers determined in sera obtained 14 days later. In mice immunized with HSA1 alone, anti-HSA Ab titers were only detectable in sera from 2 of 5 mice at the 1:200 cutoff threshold employed (FIG. 10A, 10B). Ab titers of mice given HSA1R4 averaged 1100 times those of mice given HSA1 (p0.001), and were seven-fold those of mice given HSA1Fc (p=0.01). Isotype analysis revealed that both IgG1 and IgG2, Ab titers rose following immunization with HSA1R4.
A 200 fold lower dosage of immunogens (250 ng) was next tested, again in Ribi adjuvant, with Ab measured in sera obtained 14 days post-immunization. Mice given HSA1R4 developed 130 times as much HSA1 specific Ab as mice immunized with HSA1Fc (p0.001; FIG. 10B).
Since SJL mice can produce abnormally high levels of IgG (Jiang et al., 2000), we next determined responses in C57BL6 mice that have lower antibody responses to vaccination. C57BL6 mice were immunized with HSA1R4, HSA1Fc, and HSA1 all in Ribi adjuvant. Sera were collected 14 days later and assayed for HSA-specific Ab titers. C57BL6 mice developed Ab titers that were substantially lower than those observed in SJL mice. Nonetheless, anti-HSA Ab titers in C57BL6 mice receiving HSA1R4 were 10 fold those receiving HSA1Fc (p0.05) and 50 fold higher than those receiving HSA1 (p0.005) (FIG. 11).
This example shows that in mice immunized with an antigen linked to R4, the primary T cell response to that antigen is augmented. In this example the antigen is HSA1, and the mice are immunized with HSA1R4 in Ribi adjuvant and also with HSA1Fc in Ribi adjuvant as a comparator.
Mice. SJL/J mice were purchased and maintained as in Example 13.
Immunization. Mice were immunized with HSA1Fc and HSA1R4 as in Example 14. To generate HSA1-reactive T cells for in vitro use, mice were injected with 0.1 ml of an emulsion consisting of 0.05 ml saline containing 100 g of HSA (Sigma) and 0.05 ml of CFA distributed intradermally with 0.025 ml given in each flank and over each scapula.
Proliferative Responses. LNs and spleens were harvested from mice immunized 14 days earlier with: 1) HSA in CFA or with; 2) HSA1R4 or HSA1Fc in Ribi adjuvant. LN and spleen fragments were placed in saline, and disrupted mechanically using a tissue homogenizer to obtain a single cell suspension. RBCs were removed by centrifugation on ficoll-hypaque gradients. Buffy layers were harvested from the gradients, cells were washed with HBSS, and resuspended in HL-1 Ventrex medium (Fisher Scientific, Pittsburgh, Pa.) supplemented with 2 mM L-Glutamax, 50 m 2-mercaptoethanol, 1MEM amino acids, and 10 g/ml gentamicin (Invitrogen). Cells were plated in 96 well flat bottom plates at 6105 splenocytes/well or 4105 LN-derived cells/well and HSA1Fc or HSA1R4 added as indicated in the results section. As a source of APCs, splenocytes from native mice, processed as described above, were incubated at 37 C. in RPMI containing 75 g/ml of mitomycin C (Sigma) for 20 min, washed five times in saline, and added (3105 cells/well) to wells containing LN-derived cells. Cells were incubated at 37 C. for 72 h and pulsed for an additional 8 h with 1 Ci/well of 3H-Thymidine. Cells were harvested using a Cambridge Phd cell harvester and radioactivity determined by liquid scintillography.
Statistics. Proliferative responses of HSA-reactive LN cells, were compared using Student's unpaired T test. Proliferative responses of splenocytes were compared using Chi-square analysis of values above or below a SI of 3.
