Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/521—Chemokines
  • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/385—Haptens or antigens, bound to carriers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55522—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention relates to compositions and methods relating to enhancing or modulating immune responses, such as those elicited by vaccination.
• the compositions and methods are useful for, among other things, vaccine formulation for therapeutic and prophylactic vaccination (immunization) and for production of useful antibodies (e.g., monoclonal antibodies, for therapeutic or diagnostic use).
• HIV human immunodeficiency virus
• Vaccination exploits the immune system, which comprises leukocytes (white blood cells (WBCs): T and B lymphocytes, monocytes, eosinophils, basophils, and neutrophils), lymphoid tissues and lymphoid vessels.
• WBCs white blood cells
• B and T lymphocytes circulate throughout the body, interact with antigen-presenting cells and detect pathogens. Once an invader is detected, cytotoxic T cells or antibody-secreting B cells specific for the foreign agent are recruited to the infection site to destroy it.
• the concept of vaccination is to generate the same types of host-protective immune responses without exposing the individual to the pathology-inducing foreign agent (such as a pathogen or tumor).
• Such immune responses may be, for example, cell-mediated and/or antibody based.
• APCs antigen presenting cells
• macrophages such as macrophages, activated B cells and dendritic cells.
• dendritic cells are especially important in the immune response. Immature or resting dendritic cells reside in epithelial layers, phagocytosing foreign material (called antigens). These dendritic cells become activated by tumor necrosis factor (TNF) secreted by nearby macrophages that have been stimulated by the foreign material. These activated dendritic cells, laden with foreign antigens, travel through the lymphatic system to the nearest lymph node. There, resting na ⁇ ve (unexposed to antigen) T cells whose antigen-specific receptors recognize the foreign antigen are activated, and the immune system is triggered into action.
• TNF tumor necrosis factor
• the safest vaccinations are those that provoke an immune response to a subset of isolated antigens or epitopes, expressed by the foreign agent.
• many such antigens are by themselves are weakly immunogenic or incompetent for instigating a strong immune response.
• adjuvants are often added to vaccine compositions. Examples of adjuvants include oil emulsions of dead mycobacteria (Freund's complete), other dead bacteria (e.g., B. pertussis ), bacterial polysaccharides, bacterial heat-shock proteins or bacterial DNA. While effective, many of these adjuvants cause significant inflammation and are not suitable for human administration.
• the invention provides methods for eliciting an immune response to an antigen in a subject, such as in a human, wherein a polypeptide with at least a part of a sequence of SEQ ID NOS:1-6 or 13 (“SHAAGtides”) is administered with an antigen to a subject.
• a polypeptide with at least a part of a sequence of SEQ ID NOS:1-6 or 13 (“SHAAGtides”) is administered with an antigen to a subject.
• SHAAGtides polypeptide with at least a part of a sequence of SEQ ID NOS:1-6 or 13
• the immune response may be antibody mediated, and upon administration, the titer of antigen-specific antibodies increases at least two-fold.
• the immune response is cell-mediated and the polypeptide having at least a portion of SEQ ID NOS:1-6 or 13 attracts and/or activates various leukocytes, including dendritic cells.
• the invention provides methods of eliciting an immune response by co-administering a polypeptide having at least a portion of the sequence SEQ ID NOS:1-6 or 13 with an antigen; in other aspects, the antigen and polypeptide may be administered separately. In yet other aspects, more than one polypeptide of SEQ ID NOS:1-6 or 13 may be administered, either separately or as concatamers or fusion proteins. In all cases, variants of SEQ ID NOS:1-6 or 13 may be used.
• polypeptides having at least a portion of SEQ ID NOS:1-6 or 13 may be administered as polynucleotides (SEQ ID NOS:7-12 or 14) operably-linked such that they are expressed by the subject upon or after administration.
• antigens may also be administered as polynucleotides that are expressed after administration.
• the administered antigen is a polypeptide from a pathogen, such as Hepatitis, Influenza, tumor antigens or allergens.
• the methods also provide the use of compositions containing the various SHAAGtide sequences incorporated into sustained release formulations.
• the methods also provide for the use of adjuvants in the administered compositioins.
• adjuvants include alum, incomplete Freund's adjuvant, a bacterial capsular polysaccharide, bacterial DNA, dextran, IL-12, GM-CSF, CD40 ligand, IFN- ⁇ , IL-1, IL-2, IL-3, IL-4, IL-10, IL-13, IL-18 or a cytokine, or fragments thereof.
• the methods of the invention also provide for the administration of multivalent carriers with the SHAAGtide and antigen molecules.
• the multivalent carrier may be linked to a SHAAGtide polypeptide, the antigen or an adjuvant.
• multivariant carriers include bacterial capsular polysaccharide (such as Pneumococci, Streptococci or Meningococci polysaccharides), a dextran and polynucleotide vectors.
• the methods of invention provide for the administration of a pharmaceutical carrier with the SHAAGtide and antigen molecules.
• sites of administration include solid tumors or tissues surrounding such tumors. Administration may be accomplished by any number of means, including injection, inhalation, or oral. Suppositories may also be used.
• the methods of the invention also provide for multiple administrations of the SHAAGtide-containing compositions; administrations may of course be at the same or different site. In some instances, the administration site is removed from a target site of polypeptide delivery.
• liposomes may be administered containing SHAAGtides and antigens, as well as incorporating molecules that enable the liposome to be targeted to specific tissues or cells.
• the invention provides compositions having at least one SHAAGtide-containing polypeptide or fragment thereof; and at least one antigen.
• two different SHAAGtide peptides may be used.
• Such compositions may be formulated in sustained release formulations.
• the compositions of the invention may also incorporate a pharmaceutically acceptable carrier, which may be an adjuvant in some cases.
• Other pharmaceutically acceptable carriers include water, oil, saline, aqueous dextrose and glycerol.
• compositions may incorporate a cell, a microbial vector or viral vector expressing a polynucleotide, such as one encoding SHAAGtide sequences.
• the cell may be allogeneic or autologous.
• the compositions may also include tumor-associated antigens (which may be obtained from autologous cells), cancer cells, cells from cancer cell lines (such as human ovarian or human brain cancer).
• the invention provides compositions formulated with at least one tumor cell; and at least one cell exogenously expressing at least one SHAAGtide polynucleotide sequence.
• the tumor cell may be a primary, autologous or allogenic.
• the tumor cell may also be a glioma, glioblastoma, gliosarcoma, astrocytoma, melanoma, breast cancer cell or an ovarian cancer cell.
• the tumor cell is a cancer cell.
• a pharmaceutical composition incorporating at least one SHAAGtide molecule (polypeptide and/or polynucleotide) and a syringe.
• SHAAGtides novel peptides
• truncation mutants of a splice variant of the CC chemokine CCL23, CK ⁇ 8-1 that is able to modulate and/or enhance an immune responses in vitro and in vivo.
• To modulate an immune response is to influence the classes and subtypes of produced immunoglobulins (Ig's) and the number and type of cells (e.g., cytotoxic T cells, eosinophils, and mast cells) that localize to the site of infection.
• Ig's produced immunoglobulins
• cells e.g., cytotoxic T cells, eosinophils, and mast cells
• SHAAGtides act as ligands to a receptor because calcium flux in leukocytes is seen upon addition of these peptides.
• SHAAGtides effectively attract monocytes, neutrophils and mature dendritic cells (mDCs), as well as immature dendritic cells (iDCs).
• CK ⁇ 8 CCL23, also known as myeloid progenitor inhibitor factor 1 or MPIF-1; 99 amino acids
• MPIF-1 myeloid progenitor inhibitor factor 1
• CK ⁇ 8-1 (residues 1-116), an alternative spliced form of CK ⁇ 8 is a functional ligand for the CCR1 receptor, as is CK ⁇ 8 (Youn et al., 1998).