Results and Discussion. These data show that HSA1R4 is a potent Ag delivery vehicle for induction of T cell responses. Mice were immunized with 50 ug of HSA1R4 or HSA1Fc in Ribi adjuvant. HSA1-specific splenic T cell proliferative responses were measured 14 days later. Taking a stimulation index (SI) of 3 as indicative of response, T cells from mice given HSA1R4 responded to a 20 fold lower concentration of HSA1 than T cells from mice given HSA1Fc (FIG. 12A). T cells from mice given HSA1R4 responded better to all 6 concentrations of HSA1 tested than T cells from mice given HSA1Fc (p0.004) (FIG. 12A). The potency of HSA1R4 may be understated in our assay as mice given HSA1Fc received 2.5 times as much HSA1 as those given HSA1R4.
HSA1R4 presents Ag to T cells more efficiently than HSA1Fc (FIG. 12B). Targeting of Ag to FcRs increases Ag uptake by APCs, Ag processing by them, and Ag presentation to T cells (Zaghouani et al., 1993; Brumeanu et al., 1993). Accordingly, studies were conducted to determine whether HSA1R4 could increase Ag presentation to T cells. Cells isolated from draining LNs of mice immunized with HSA in CFA 14 days earlier were used as a source of HSA-reactive T cells. Mitomycin C treated splenocytes from nave mice served as a source of APCs. HSA-reactive T cells respond more briskly to HSA1R4 than to HSA1Fc (p0.008) or to HSA1 (p0.001) at molar equivalents (1.6109 M) (FIG. 12B).
These results demonstrate that robust Ab responses to HSA1, a weakly antigenic peptide, can be obtained by coupling it to the HCH2 polymer R4. HSA1R4 has 10 potential FcR binding regions and 2 copies of HSA1. ICs in Ab excess, known facilitators of Ab responses to weak Ags, bind to FcRs expressed by APCs (Marusic-Galesci et al., 1991; Marusic-Galesci et al., 1992). Binding of ICs to FcRs triggers IC internalization so that more Ag enters the APC than when Ag alone is given. Augmented Ag processing, and increased presentation of processed Ag to T cells, ensue. In HSA1R4, these properties of ICs have been integrated into a single defined molecule. Additionally, placement of Ag at the amino terminus of the HSA1R4 molecule renders the Ag fully accessible to processing enzymes. Thus, any antigenic peptide, of any size, or more than one when the goal is to develop a polyvalent vaccine, can be linked to R4 for Ag delivery.
BoNT/A activities map to discrete regions within the polypeptide chains: Endoprotease activity resides within the light chain. The heavy chain is responsible for receptor binding and translocation. The heavy chain can be further subdivided both functionally and proteolytically into an amino-terminal fragment (HN), involved in ion-channel formation and light chain translocation, and a carboxyl-terminal fragment (Hc) involved in receptor binding The Hc fragment is composed of two 200 amino acid sub-domains that are structurally distinct. The amino-terminal portion, HcN (residues 871 to 1078 of the holotoxin) forms a lectin-like sub-domain. The carboxyl-terminal portion, HcC (residues 1090 to 1296 of the holotoxin) adopts a -trefoil structure. The respective roles of HcN and HcC in receptor recognition and binding to neurons are not fully understood (FIG. 13).
Expression of Hc antigens in heterologous systems has previously proven problematic due to the codon bias in the C. Botulinum gene. To circumvent this limitation a synthetic gene approach was pursued. Designing the gene segments de novo permitted the introduction of restriction sites to facilitate the subcloning of the Hc fragments into expression vectors. The strategy was to synthesize the HcN and HcC gene segments separately and to combine segments to produce the Hc gene segment. The C. Botulinum Hc codon usage was optimized for expression in the spodoptera frugiperda (SF) cell lines using the UPGENE codon optimization algorithm (http://www.vectorcore.pitt.edu/upgene/upgene.html). The codon usage data set was derived from highly expressed genes in SF cells (http://www.kazusa.or.jp/codon/). The optimization scheme resulted in the resolution of two types of codon bias: First was to reduce or eliminate the use of rare codons, as seen for example in the almost exclusive use of the TTA codon (LEU) in C. Botulinum. The second was the balanced use of codons when multiple codons were available, as exemplified by the AAT codon (ASN).