• CK ⁇ 8-1 (1-116) does not exert its functions through the SHAAGtide sequences.
• the SHAAGtide sequences represent cryptic functional peptides that are therefore surprisingly effective as adjuvants and immunomodulators.
• the SHAAGtide polypeptides promote an immune reaction to the immunogen by recruiting APCs to site of administration.
• Immunogens antigens
• APCs are ingested by APCs and partially degraded.
• a fraction of the degraded antigen is presented associated with MHC class I or II molecules on the surface of the APC.
• proliferation of cytotoxic T cells or helper T cells is stimulated, or antibody production and secretion by B cells is activated.
• SHAAGtides act as effective molecular beacons to attract cells of the immune system, the immune response is enhanced and/or modulated.
• SHAAGtides enhance the immune response such that antigens that usually do not elicit (or weakly elicit) such a response do so; use of SHAAGtides can also decrease the need for subsequent booster injections.
• SHAAGtides can also modify the type of generated immune response.
• the invention encompasses compositions containing SHAAGtide or nucleic acids encoding SHAAGtides and their prophylactic uses, as well as treating disease conditions.
• the SHAAGtide polypeptide sequence (SEQ ID NO:1) and some active variants (SEQ ID NOS:2-6) are shown in Tables 1 and 3; the polynucleotide sequences that encode SEQ ID NOS:1-6, respectively, are shown in Table 2 (SEQ ID NOS:7-12).
• CK ⁇ 8-1 Another derivative of CK ⁇ 8-1 that has SHAAGtide-like activity (CK ⁇ 8-1 (25-116; SEQ ID NO:13), is shown in Table 3; the nucleotide sequence that encodes SEQ ID NO:13 is shown in Table 4. The sequences corresponding to SEQ ID NOS:1 and 7 are underlined.
• SEQ ID NOS:1-14 The “parent” sequences of SEQ ID NOS:1-14 are shown in Table 5 (SEQ ID NO:15; CK ⁇ 8-1 polypeptide) and Table 6 (SEQ ID NO:16, CK ⁇ 8-1 polynucleotide). SHAAGtide sequences are underlined. Note that CK ⁇ 8-1 (SEQ ID NO:15), while containing the SHAAGtide sequence (SEQ ID NO:1), does not possess the same activities as SEQ ID NO:1 by itself.
• compositions that include SHAAGtide polypeptide or polynucleotide include those suitable for administration to a subject to enhance an immune response, such as in response to vaccination. Also included are kits that include SHAAGtide polypeptide and/or SHAAGtide nucleotide. Such kits may be assembled to facilitate administration of, for example, pharmaceutical compositions.
• the methods of the invention include administering a SHAAGtide (SEQ ID NOS:1-6, 13) or SHAAGtide nucleic acid (SEQ ID NOS:7-12, 14) composition to a subject.
• a SHAAGtide SEQ ID NOS:1-6, 13
• SHAAGtide nucleic acid SEQ ID NOS:7-12, 14
• SHAAGtides When used to enhance or modulate an immune response, SHAAGtides may be administered as polypeptide or as polynucleotides that are expressed in vivo. To further facilitate such methods, SHAAGtide polypeptides may be associated (covalently or non-covalently) to the antigen of interest. In some instances, SHAAGtides in either form may be administered prior to or after administration of the antigen. When SHAAGtide compositions are administered separately from antigen (immunogen) compositions, the compositions are administered at the same physical location in a subject.
• the methods of the invention when enhancing, eliciting or modulating an immune response, include administering SHAAGtide compositions containing the immunogens of interest.
• SHAAGtide compositions may be administered in the absence of immunogens.
• a SHAAGtide composition is first administered, followed by a second administration of immunogen, with or without SHAAGtide polypeptides.
• the immunogen-containing composition is administered first, followed by administration of a SHAAGtide-containing composition.
• the different compositions may be administered simultaneously, closely in sequence, or separated in time, e.g., one hour to two weeks or more.
• SHAAGtide compositions are administered at the sites of abnormal growth or directly into the tissue (i.e., a tumor). Tumor or cancer antigens are then detected by the SHAAGtide-recruited or activated leukocytes, such as dendritic cells. By provoking an immune response to these antigens, tumors and cancers are attacked by the body and are reduced or eliminated. As such, these methods represent treatments for conditions involving uncontrolled or abnormal cell growth, e.g., tumors and cancers. Immune responses to tumors and cancers may also be promoted and/or modulated by administering isolated polypeptide tumor antigens with SHAAGtides. SHAAGtides may either be conjugated to the antigen or unconjugated.
• the SHAAGtide polypeptides are truncations of a splice variant of the CK ⁇ 8-1 chemokine.
• Chemokines act as molecular beacons for the recruitment and activation of T lymphocytes, neutrophils, monocytes and macrophages, flagging pathogen battlegrounds.
• Chemokines a group of greater than 40 small peptides (7-10 kD), ligate receptors expressed on WBCs that signal through G-protein-coupled signaling cascades to mediate their chemotractant and chemostimulant functions.
• Receptors may bind more than one ligand; for example, the receptor CCR1 ligates RANTES (regulated on activation normal T cell expressed), MIP-1 ⁇ (macrophage inflammatory protein) and MIP-1 ⁇ chemokines.
• RANTES regulated on activation normal T cell expressed
• MIP-1 ⁇ macrophage inflammatory protein
• MIP-1 ⁇ chemokines MIP-1 ⁇ chemokines.
• 24 chemokine receptors are known. The sheer number of chemokines, multiple ligand binding receptors, and different receptor profiles on WBCs allow for tightly controlled and specific immune responses (Rossi and Zlotnik, 2000). Chemokine activity can be controlled through the modulation of their corresponding receptors, treating related inflammatory and immunological diseases and enabling organ and tissue transplants.
• the immune response such as elicited during vaccination can be enhanced and/or modulated. That is, the vigor and/or magnitude and/or quality of the immune response is increased. For example, the early appearance and/or a high titer and/or avidity of antigen-specific antibodies indicates a vigorous immune response. The magnitude is augmented at least two-fold up to ten-fold or even hundred-fold compared to traditional vaccination methods. Enhancing or modulating the immune response's quality includes the production of high affinity antibodies to the immunogen and/or a higher concentration of preferred immunoglobulin classes, e.g., IgGs.
• preferred immunoglobulin classes e.g., IgGs.
• Modulating the quality of the immune response also includes inducing different subsets of T lymphocytes that are distinguished by different subsets of cytokines and/or chemokines and/or the co-stimulatory molecules they produce. Modulating the quality of the immune response also includes inducing antigen-specific cytotoxic T cells and/or antibodies of different isotypes.
• SHAAGtide or SHAAGtide refers to the nucleotide sequence that encodes SHAAGtide.
• Control sequences are DNA sequences that enable the expression of an operably-linked coding sequence in a particular host organism.
• Prokaryotic control sequences include promoters, operator sequences, and ribosome binding sites.
• Eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.
• Nucleic acid is “operably-linked” when it is placed into a functional relationship with another nucleic acid sequence.
• a promoter or enhancer is operably-linked to a coding sequence if it affects the transcription of the sequence, or a ribosome-binding site is operably-linked to a coding sequence if positioned to facilitate translation.
• An “isolated” nucleic acid molecule is purified from the setting in which it is found in nature and is separated from at least one contaminant nucleic acid molecule.
• Isolated SHAAGtide molecules are distinguished from the specific SHAAGtide molecule, as it exists in cells.
• An isolated SHAAGtide nucleic acid molecule comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NOS:7-12, 14, or a portion of this nucleotide sequence.
• a “complementary nucleic acid molecule” is one that is sufficiently complementary to a sequence, e.g., SEQ ID NOS:7-12, such that hydrogen bonds are formed with few mismatches, forming a stable duplex. “Complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides.
• “Derivatives” are nucleic acid sequences (or amino acid sequences) formed from native compounds either directly or by modification or partial substitution. “Analogs” are nucleic acid sequences or amino acid sequences that have a structure similar, but not identical, to the native compound but differ from it in respect to certain components or side chains. Analogs may be synthesized or from a different evolutionary origin. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
• Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid.
• Derivatives or analogs of the nucleic acids or proteins of SHAAGtide include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of SHAAGtide by at least about 70%, 80%, or 95% identity over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a homology algorithm, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions (Ausubel et al., 1987).
• homologous nucleotide sequences encode those sequences coding for isoforms of SHAAGtide.
• homologous nucleotide sequences include nucleotide sequences encoding for a SHAAGtide of species other than humans, such as vertebrates, e.g., frog, mouse, rat, rabbit, dog, cat, cow and horse.
• homologous nucleotide sequences also include naturally occurring allelic variations and mutations of the nucleotide sequences.
• a homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human SHAAGtide.
• Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions as well as a polypeptide possessing SHAAGtide biological activity.
• DNA sequence polymorphisms that change the amino acid sequences of the SHAAGtide may exist within a population. For example, allelic variation among individuals will exhibit genetic polymorphism in SHAAGtide.
• the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORF) encoding SHAAGtide, preferably a vertebrate SHAAGtide. Such natural allelic variations can typically result in 1-5% variance in SHAAGtide.
• SHAAGtide variant polynucleotide or “SHAAGtide variant nucleic acid sequence” means a nucleic acid molecule which encodes an active SHAAGtide that (1) has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native SHAAGtide, (2) a full-length native SHAAGtide lacking the signal peptide, or (3) any other fragment of a full-length SHAAGtide.
• an SHAAGtide variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with the nucleic acid sequence encoding a full-length native SHAAGtide.
• a SHAAGtide variant polynucleotide may encode full-length native SHAAGtide lacking the signal peptide, or any other fragment of a full-length SHAAGtide. Variants do not encompass the native nucleotide sequence.
• SHAAGtide variant polynucleotides are at least about 30 nucleotides in length, often at least about 60, 90, 120, 150, 180, 210, 240, 270, 300, 450, 600 nucleotides in length, more often at least about 900 nucleotides in length, or more.
• Percent (%) nucleic acid sequence identity with respect to SHAAGtide-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in SHAAGtide that are identical with the nucleotides in a candidate sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining % nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
• the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) can be calculated as follows:
• W is the number of nucleotides scored as identical matches by the sequence alignment program's or algorithm's alignment of C and D
• Z is the total number of nucleotides in D.
• nucleic acid sequence C is not equal to the length of nucleic acid sequence D
• the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
• Homologs i.e., nucleic acids encoding SHAAGtide derived from species other than human
• other related sequences e.g., paralogs
• hybridization stringency increases as the propensity to form DNA duplexes decreases.
• the stringency can be chosen to either favor specific hybridizations (high stringency), which can be used to identify, for example, full-length clones from a library. Less-specific hybridizations (low stringency) can be used to identify related, but not exact, DNA molecules (homologous, but not identical) or segments.
• DNA duplexes are stabilized by: (1) the number of complementary base pairs, (2) the type of base pairs, (3) salt concentration (ionic strength) of the reaction mixture, (4) the temperature of the reaction, and (5) the presence of certain organic solvents, such as formamide which decreases DNA duplex stability.
• the longer the probe the higher the temperature required for proper annealing.
• a common approach is to vary the temperature: higher relative temperatures result in more stringent reaction conditions. (Ausubel et al., 1987) provide an excellent explanation of stringency of hybridization reactions.
• stringent conditions describes hybridization protocols in which nucleotide sequences at least 60% homologous to each other remain hybridized.
• stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
• Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
• Stringent hybridization conditions enable a probe, primer or oligonucleotide to hybridize only to its target sequence. Stringent conditions are sequence-dependent and will differ. Stringent conditions comprise: (1) low ionic strength and high temperature washes (e.g. 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1% sodium dodecyl sulfate at 50° C.); (2) a denaturing agent during hybridization (e.g.
• washes typically also comprise 5 ⁇ SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 ⁇ Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C.
• the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. These conditions are presented as examples and are not meant to be limiting.
• Modely stringent conditions use washing solutions and hybridization conditions that are less stringent (Sambrook, 1989), such that a polynucleotide will hybridize to the entire, fragments, derivatives or analogs of SEQ ID NOS:7-12, 14.
• One example comprises hybridization in 6 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55° C., followed by one or more washes in 1 ⁇ SSC, 0.1% SDS at 37° C.
• the temperature, ionic strength, etc. can be adjusted to accommodate experimental factors such as probe length.
• Other moderate stringency conditions have been described (Ausubel et al., 1987; Kriegler, 1990).
• Low stringent conditions use washing solutions and hybridization conditions that are less stringent than those for moderate stringency (Sambrook, 1989), such that a polynucleotide will hybridize to the entire, fragments, derivatives or analogs of SEQ ID NOS:7-12, 14,.
• low stringency hybridization conditions are hybridization in 35% formamide, 5 ⁇ SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40° C., followed by one or more washes in 2 ⁇ SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50° C.
• Other conditions of low stringency such as those for cross-species hybridizations are well-described (Ausubel et al., 1987; Kriegler, 1990; Shilo and Weinberg, 1981).
• allelic variants of SHAAGtide changes can be introduced by mutation into SEQ ID NOS:7-12, 14 that incur alterations in the amino acid sequences of the encoded SHAAGtide that do not alter SHAAGtide function.
• nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of SEQ ID NOS:3 or 4.
• a “non-essential” amino acid residue is a residue that can be altered from the wild-type sequences of the SHAAGtide without altering biological activity, whereas an “essential” amino acid residue is required for such biological activity.
• amino acid residues that are conserved among the SHAAGtide of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well known in the art.
• substitutions Preferred Original residue Exemplary substitutions substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L) Norleucine, Ile, Val, Met, Ala, Ile Phe Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr
• Non-conservative substitutions that effect (1) the structure of the polypeptide backbone, such as a ⁇ -sheet or ⁇ -helical conformation, (2) the charge (3) hydrophobicity, or (4) the bulk of the side chain of the target site can modify SHAAGtide polypeptide function.
• Residues are divided into groups based on common side-chain properties as denoted in Table B.
• Non-conservative substitutions entail exchanging a member of one of these classes for another class. Substitutions may be introduced into conservative substitution sites or more preferably into non-conserved sites.
• the variant polypeptides can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
• Site-directed mutagenesis Carter, 1986; Zoller and Smith, 1987
• cassette mutagenesis restriction selection mutagenesis
• Wells et al., 1985 or other known techniques can be performed on the cloned DNA to produce the SHAAGtide variant DNA (Ausubel et al., 1987; Sambrook, 1989).
• Isolated polypeptide, protein or biologically active fragment is separated and/or recovered from a component of its natural environment.
• Isolated polypeptides include those expressed heterologously in genetically engineered cells or expressed in vitro.
• Contaminant components include materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide.
• Polypeptides and fragments of interest can be produced by any method well known in the art, such as by expression via vectors such as bacteria, viruses and eukaryotic cells.
• vectors such as bacteria, viruses and eukaryotic cells.
• in vitro synthesis such as peptide synthesis, may be also used.
• an “active” polypeptide or polypeptide fragment retains a biological and/or an immunological activity similar, but not necessarily identical, to an activity of the SHAAGtide polypeptide shown in Tables 1 and 3.