The HcN and HcC gene segments were either subcloned individually or combined at internal restriction sites to produce the Hc gene. The three gene segments (SEQ ID NO: 22 for Hc; SEQ ID NO: 24 for HcN; SEQ ID NO: 26 for HcC) were subcloned into the pFastBac expression vector. The leader sequence from human IgG1 was cloned upstream and in-frame to the synthetic genes to direct their secretion from the cell into the medium. The HcN, HcC, and Hc genes were followed in frame either by a short sequence coding for a 6 histidine tag or by the R4 ligand coding sequences. The R4 ligands are based on human IgG1 sequences as described in Example 3. We have also developed identical ligands, mR4, based on murine IgG2a sequences (Example 7) for use in mice. The murine Ig2a sequences are syntenic to human IgG1. Accordingly we subcloned the Hc gene segments in-frame with the mR4 sequences.
Recombinant virus was derived and used to infect SF9 cells as we have described in Example 5 and Jensen et al., 2007). Conditioned media containing the 6His tagged recombinant antigens, HcNHis, HcCHis, and HcHis control antigens were purified in a single step by passage of the conditioned medium over a Nickel affinity column. The Hc antigens fused to the R4 ligand, HcNR4, HcCR4, and HcR4, were purified in a single step from conditioned medium by affinity chromatography using protein-G Sepharose columns. Table 8 lists the number of amino acids in each of the recombinant Hc antigens as well as their apparent molecular weights as estimated from SDS-PAGE. Sizes and weights for HcNR4, HcCR4, and HcR4 reflect the dimerization that is a feature of the R4 ligands. Also listed in Table 8 are the antigen percentages in each of the HcR4 ligands. Antigen percentage is used to calculate the antigen load for vaccination, for example an immunization with 5 ug of HcR4 represents a load of 1.65 ug of Hc antigen.
In this study we determined if HcR4 ligand can target Hc antigens to FcR. We used the receptor binding assay introduced in Example 10. HcR4 was incubated with the immobilized receptors, plates were washed and residual ligand binding was determined. Hc antigen served as a control in these studies. Results: HcR4 binds exceptionally well to both low-affinity and high-affinity FcRs (FIG. 14). Hc antigens alone fail to engage the Fc receptors. Antigens delivered as HCH2 polymers engage Fc receptors directly and do not need to be incorporated into immune complexes.
HCH2 polymers that deliver Hc region from Botulinum neurotoxin subtype A worked as well as HCH2 polymers that delivered domain I of human serum albumin. Thus it is the HCH2 polymer that confers enhanced binding to the low affinity Fc receptors. HCH2 polymers also avidly bind FcRI, the high affinity receptor for the Fc region of IgG
The goal of this study was to determine if the Hc, HcR4, and HcmR4 antigens could direct antigen-specific responses. The SJL strain has been identified as a high responder to Hc immunizations whereas the C57BL/6 strain is a poor/non-responder to Hc antigens. Thus we examined responses in the SJL mice to single dose SC immunizations with Hc and HcR4 at the 0.5 g and 1 g doses. The immunization protocol was identical to that described in Example 14 above; antigens were administered as an emulsion in Ribi adjuvant. Mice were immunized in their flanks and bled 14 days later. To detect antigen specific responses we developed an ELISA employing recombinant Hc as capture antigen. Hc-based ELISA has been validated as being predictive for the presence of neutralizing antibodies and correlates with an in vivo lethal challenge toxin model. Rabbit anti-sera produced against native BoNT/A binds avidly to the immobilized recombinant Hc and serves as a positive control.
Results for SJL: Immunization with 0.5 ug HcmR4 produced robust antigen-specific responses by day 14. In contrast, immunization with Hc alone resulted in poor antigen-specific responses at 14 days. To further characterize the antibody responses, antigen specific antibody titers were determined using the ELISA described above. Immunization with a single 0.5 g of HcR4, or its murine analog, HcmR4 results in large Hc-specific antibody titers whereas immunization with a similar dose of Hc results in poor antibody titers (FIG. 15A.). Similar trends are observed at the 1 ug dose (FIG. 15B.).