• Immunological activity in the context of this immediate discussion of the polypeptide per se, and not an actual biological role for SHAAGtide in eliciting or enhancing an immune response, refers to an aspect of a SHAAGtide polypeptide in that a specific antibody against a SHAAGtide antigenic epitope binds SHAAGtide.
• Biological activity refers to a function, either inhibitory or stimulatory, caused by a native SHAAGtide polypeptide.
• a biological activity of SHAAGtide polypeptide includes, for example, chemotaxis, inducing, enhancing or aiding an immune response.
• a particular biological assay (see Examples), with or without dose dependency, can be used to determine SHAAGtide activity.
• a nucleic acid fragment encoding a biologically-active portion of SHAAGtide can be prepared by isolating a polynucleotide sequence that encodes a polypeptide having a SHAAGtide biological activity, expressing the encoded portion of SHAAGtide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of SHAAGtide polypeptide.
• a SHAAGtide polypeptide variant that preserves SHAAGtide polypeptide-like function and includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further includes the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence.
• Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
• SHAAGtide polypeptide variant means an active SHAAGtide polypeptide having at least: (1) about 70% amino acid sequence identity with a full-length SHAAGtide sequence or (2) any fragment of a full-length SHAAGtide sequence.
• SHAAGtide variants include SHAAGtide polypeptides wherein one or more amino acid residues are added or deleted at the N- or C-terminus of the sequences of SEQ ID NOS:1-6, 13.
• a SHAAGtide polypeptide variant will have at least about 70% amino acid sequence identity, preferably at least about 71% amino acid sequence identity, more preferably at least about 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% amino acid sequence identity and most preferably at least about 99% amino acid sequence identity with SHAAGtide polypeptide sequence.
• Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues that are identical with amino acid residues in a SHAAGtide sequence in a candidate sequence when the two sequences are aligned. To determine % amino acid identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum % sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures to determine percent identity are well known to those of skill in the art. Publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) can be used to align polypeptide sequences. Parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared, can be determined.
• the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B can be calculated as:
• X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B
• Y is the total number of amino acid residues in B.
• Fusion polypeptides are useful in expression studies, cell-localization, bioassays, SHAAGtide purification and importantly in adjuvant applications when the peptide may be fused to the antigen(s) of interest.
• a SHAAGtide “chimeric polypeptide” or “fusion polypeptide” comprises SHAAGtide fused to a non-SHAAGtide polypeptide.
• a non-SHAAGtide polypeptide is not substantially homologous to SHAAGtide (SEQ ID NOS:1-6, 13).
• a SHAAGtide fusion polypeptide may include any portion to an entire SHAAGtide, including any number of biologically active portions. In some host cells, heterologous signal sequence fusions may ameliorate SHAAGtide expression and/or secretion.
• Fusion partners can be used to adapt SHAAGtide therapeutically.
• SHAAGtide-Ig fusion polypeptides can be used as immunogens to produce anti-SHAAGtide Abs in a subject, to purify SHAAGtide ligands, and to screen for molecules that inhibit interactions of SHAAGtide with other molecules. Additionally, fusions with antigens of interest can be used to facilitate vaccination/immunication proceduresz
• Fusion polypeptides can be easily created using recombinant methods.
• a nucleic acid encoding SHAAGtide can be fused in-frame with a non-SHAAGtide encoding nucleic acid, e.g., antigen(s) with which to immunize, to the SHAAGtide NH 2 - or COO-terminus, or internally.
• Fusion genes may also be synthesized by conventional techniques, including automated DNA synthesizers. PCR amplification using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (Ausubel et al., 1987). Many vectors are commercially available that facilitate sub-cloning SHAAGtide in-frame to a fusion moiety.
• Polypeptide mimetics of SHAAGtide may also be used.
• the terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics as a SHAAGtide polypeptide. Mimetics can be either entirely composed of synthetic, non-natural analogues of amino acids, or a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. Mimetics can also incorporate any amount of natural amino acid conservative substitutions.
• Polypeptide mimetic compositions can contain any combination of nonnatural structural components, which are typically from three structural groups: (a) residue linkage groups other than the natural amide bond (“peptide bond”) linkages; (b) non-natural residues in place of naturally occurring amino acid residues; or (c) residues which induce secondary structural mimicry, i.e., inducing or stabilizing a secondary structure, e.g., a ⁇ turn, ⁇ turn, ⁇ sheet, ⁇ helix conformation, and the like.
• a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
• Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide (DIC).
• Linking groups that can be an alternative to the traditional amide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g., —C( ⁇ O)—CH 2 — for —C( ⁇ O)—NH—), aminomethylene (CH 2 —NH), ethylene, olefin (CH ⁇ CH), ether (CH 2 —O), thioether (CH 2 —S), tetrazole (CN 4 —), thiazole, retroamide, thioamide, or ester (Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).
• ketomethylene e.g., —C( ⁇ O)—CH 2 — for —C( ⁇ O)—NH—
• aminomethylene e.g., —NH
• ethylene olefin
• ether CH 2 —
• a polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues.
• Nonnatural residues, as well as appropriate substitutions for each class of amino acids are well known.
• mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3- or 4-pyreneylalanine, etc.
• mimetics include those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the ⁇ -amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.
• a component of a natural polypeptide can also be replaced by an amino acid or peptidomimetic residue of the opposite chirality.
• Mimetics also include compositions that contain a structural mimetic residue, particularly a residue that induces or mimics secondary structures, such as a ⁇ turn, ⁇ sheet, ⁇ helix structures, ⁇ turns, and the like.
• a structural mimetic residue particularly a residue that induces or mimics secondary structures, such as a ⁇ turn, ⁇ sheet, ⁇ helix structures, ⁇ turns, and the like.
• substitution of natural amino acid residues with D-amino acids; N- ⁇ -methyl amino acids; C- ⁇ -methyl amino acids; or dehydroamino acids within a peptide can induce or stabilize ⁇ turns, ⁇ turns, ⁇ sheets or ⁇ helix conformations.
• cyclic SHAAGtide peptides may be advantageous.
• cysteine residues included in a peptide can be oxidized to form —S—S-dimers or larger multimer (trimers, etc.) by oxidization.
• Two cysteines placed distal to each other in a peptide can be oxidized to prepare a cyclic peptide containing one or more functional amino acid sequences.
• SHAAGtides have certain properties when used as an adjuvant; namely, enhancing, eliciting or modulating an immune response. Other activities of the SHAAGtides are known, including inducing chemotaxis on certain cells, including those expressing the formyl-peptide receptor-like-1 (FPRL1) receptor.
• FPRL1 formyl-peptide receptor-like-1
• In vitro chemotaxis (cell migration) assays can be used to identify SHAAGtide chemotactic properties. Such assays physically separate the cells from the candidate chemoattractant using a porous membrane and assaying the cell migration from one side of the membrane to the other, indicating cell migration.
• a conventional cell migration assay such as the ChemoTx® system (NeuroProbe, Rockville, Md.; (Goodwin, U.S. Pat. No. 5,284,753, 1994)) or any other suitable device or system (Bacon et al., 1988; Penfold et al., 1999) may be used.
• Cells expressing the target receptor are gathered.
• a candidate compound such as SHAAGtide peptides or other chemokine/chemokine-like compound is prepared, usually in a concentration series by serial dilution in a buffer. The concentration range is typically between 0.1 nM and 10 mM, but will vary with the compound being tested.
• non-migrating cells on the upper chamber of the apparatus are removed using a rubber scraper or other manual method, enzymatically or chemically, e.g., EDTA and EGTA solutions.
• the membrane that separates the two chambers is then removed from the apparatus and rinsed with Dulbecco's phosphate buffered saline (DPBS) or water.
• DPBS Dulbecco's phosphate buffered saline
• the number of cells that migrated into the lower chamber is then determined. Cell migration at levels above background (without a chemotactic or candidate compound), indicate that the candidate compound is chemotactic for the tested cells.
• a candidate compound is considered chemotactic for a particular cell type if, at a concentration of about 1 pM to about 1 ⁇ m (e.g., between about 1 nM and 500 nM, e.g., 1 nM, about 10 nM, about 100 nM, or between about 1 pg/ml and about 10 ⁇ g/ml, e.g., between about 1 ng/ml and 1 ⁇ g/ml, e.g., about 10 ng/ml, about 100 ng/ml or about 1 ⁇ g/ml) attracts the cell at least 2-fold to 8-fold or more than a negative control.
• a concentration of about 1 pM to about 1 ⁇ m e.g., between about 1 nM and 500 nM, e.g., 1 nM, about 10 nM, about 100 nM, or between about 1 pg/ml and about 10 ⁇ g/ml, e.g.,
• Chemotactic properties of a compound can be determined in animals, e.g., mammals such as non-human primates and mice.
• the candidate compound e.g., 2-20 ⁇ g in PBS
• the presence or absence of cell infiltration is determined, using routine histological techniques. If an infiltrate is present, the cells are identified by type (mononuclear, neutrophil, dendritic, etc.) and are quantified.
• SHAAGtide polypeptides (SEQ ID NOS:1-6, 13), or derivatives, analogs, etc. may be administered in compositions, such as those used to elicit, enhance or modulate an immune response; one or more of the SHAAGtide polypeptides (SEQ ID NOS:1-6, 13) may be included.
• the compositions may include antigens of interest; however, SHAAGtide polypeptides may be administered by themselves.
• the SHAAGtide polypeptides are administered in sequence with other administrations containing other molecules, such as polypeptide or polysaccharide immunogens.
• the methods of the invention involve administration of an immunogen, in addition to a SHAAGtide composition.
• an immunogen in addition to a SHAAGtide composition.
• these compositions are administered at the same physical site in the subject.
• the immunogen may be combined with a SHAAGtide composition, and the mixture administered (e.g., injected) together.
• the composition and the antigen are administered separately to the same area of the subject (e.g., injected to the same site, applied topically to the same site, etc.).
• the different compositions are administered at different times.
• SHAAGtide compositions can also be administered without an accompanying antigen (e.g., injection into a solid tumor to elicit an immune response to cancer cells, or injection in tissue surrounding a solid tumor, e.g., within 2 cm, of a solid tumor).
• an accompanying antigen e.g., injection into a solid tumor to elicit an immune response to cancer cells, or injection in tissue surrounding a solid tumor, e.g., within 2 cm, of a solid tumor.
• SHAAGtide compositions may additionally contain an excipient or carrier.
• SHAAGtide compositions may also include one or more immunogens (antigens; i.e., the antigen to which it is desired to induce, enhance or modulate an immune response).
• SHAAGtide compositions may contain a conventional adjuvant.
• Conventional adjuvants typically convert soluble protein antigens into particulate material.
• Conventional adjuvants include Freund's incomplete, Freund's complete, Merck 65, AS-2, alum, aluminum phosphate, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
• bacterial capsular polysaccharides include, but are not limited to, bacterial capsular polysaccharides, dextran, IL-12, GM-CSF, CD40 ligand, IFN- ⁇ , IL-1, IL-2, IL-3, IL-4, IL-10, IL-13, IL-18 or any cytokine or bacterial DNA fragment.
• the present invention provides a method of eliciting or enhancing an immune response to an antigen, e.g., a predetermined or specified antigen.
• An antigen is a molecule that reacts with an antibody.
• the antigen is an immunogen.
• the antigen is linked to a protein carrier.
• a SHAAGtide and an antigen may be physically linked, such as by a fusion protein, chemically cross-linking or complexes such as biotin and streptavidin.
• An antigen is typically a peptide, a polypeptide, chemical compound, microbial pathogen, bacteria (e.g., live, attenuated, or inactivated), a virus (including inactivated virus particles, modified live viral particles, and recombinant virus particles), a recombinant cell, glycoproteins, lipoproteins, glycopeptides, lipopeptides, toxoids, carbohydrates, tumor-specific antigens, and other immunogenic components of pathogens. Mixtures of two or more antigens may be used. The antigen may be purified. In some embodiments, the antigen may be associated (covalently or non-covalently) with a SHAAGtide polypeptide.
• the invention is used to provide protection from exogenous foreign infectious pathogenic agents prior to exposure.
• the invention can be used to provide therapeutic effects against exogenous foreign pathogens to which an individual has been exposed or to individual displaying symptoms of exposure.
• the invention can be used to treat cancers, including, but not limited to, melanomas, lung cancers, thyroid carcinomas, breast cancers, renal cell carcinomas, squamous cell carcinomas, brain tumors and skin cancers.
• the antigen may be a tumor-associated antigen (tumor specific-antigen).
• Tumor antigens are molecules, especially cell surface proteins, which are differentially expressed in tumor cells relative to non-tumor tissues.
• compositions containing SHAAGtides are administered (e.g., in conjunction with immunogens) to a subject.
• compositions containing the SHAAGtides are administered to a subject once a disease is detected, diagnosed or even treated, such as after surgical removal of a tumor.
• antigens or vaccine components of the invention include antigens derived from microbial pathogens such as bacteria [e.g., Pertussis ( Bordetella pertussis, inactivated whole organism); Cholera ( Vibrio cholerae, whole killed organism); Meningitis ( Neisseria meningitidis, polysaccharide from organism); Lyme Disease ( Borrelia burgdorferi, lipoprotein OspA); Haemophilus B ( Haemophilus influenza B polysaccharide, Tetanus conjugate or OmpC); Pneumonia ( Streptococcs pneumoniae capsular polysaccharide) Typhoid ( Salmonella typhi polysaccharide vaccine, killed whole organism)], viruses including inactivated virus particles, modified live viral particles, and recombinant virus particles to Influenza virus; Hepatitis A; Hepatitis B; Hepatitis C; Measles; Rubella virus; Mumps; Rabies; Poliovirus
• the SHAAGtide, the antigen, or both may be delivered as polynucleotides, such that the polypeptides are generated in situ.
• uptake by cells can be increased by coating the polynucleotide onto a carrier, e.g. biodegradable beads, which is efficiently transported into cells.
• the polynucleotides may be present within any of a variety of delivery systems, including nucleic acid expression systems, bacterial and viral expression systems.
• Vectors used to shuttle genetic material from organism to organism, can be divided into two general classes: Cloning vectors are replicating plasmid or phage with regions that are non-essential for propagation in an appropriate host cell and into which foreign DNA can be inserted; the foreign DNA is replicated and propagated as if it were a component of the vector.
• An expression vector (such as a plasmid, yeast, or animal virus genome) is used to introduce foreign genetic material into a host cell or tissue in order to transcribe and translate the foreign DNA, such as SHAAGtide.
• the introduced DNA is operably-linked to elements such as promoters that signal to the host cell to transcribe the inserted DNA.
• Inducible promoters that control gene transcription in response to specific factors can be exceptionally useful. Operably-linking a SHAAGtide and/or antigen polynucleotide to an inducible promoter can control the expression of a SHAAGtide and/or antigen polypeptide or fragments.
• Examples of classic inducible promoters include those that are responsive to ⁇ -interferon, heat shock, heavy metal ions, and steroids such as glucocorticoids (Kaufman, 1990), and tetracycline.
• Other desirable inducible promoters include those that are not endogenous to the cells in which the construct is being introduced, but are responsive in those cells when the induction agent is exogenously supplied.
• useful expression vectors are often plasmids. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) are contemplated.
• Vector choice is dictated by the organism or cells being used and the desired fate of the vector.
• Vectors may replicate once in the target cells, or may be “suicide” vectors.