Results for C57BL/6: The C57BL/6 strain is a poor/non-responder to Hc antigens. To determine the responses of the C57BL/6 strain to Hc and HcR4, mice received single dose SC immunizations of either 5 g or 10 g of HcR4 and Hc. Responses were determined 14 days later. C57BL/6 mice responded poorly to immunization with 5 g of Hc with only a single mouse of ten immunized having shown responses whereas 6 of 10 mice immunized with 5 g HcR4 had meaningful responses (FIG. 16). The superior results with HcR4 were achieved using 5 g of HcR4 that delivers a dose of 1.67 g of Hc antigen (See Table 8). A similar trend was observed when the immunization dose is increased to 10 ug.
Conclusions: Immunization with either HcR4 or HcmR4 results in larger anti-Hc antibody titers than can be achieved by immunization with Hc alone. The data indicate that the murine ligand, HcmR4, induces higher Hc-specific antibody responses in mice than its human R4 counterpart. These results might be expected as the murine ligand is likely better at engaging murine Fc receptors than the human ligand. Nevertheless, the results validate the use of the human ligand, HcR4, as an immunogen to achieve large and rapid responses to Hc in mice. It is also likely that the human HcR4 will work even better in humans than seen here with mice, just as the murine ligand, HcmR4, results in higher antibody titers in mice. The use of equal mass dosing in these experiments actually understates the efficacy of HcR4 and HcmR4 in comparison to Hc as 1 g of HcR4 (or HcmR4) delivers an Hc antigen load of 0.33 g (Table 8). Taken together these results indicate that Hc delivered as a R4 ligand directs better antigen-specific responses especially at lower antigen doses.
In this study we establish that BoNT/A antigens delivered using polypeptides that include HCH2 polymers are better at inducing antigen-specific T cell responses than antigen alone.
Methods: To establish that use of HcR4 and HcmR4 leads to more efficient presentation of Hc antigens on APCs, we used the in vitro T cell assay introduced in Example 15. Mice were immunized with recombinant Hc and 14 days later T cells were isolated from draining lymph nodes as a source of Hc-reactive T cells. APCs were loaded with equal concentrations of Hc, HcR4 and HcmR4 ligands in the presence of Hc-reactive T cells to serve as a read out for the assay.
Results: Hc-reactive T cells respond more strongly to Hc antigens when APCs are primed with 3.6108 M of HcR4 than when APCs are primed with 3.6108 M of Hc alone (FIG. 17A). Similar trends are observed at low dose (1.2108 M) (FIG. 17B). The data in FIG. 17B support a trend seen in antibody titer data indicating that HcmR4 performs better in mice than HcR4, due to a more favorable interaction between the murine ligand and murine Fc receptors.
Mucosal administration of HcR4 ligands results in large and rapid Botulinum neurotoxin subtype A (BoNT/A) Hc-specific antibody responses: the second route for delivery of antigens using HCH2 polymers for the induction of immune responses to delivered antigens.
In this study we sought to determine if HCH2 polymers can deliver antigen when administered mucosally. Induction of mucosal immune responses has the advantage of inducing both serum IgG and IgA as well as increased protection at the mucosal surfaces due primarily to locally expressed antigen-specific IgA. Mucosal immunity might prove to be critical in those circumstances where there is a potential for exposure to aerosolized BoNT/A, such as in a biothreat scenario. Mucosal vaccination has the additional advantage of needle-free administration. The R4 ligands may target APCs in the mucosal epithelium by several routes; FcR-bearing DCs can directly sample mucosal ICs through mucosal epithelial barriers or in collaboration with M cells within the nasal-associated lymphoid tissues. A second mechanism may involve the transport of the R4 polymer across the mucosal epithelia by FcRn. The FcRn binding site encompasses parts of both the CH2 and CH3 domains of IgG1. These sequences are present in the Fc region at the carboxyl end of the R4 polypeptide but absent from the HCH2 polymer. Antigen transcytosed by vesicular transport or FcRn could then be captured by macrophages and DCs and transported to draining lymph nodes. Hc itself binds to epithelial cells and is transcytosed by them. The Hc contained in HcR4 could contribute to transcytosis. Mucosal administration of Hc results in systemic IgG and IgA titers as well as induction of antigen specific mucosal IgA responses. As is the case for Hc administered in adjuvant SC, immune responses to mucosally administered Hc are modest.