• vectors comprise signal sequences, origins of replication, marker genes, enhancer elements, promoters, and transcription termination sequences.
• SHAAGtide compositions may contain one or more antigens or antigen-encoding polynucleotides.
• Antigens can be administered in combination with SHAAGtides (i.e., in the same mixture). Alternatively, they can be administered separately.
• the invention provides an immunization method in which a combination of one or more antigens (or antigen-encoding polynucleotides) and one or more SHAAGtides (or SHAAGtide-encoding polynucleotides) are administered to a subject.
• the antigen or SHAAGtide may be administered in a delivery vehicle such as a physiologically acceptable excipient.
• the antigen may be administered simultaneously with the SHAAGtide composition or the antigen and the SHAAGtide composition is administered at different times, typically to the same site.
• the chemotactic composition (without the antigen) can be administered between about 15 minutes and about 96 hours prior to the administration of the antigen, more often between about 15 minutes and about 48 hours, more often between 24 hours and 96 hours, often between about 48 hours and 72 hours or between 72 hours and 96 hours prior to the administration of the antigen.
• a SHAAGtide composition and an antigen composition are injected at the same site in a subject, preferably the injections are within 2 cm of each other, preferably within 1 cm or preferably within 0.5 cm of each other on the two dimensional surface of the body.
• the administrations should also be done to a similar depth and to the same tissue layers.
• the depth should be more precisely monitored to achieve a three dimensional equivalent placement of the SHAAGtide and the antigen to within 2 cm of each other, preferably to within 1 cm, and more preferably to within 0.5 cm.
• the injection site can be marked with an indelible ink to assist the physician.
• One dose (administration) of the composition may be given. However, the first administration may be followed by boosting doses.
• the SHAAGtide composition is administered in multiple doses, often in combination with an antigen (e.g., by co-administration).
• the SHAAGtide composition (optionally including antigen) may be administered once, twice, three times, or more.
• the number of doses administered to a subject is dependent upon the antigen, the extent of the disease, and the response of a subject to the SHAAGtide composition.
• a suitable number of doses includes any number required to immunize an animal to a predetermined antigen.
• a second administration (booster) of the SHAAGtide composition and antigen may be given between about 7 days and 1 year after the first administration.
• the time between the first and second administrations may be 14 days to 6 months, 21 days and 3 months, often between about 28 days and 2 months after the original administration.
• a third administration (second booster) may be given between about 14 days and 10 years after the first administration, e.g., between about 14 days and 3 years, often between about 21 days and 1 year, very often between about 28 days and 6 months after the first administration.
• Subsequent boosters may be administered at 2 week intervals, or 1 month, 3 month or 6 month to 10 year intervals.
• a variety of vaccine administration doses and schedules can be developed easily; the determination of an effective amount and number of doses of SHAAGtides of the invention, antigens, or some combination of SHAAGtides and antigens for administration is also well within the capabilities of those skilled in the art.
• the amount of SHAAGtide and antigen will be administered to a subject that is sufficient to immunize an animal against an antigen (i.e., an “immunologically effective dose” or a “therapeutically effective dose”).
• an amount adequate to accomplish an “immunologically effective dose” will depend in part on the SHAAGtide and antigen composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the subject, and the judgment of the prescribing physician or other qualified personnel.
• the effective dose of antigen and SHAAGtide can be formulated in animal models to achieve an induction of an immune response; such data can be used to readily optimize administration to humans based on animal data (see Examples).
• a dose will typically be between about 1 fg and about 100 ⁇ g, often between about 1 pg and about 100 ⁇ g, more often between about 1 ng and about 50 ⁇ g, and usually between about 100 ng and about 50 ⁇ g.
• the dose is between about 1 fg and about 100 ⁇ g per kg subject body weight, often between about 1 pg and about 100 ⁇ g, more often between about 1 ng and about 50 ⁇ g, and usually between about 100 ng and about 50 ⁇ g per kg subject body weight.
• a SHAAGtide composition may contain one or more antigens and one or more SHAAGtides at a molar or weight ratio of about 1:1000 or greater, SHAAGtide to antigen. Other useful ratios are between about 1:10 and 1:1000, between about 1:10 and 1:1000, or greater than 1:1000. The ratio of antigen to SHAAGtide in the composition may vary between about 1:10 and 10:1.
• the SHAAGtide-containing compositions of the invention may be administered in a variety of ways and in various forms.
• the SHAAGtide composition may include carriers and excipients, such as buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives; water, oils, saline solutions, aqueous dextrose and glycerol solutions, other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, etc..
• a conventional adjuvant may also be incorporated into the composition.
• any suitable carrier may be used to administer the compositions of the invention, the type of carrier will vary depending on the mode of administration.
• Compounds may also be encapsulated within liposomes.
• Biodegradable microspheres are convenient in some instances as carriers; for example, such as those described in (Tice et al., U.S. Pat. No. 5,942,252, 1999).
• compositions are desirable, such as that accomplished by conventional techniques or sterile filtering.
• aqueous solutions may be packaged for use as is, or lyophilized.
• the SHAAGtide compositions of the invention may be administered in a variety of ways, including by injection (e.g., intradermal, subcutaneous, intramuscular, intraperitoneal etc.), by inhalation, by topical administration, by suppository, by using a transdermal patch or by mouth.
• injection e.g., intradermal, subcutaneous, intramuscular, intraperitoneal etc.
• inhalation e.g., by inhalation
• topical administration e.g., by topical administration, by suppository, by using a transdermal patch or by mouth.
• compositions When administration is by injection, compositions may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer.
• the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• the chemotactic composition may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• Inhalation-delivered compositions may be as aerosol sprays from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, carbon dioxide or other suitable gas.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the proteins and a suitable powder base such as lactose or starch.
• the compositions may be formulated as solutions, gels, ointments, creams, suspensions, and the like, as are well known in the art. In some embodiments, administration is by means of a transdermal patch.
• Suppository compositions may also be formulated to contain conventional suppository bases.
• compositions can be readily formulated by combining the composition with pharmaceutically acceptable carriers.
• Solid carriers include mannitol, lactose, magnesium stearate, etc.; such carriers enable the formation of tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions etc., for oral ingestion.
• Such formulations may be powders, capsules and tablets; suitable excipients include fillers such as sugars, cellulose preparation, granulating agents, and binding agents.
• Nucleic acid molecules such as those encoding SHAAGtides, can be inserted into vectors and used as gene therapy vectors. Gene therapy techniques have recently become quite advanced and are meeting enviable success (Meikle, 2002). Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (Nabel and Nabel, U.S. Pat. No. 5,328,470, 1994), or by stereotactic injection (Chen et al., 1994).
• the pharmaceutical preparation of a gene therapy vector can include an acceptable diluent or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
• sustained-release formulations include, for example, SHAAGtide molecules and/or antigens, allowing for the release of SHAAGtides and/or antigens over extended periods of time, such that without the sustained release formulation, the SHAAGtides and/or antigens would be cleared from a subject's system or degraded.
• polyclonal and monoclonal antibodies including binding fragments (e.g., F (ab)2 ) and single chain versions are well known.
• binding fragments e.g., F (ab)2
• single chain versions many antigens are incapable of triggering an adequate antibody response.
• a composition comprising a SHAAGtide of the invention and an antigen is administered to an animal, thus inducing or enhancing the immune response in the animal.
• Polyclonal or monoclonal antibodies are subsequently prepared by standard techniques.
• compositions of the invention are administered to a subject to stimulate the innate immune response.
• the innate immune response is body's initial defense against pathogens and is elicited by a variety of cells including APCs. These cells express surface and cytoplasmic receptors that recognize molecules of foreign origin (e.g., bacterial and viral nucleic acids, proteins, carbohydrates). Upon detecting these signals, the dendritic cells and macrophage elicit a defensive response that includes the release of cytokines (including interferons, TNF- ⁇ , and IL-12) and chemokines that attract cells such as immature dendritic cells, macrophage, NK cells, and granulocytes, to the site of challenge.