Methods: To determine if linking Hc to the R4 ligand can improve mucosal immune response, HcR4 was instilled nasally into SJL mice at a dose of 25 g/nostril on days 0, 7, and 14. Serum was obtained on days 21 and 28 and assessed for Hc specific IgG titers.
Results: Intranasal (IN) administration of HcR4 resulted in large and rapid induction of systemic antigen specific antibody titers (FIG. 18). The magnitude of the response is 2030 fold larger, achieved with fewer IN immunizations and two weeks earlier than in a published reports employing Hc alone. The rapidity and magnitude of the response suggest a synergism between the FcR targeting capacity of the R4 ligand and the intrinsic binding capacity of the Hc domain.
Fatty acid binding proteins (FABPs) are a family of small generally cytosolic proteins with high affinity for long chain fatty acids and their CoA derivatives. Fatty acid binding proteins are involved in the uptake and transport of fatty acids and as such impact fatty acid metabolism and lipid biosynthesis. FABPs are also involved in the modulation of other cellular functions including gene expression, differentiation and signal transduction.
The brain form of fatty acid binding protein, FABP7, is mainly expressed early in the development of the CNS but sparsely in the adult brain. FABP7 is expressed in a subset of adult glial tumors or gliomas, FABP7 expression enhances glioma cell migration and may therefore contribute to tumor spreading. In addition to cancers of the brain, FABP7 is frequently over expressed in melanoma where it also contributes to extracellular matrix invasion. FABP7 has characteristics favorable for targeted immunotherapy: it is expressed in the cancer cells but not in normal adult tissue and its expression in the cancer cells contributes to the malignant properties of the cancer.
As a first step in the evaluation of an FABP7 therapeutic vaccine, FABP7 was expressed as a fusion to the R4 HCH2 polymer. We have expressed murine FABP7 fused to the murine IgG2a R4, mR4, for use in mouse cancer models. We have expressed human FABP7 fused to human IgG1 based R4 for evaluation in humans.
Murine FABP7: The full-length cDNA for murine FABP7 is present in IMAGE clone 5700428, Genbank Accession # BC057090, and was used as template for PCR reactions. The coding sequences were amplified from the full-length cDNA using PCR and the oligonucleotides primers Mu_FABP7-F1,
The primers direct amplification of the coding region of murine FABP7 absent the initiation ATG and stop codon and introduce a 5 flanking Xho I site and a 3 flanking Eco RI site. The PCR products were digested with Eco RI and Xho I and ligated into like digested mR4 pFastBac expression vector.
Human FABP7: The full-length cDNA for human FABP7 is present in IMAGE clone IMAGE:4707233, Genbank Accession # BC012299, and was used as template for PCR reactions. The coding sequences were amplified from the full-length cDNA using PCR and the oligonucleotides primers Hu_FABP7-F1,
The primers direct amplification of the coding region of human FABP7 absent the initiation ATG or the stop codon and introduce a 5 flanking Xho I site and a 3 flanking Eco RI site. The PCR products were digested with Eco RI and Xho I and ligated into like digested human IgG1 R4 pFastBac expression vector.
The recombinant FABP7-R4 fusion proteins were directed into the secretory pathway by proceeding the FABP7 coding region with the leader sequence from human IgG1 (MEFGLSWVFLVAILKGVQC) (SEQ ID NO: 45). When the IgG1 leader sequence precedes the FABP7-R4 coding region, it directs secretion of the expressed proteins from the cell into the medium. The recombinant proteins are purified from the conditioned medium.
Proteolipid protein (PLP) and myelin basic protein (MBP) are components of the myelin sheath that surrounds the axons of nerve cells. PLP and MBP are targets for autoimmune reactions in multiple sclerosis in humans and in experimental autoimmune encephalomyelitis (EAE), the mouse model of the disease. Specific peptide antigens have been identified within PLP and MBP that are encephalitogenic T cell epitopes capable of inducing EAE in mice.