• cytokines including interferons, TNF- ⁇ , and IL-12
• chemokines that attract cells such as immature dendritic cells, macrophage, NK cells, and granulocytes
• compositions of the invention can be used to attract dendritic cells and other cells to the site of administration, but also to stimulate these cells into eliciting elements of the innate immune response to confer non-specific protection while the body is generating the adaptive response.
• a SHAAGtide composition is administered (without antigen) prior to or post exposure of an anticipated infection, including those that are sinisterly applied, such as in bioterrorism.
• SHAAGtides are administered with “foreign” molecules (e.g., bacterial or viral nucleic acids, proteins, carbohydrates, or synthetic elements which mimic these elements).
• kits containing one or more of the following in a package or container: (1) a SHAAGtide composition of the invention; (2) a pharmaceutically acceptable adjuvant or excipient; (3) an antigen (e.g., a biologically pure antigen); (4) a vehicle for administration, such as a syringe; (5) instructions for administration.
• a SHAAGtide composition of the invention (2) a pharmaceutically acceptable adjuvant or excipient; (3) an antigen (e.g., a biologically pure antigen); (4) a vehicle for administration, such as a syringe; (5) instructions for administration.
• an antigen e.g., a biologically pure antigen
• kits When a kit is supplied, the different components of the composition may be packaged in separate containers and admixed immediately before use. Such packaging of the components separately may permit long-term storage without losing the activity.
• the reagents included in the kits can be supplied in containers of any sort such that the life of the different components are preserved and are not adsorbed or altered by the materials of the container.
• sealed glass ampules may contain lyophilized SHAAGtide polypeptides or polynucleotides, or buffers that have been packaged under a neutral, non-reacting gas, such as nitrogen.
• Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc.; ceramic, metal or any other material typically employed to hold similar reagents.
• suitable containers include simple bottles that may be fabricated from similar substances as ampules, and envelopes that may comprise foil-lined interiors, such as aluminum or an alloy.
• Other containers include test tubes, vials, flasks, bottles, syringes, or the like.
• Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
• Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to be mixed. Removable membranes may be glass, plastic, rubber, etc.
• Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
• SHAAYtide (SEQ ID NO:4) peptide preparation
• the peptide of SEQ ID NO:4, “SHAAYtide” was chemically synthesized and purified (Phoenix Pharmaceuticals; Belmont, Calif.). The material was suspended in phosphate-buffered saline (PBS) at a concentration of approximately 1 mg/ml and stored at ⁇ 20° C.
• PBS phosphate-buffered saline
• Enzyme-linked immunosorbent assays First, 96-well U-bottom plastic dishes were coated overnight with 1 ⁇ g ovalbumin (OVA) in 100 ⁇ l PBS per well. The next day, the dishes were rinsed with PBS, blocked with PBS containing 5% fetal bovine serum (FBS), and rinsed with PBS again. Plasma samples from experimental animals (see below) were diluted 10 2 - to 10 5 -fold and added to the dishes for 2 hours, after which the dishes were again rinsed with PBS.
• OVA ovalbumin
• FBS fetal bovine serum
• the dishes were then incubated with biotinylated goat anti-monkey IgG detection antibodies, then rinsed with PBS and incubated with streptavidin-linked horseradish peroxidase (SA-HRP). After a final rinsing with PBS, the HRP substrate 2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt was added.
• Dendritic cell purification Substantially purified dendritic cells (including subpopulations of mature or immature cells) can be prepared. Subpopulations of dendritic cells include: (1) immature peripheral blood monocyte derived cells, (2) mature peripheral blood monocyte derived cells, and (3) cells derived from CD34-expressing precursors.
• Human or macaque dendritic cells of various developmental stages can be generated in culture from CD14-expressing blood progenitors using specific cytokines.
• a separate lineage of dendritic cells can be differentiated from CD34-expressing precursor cells from cord blood or bone marrow.
• immature and mature dendritic cells from peripheral blood mononuclear cells (PMBCs) can also be produced (Bender et al., 1996).
• Mature dendritic cells can be made using macrophage conditioned medium and double stranded RNA-ploy (I:C) stimulation (Cella et al., 1999; Romani et al., 1996; Verdijk et al., 1999).
• Chemokine receptor expression also changes dramatically during maturation; e.g., CCR1 and CCR5 are down-regulated in mature cells while CCR7 is up-regulated. Functional characteristics may also be exploited to confirm a cell type. For example, mature dendritic cells are incapable of taking up antigen efficiently, but gain the ability to stimulate the proliferation of naive T cells and B cells. Mature dendritic cells also change their migratory behaviors, being unresponsive to CCR1, CCR2 and CCR5 ligands while being newly responsive to CCR7 ligands.
• This example describes an in vivo assay in which the ability of several chemokines and SHAAYtide (SEQ ID NO:2) to attract dendritic cells was demonstrated.
• chemokines were obtained from R&D Systems (Minneapolis, Minn.): vMCK-2, mC10, and GM-CSF.
• the following peptides were synthesized at Phoenix Pharmaceuticals (San Carlos, Calif.): SHAAYtide (SEQ ID NO:4), several STRUCTURALLY MODIFIED peptides of SHAAGtide variant (SEQ ID NO:) (i.e.
• mice received an injection of PBS only as a negative control.
• the mice were euthanized, and the area around the injection site was excised and subjected to immunohistology. Frozen sections were stained with anti-DEC-205 antibody (Bio-Whittaker Molecular Applications; Rockland, Me.) that recognizes a dendritic cell-specific molecule (Kraal et al., 1986). A relative staining number on a scale of 0 to 5 was assigned to each section (0, none; 1, slight; 2, mild; 3, moderate; 4, severe). Results are shown in Tables 7, 8 and 9.
• Mononuclear cell infiltration was scored on a scale of 0 to 5: 0, very mild perivascular mononuclear inflammatory infiltration throughout the dermis; 1, a mild perivascular mononuclear inflammatory infiltrate seen throughout the dermis; 2, a mild/moderate perivascular mononuclear inflammatory infiltrate seen throughout the dermis; 3, a moderate perivascular mononuclear inflammatory infiltrate seen throughout the dermis; 4, an extensive perivascular mononuclear inflammatory infiltrate seen throughout the dermis; 5, a florid perivascular mononuclear inflammatory infiltrate seen throughout the dermis.
• intermediate scores are indicates, e.g., “2/3” represents a score between 2 and 3.
• SHAAYtide SEQ ID NO:4
• vMCK-2 caused a dramatic infiltration of cell.
• the 20 ⁇ g administration caused more infiltration than did the 60 ⁇ g and 8 ⁇ g administration.
• vMIP-1 caused a mild infiltration at all doses tested.
• mC10 caused little to no infiltration in this experiment.
• VKB8-1 caused no infiltration in this experiment.
• Example 3 To better define the identity of the infiltrating cells seen in Example 3 (Table 10), the same samples were analyzed by immunohistochemistry using antibodies specific for different cell types. These antibodies included: CD68 (expressed on macrophages, neutrophils and dendritic cells), MHC II (antigen-presenting cells, e.g. macrophages and dendritic cells), HAM-56 (macrophages), fascin (dendritic cells, endothelial cells and epithelial cells), elastase (neutrophils), cytokeratin (epithelial cells), CD3 (T cells), CD20 (B cells), and CD1a (Langerhans cells).
• CD68 expressed on macrophages, neutrophils and dendritic cells
• MHC II antigen-presenting cells, e.g. macrophages and dendritic cells
• HAM-56 macrophages
• fascin dendritic cells, endothelial cells and epithelial cells
• the vMCK-2-injected skin samples contained primarily neutrophils and antigen-presenting cells, including macrophages and dendritic cells.
• the mC10-injected skin samples contained primarily antigen-presenting cells, including macrophages and dendritic cells, but few neutrophils.
• the vMIP-1-injected skin samples contained primarily neutrophils and macrophages, with few dendritic cells. Few T cells, and no B cells, were found in the skin samples for each of the three chemokines.
• the third group contained OVA, IFA, and vMCK-2
• the fourth group contained OVA, IFA, mC10
• the fifth group contained OVA, IFA, and SEQ ID NO:4.
• the formulations (containing 2 mg OVA and 16 ⁇ g polypeptide) were injected intradermally in 100 ⁇ l. Ten ml of peripheral blood was drawn from each monkey twice a week for three weeks, and the blood samples were then subjected to centrifugation over Ficoll to remove erythrocytes and granulocytes. The plasma supernatant was analyzed by sandwich ELISA to determine the levels of anti-OVA antibodies using OVA-coated plastic dishes and a biotinylated anti-monkey IgG detection antibody.
• monkeys injected with OVA and IFA developed a significant antibody to OVA, as demonstrated by development of circulating anti-OVA IgG, commencing on day 12.
• the levels of OVA-specific IgG in monkeys injected with OVA, mC10 and IFA, or OVA, shaag and IFA were substantially greater than those in monkeys not receiving mC10 or SEQ ID NO:4 respectively.
• Example 5 The experiment described in Example 5 was repeated, except the formulations were administered to BALB/c mice with 10 ⁇ g (Table 12) or 500 ⁇ g (Table 13) of OVA with in 100 ⁇ l. IFA was not used.
• mice were given OVA with or without SHAAYtide (SEQ ID NO:4) either intraperitoneally on days 0 and 21 (Table 12), or sub-cutaneously on days 0 and 14 (Table 13). Blood samples were collected at the indicated time points. The results, reported in antibody units, are given in Tables 12 and 13, showing IgG levels.
• SHAAYtide (SEQ ID NO:4) Shows Different Modulatory Effects on Different Types of Immune Responses Generated in Rhesus Monkeys
• Different types of immune responses can be induced in mammals by varying parameters such as the dose of antigen, the formulation, the route of administration, and the type of adjuvant.
• the adjuvant alum when used for vaccination purposes in humans or laboratory animals, the generated immune response is predominated by antibodies of the IgG 1 and IgGE classes, shows little generation of cytotoxic T cells, and shows augmentation of eosinophils and mast cells.
• stronger adjuvants such as Complete or Incomplete Freund's adjuvants
• IgG 2 antibodies are used.
• SHAAYtide SEQ ID NO:4
• IFA adjuvant alum adjuvant
• SHAAYtide SEQ ID NO:4
• a candidate or known chemotaxins for a specific cell type such as for dendritic cells (immature or mature)
• a specific cell type such as for dendritic cells (immature or mature)
• chemotaxins for a specific cell type such as for dendritic cells (immature or mature)
• Day 7 immature dendritic cells are harvested, washed once with chemotaxis buffer (0.1% BSA in Hank's balanced salt solution (HBSS; Invitrogen, Carlsbad, Calif.), with Ca ++ and Mg ++ ), and resuspended in chemotaxis buffer at 5 ⁇ 10 6 cells/ml.
• HBSS Hank's balanced salt solution
• DPBS Dulbecco's phosphate buffered saline
• cells that have migrated are quantified by cell staining, such as the Hema3 staining kit (Fisher Scientific; Tustin, Calif.) or the CyQuant assay (Molecular Probes; Eugene, Oreg.), a fluorescent dye method that measures nucleic acid content and microscopic observation.
• the lower chamber is inspected microscopically to determine if any cells have migrated into the wells. If significant number of cells is present in the wells, quantification is done in the wells as well as the filter. The magnitude of migration is calculated as the ratio of absorbance between the wells with chemoattractants and the wells with chemotaxis buffer alone.
• mice are injected either subcutaneously, intradermally, intranasally, or by any other mode with varying doses of the virus, bacterium, or parasite under study, using a typical immunization schedule, e.g., days 0, 7, and 14, in the presence or absence of APC chemotaxin given simultaneously with the microorganism in an appropriate formulation which may include adjuvants.
• Serum and/or mucosal secretions are collected on days ⁇ 7, 0, 7, 14, 21, 28 and 35 for antigen-specific antibody analysis by ELISA.
• Mice are sacrificed at different time intervals (such as after the last immunization to quantitate the antigen-specific antibody-forming cells and antigen-specific T cell responses (both cytotoxic and helper T cell populations)) present in immune compartments, using standard procedures.
• mice While many tumor cells express unique tumor-associated antigens, these antigens are invariably weak immunogens and fail to generate potent anti-tumor immunity during tumor progression.
• APC chemotaxins such as SHAAYtide (SEQ ID NO:4)
• SEQ ID NO:4 APC chemotaxins
• EL4 cells American Type Tissue Collection (ATTC); Manassas, Va.; no. TIB-39
• SEQ experimental protein antigen OVA
• CRL-2113 dry OVA EL4 transfectants
• OVA EL4 transfectants OVA EL4 transfectants
• the tumor grows and eventually kills the mouse.
• Animals can be at least partially protected by vaccinating them with OVA formulated in adjuvant to induce an antigen-specific immune response directed against the OVA-transfected thymoma cells (REF?).
• This model is effective to evaluate the relative efficacy of adjuvants in augmenting or modulating protective anti-tumor immunity.
• Positive controls in this model include the following adjuvants: CFA, IFA, alum and GM-CSF.
• the ability of APC chemotaxins to augment cancer immunotherapy regimens can be evaluated by comparison to these known adjuvants.
• An animal model of asthma can be induced by sensitizing rodents to an experimental antigen (e.g., OVA) by standard immunization, and then subsequently introducing that same antigen into the rodent's lung by aerosolization.
• OVA experimental antigen
• Three series of rodent groups, comprising 10 rodents per group, are actively sensitized on Day 0 by a single intraperitoneal injection with 100 ⁇ g OVA in phosphate-buffered saline (PBS), along with an IgE-selective adjuvant, such as aluminum hydroxide (“alum” adjuvant).
• PBS phosphate-buffered saline
• IgE-selective adjuvant such as aluminum hydroxide (“alum” adjuvant
• mice At 11 days after sensitization at the peak of the IgE response, the animals are placed in a Plexiglas chamber and challenged with aerosolized OVA (1%) for 30 minutes using an ultrasonic nebulizer (De Vilbliss Co.; Somerset, Pa.).
• An ultrasonic nebulizer (De Vilbliss Co.; Somerset, Pa.).
• One series of mice additionally receives phosphate buffered saline (PBS) and Tween 0.5% intraperitoneally at the initial sensitization, and at different dosing schedules thereafter, up until the aerosolized OVA challenge.
• PBS phosphate buffered saline
• a second series consists of groups of mice receiving different doses of APC chemotaxins given either intraperitoneally, intra-venously, sub-cutaneously, intramuscularly, orally, or via any other mode of administration, at the initial sensitization, and at different dosing schedules thereafter, up until the aerosolized OVA challenge.
• a third series of mice, serving as a positive control consists of groups treated with either mouse IL-10 intraperitoneally, anti-IL4 antibodies intraperitoneally, or anti-IL5 antibodies intraperitoneally at the initial sensitization, and at different dosing schedules thereafter, up until the aerosolized OVA challenge.
• 6-C-kine a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3 ⁇ receptor CCR7. J Cell Biol. 141:1053-9.
• Oligonucleotide-directed mutagenesis a simple method using two oligonucleotide primers and a single-stranded DNA template. Methods Enzymol. 154:329-50.

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