The PLP and MBP peptides were expressed as fusions to the human IgG1 R4 HCH2 polymers either as single peptides or in tandem. The PLP peptide, HSLGKWLGHPDKF (SEQ ID NO: 49), spans 13 amino acids. The cysteine present in the wild-type sequence was changed to serine to prevent unwanted disulphide bond formation. Complementary oligonucleotides coding for the peptide and that introduce a flanking 5 Xho I half-site and a flanking 3 EcoR I half-site were prepared. Most restriction enzymes, (e.g., Eco RI and Xho I) result in recessed 3 ends (5 overhangs) but blunt end restriction sites result in evenly matched ends (e.g., Sma I) and some restriction enzymes result in recessed 5 ends (e.g., Sac I and Kpn I). By designing complementary oligonucleotides with appropriate 5 or three 3 overhangs, the hybridized double-stranded oligonucleotides can be ligated directly into restriction digested expression vectors. Accordingly, the complementary oligonucleotides coding for the peptide were treated with poly-nucleotide kinase (New England Biolabs) to phosphorylate the oligonucleotides. Oligonucleotides were purified and hybridized to form double stranded DNA from the complementary oligonucleotides. The phosphorylated, hybridized oligonucleotides were ligated directly into the R4 pFastBac expression vector that had been previously prepared by digestion with Eco RI and Xho I. The MBP peptide, VHFFKNIVTPRTP (SEQ ID NO: 40), spans 13 amino acids and was introduced into the R4 pFastBac expression vector using a strategy identical to that pursued for the PLP peptide.
PLP-PLP peptides expressed in tandem: two copies of the PLP peptide were expressed separated by a 2 amino acid linker (HSLGKWLGHPDKFGTHSLGKWLGHPDKF) (SEQ ID NO: 43). To express two PLP peptides in tandem, complementary oligonucleotides were synthesized as described above that code for a single peptide but also introduce a Kpn I site proximal to the Eco RI half-site. Kpn I was chosen as it codes for gly-thr (GT) when expressed in frame. The oligonucleotides were phosphorylated, hybridized and ligated into the R4 pFastBac expression vector. The result was the introduction of as single PLP peptide with an in frame 3 unique Kpn I site between the peptide sequences and the Eco RI site. The resultant expression construct was termed PLPR4-KE to denote the addition of the internal Kpn I site in frame with the Eco RI site. To introduce the second peptide sequence, complementary oligonucleotides were once again synthesized that coded for the peptide but that introduce a flanking 5 Kpn I half-site and a flanking 3 EcoR I half-site. These oligonucleotides were phosphorylated, hybridized to make them into double-stranded DNA and ligated into the PLPR4-KE construct that had been digested with Kpn I and Eco RI. This resulted in the PLP-PLPR4 pFastBac expression vector.
Combined PLP-MBP peptides expressed in tandem: A similar strategy was pursued to make the PLP-MBP tandem peptides (HSLGKWLGHPDKFGTVHFFKNIVTPRTP) (SEQ ID NO: 50). Complementary oligonucleotides were synthesized that coded for the MBP peptide but that introduce a flanking 5 Kpn I half-site and a flanking 3 EcoR I half-site. These oligonucleotides were phosphorylated, hybridized to make them into double-stranded DNA and ligated into the PLPR4-KE construct that had been digested with Kpn I and Eco RI. This resulted in the production of PLP-MBPR4 pFastBac expression construct.
The recombinant PLPR4, MBPR4, PLP-PLPR4 and PLP-MBPR4 fusion proteins were directed into the secretory pathway by preceding the peptide coding regions with the leader sequence from human IgG1 (MEFGLSWVFLVAILKGVQC) (SEQ ID NO: 45). When the IgG1 leader sequence precedes the coding regions, it directs secretion of the expressed proteins from the cell into the medium. The recombinant proteins are purified from the conditioned medium.
The following may be relevant to the instant application.