Patent Application
20150140065
This application contains a “Sequence Listing” submitted as an electronic .txt file named “CS_ST25.txt.” The subject matter of the “Sequence Listing” is incorporated herein by reference along with the subject matter of International Publication Number WO 2010/120897 A1 (International Application Number PCT/US2010/031054) and U.S. Patent Application 61/490,050, 61/538,427 and 61/490,056.
The invention generally relates to methods for preventing or treating a disease by generating or modulating an immune response with the use of specific peptide constructs. In one embodiment, an immunomodulatory Peptide J, DLLKNGERIEKVE (SEQ ID No. 3), is part of a peptide construct to induce an antigen-specific maturation of immune cells from a mammal subject. The immunomodulator Peptide J can be linked to antigen epitopes of infectious diseases such as Type A influenza viruses (H1N1, H5N1, H3N2, etc.), including influenza viruses that originate in “swine,” “avian” or “bird” species, to provide for a method of treatment or prevention of influenza viral diseases. In other embodiments, for example, the immunomodulator Peptide J can be linked to antigen epitopes of infectious diseases such as Type such as Herpes simplex virus or other RNA or DNA single- or double-stranded viruses, bacteria, rickettsia or parasites to provide for a method of treatment or prevention.
Influenza is a common infectious disease brought about by an RNA virus of the same name. While vaccines for influenza have been available, strains of influenza evolve readily and novel viruses emerge from various species (e.g. bird to human transfer) requiring new vaccines to be provided on seasonal or sporadic basis. Similarly, other disease causing agents including various viruses such as HIV, HTLV viruses and others evolve readily and novel viruses emerge from various species (e.g. bird, rodents, livestock and other primate-to-human transfer) requiring new vaccines to be provided on seasonal or sporadic basis. Whole virus vaccines, split virus vaccines, surface antigen vaccines and live attenuated virus vaccines are available for influenza. However, presently available vaccines require constant updating because of 1) mutations, 2) re-assortment of genes between various strains, and 3) the continual emergence (or re-emergence) of different strains.
Common vaccines for influenza are designed to induce an immune response that is directed at the so-called protective antigens, hemagglutinin (HA or H) and neuramindase (NA or N), and the vaccines induce strain specific immunity.
Each year, numerous individuals are infected with different strains and types of influenza virus. Oftentimes, complications from influenza infection lead to protracted illness or death. Infants, the elderly, those without adequate health care and immuno-compromised persons are particularly vulnerable to complications arising from influenza infections. In some cases, otherwise healthy adults are at risk for severe complications including death from influenza infection. The 1918 Spanish influenza, which was genetically related to the 2009 pandemic H1N1 influenza, had the propensity for affecting individuals with healthy immune systems. The H5N1 influenza virus is also believed to have a heightened risk for affecting individuals with healthy immune systems, which may result in a cytokine storm (hypercytokimemia). A cytokine storm is caused by excessive amounts of pro-inflammatory cytokines and tends to occur in patients with stronger, “robust,” immune systems and leads to an increased risk for death in otherwise healthy adults. There is a need for a formulation and a method of vaccination and/or treatment to combat a deadly pandemic and to protect against new strains of Type A influenza that may present an elevated risk for even those with healthy immune systems. Emergent influenza viruses can be the most deadly for people in their prime, rather than affecting only the very young, the very old, or the most severely immuno-compromised.
Appropriate formulations of peptide constructs can stimulate and produce a systemic immune response. Peptide construct technology has provided the ability to produce vaccines using genetic engineering (recombinant vaccines). Such vaccines are typically created using antigenic moieties of the newly emergent virus strains when polypeptides and polynucleotides of novel, newly emergent, or newly re-emergent virus strains are desired. The focus on most current vaccines is not on conserved proteins and, especially, essential regions of such conserved proteins.
Peptides and compositions for use in treatment for Type A influenza infections and other infectious diseases are disclosed, including the treatment of HSV I, II, EBV, VZV, CMV, KHSV (HSV-VIII), HTLV-I, HTLV-II, HBV, RSV, HPV, TB, and the causative agent of Lyme disease. Peptides and compositions disclosed herein are competent for the ex vivo treatment of immune cells for maturation and/or activation of immunity infection as well as methods for the use of such matured immune cells for the prevention and treatment of many of these agents and related diseases. The peptide constructs disclosed herein are based on a Ligand Epitope Antigen Presentation System (LEAPS™) technology that can convert small peptides, which typically do not elicit strong and protective immune responses, into immunogens that do elicit an immune response.
In certain embodiments, peptides and compositions disclosed herein are competent for the ex vivo treatment of immune cells for maturation and/or activation of immunity against Type A influenza infection as well as methods for the use of such matured immune cells for the prevention and treatment of Type A influenza infections. The peptide constructs disclosed herein are based on a Ligand Epitope Antigen Presentation System (LEAPS™) technology that can convert small peptides that typically do not elicit strong and protective immune responses, into immunogens that do elicit an immune response.
In certain embodiments, the novel heteroconjugates or peptide constructs disclosed herein are based upon highly-conserved sequences common to various strains of Type A Influenza viruses (H1N1, H5N1, H3N2, etc.), including “swine,” “avian” or “bird,” and “Spanish Influenza,” in order to minimize the chance of insufficient immunity due to mutation. As such, the heteroconjugates or peptide constructs provide immunity to more than one sub-type and/or strain of type A influenza virus. That is, the novel heteroconjugates or peptide constructs promote immune recognition of antigens and/or epitopes common to different sub-types and strains of Type A influenza viruses and common between different Type A influenza viruses.
In certain embodiments, a composition contains one or more heteroconjugates or peptide constructs or dendritic cells treated with one or more heteroconjugates or peptide constructs for treatment or immunization to several or multiple subtypes of type A influenza.
In certain embodiments, a composition containing one or more heteroconjugates or peptide constructs or dendritic cells treated with one or more heteroconjugates or peptide constructs for treatment or immunization to several or multiple subtypes of type A influenza.
In certain embodiments, a composition containing one or more heteroconjugates or peptide constructs or dendritic cells treated with one or more heteroconjugates or peptide constructs acts as a multi-strain influenza vaccine or as a treatment for more than one influenza sub-type and/or strain.
In certain embodiments, a composition containing one or more heteroconjugates or peptide constructs or dendritic cells treated with one or more heteroconjugates or peptide constructs acts as a multi-subtype influenza vaccine or as a treatment more than one influenza sub-type and/or strain.
In certain embodiments, a composition for use as a treatment or as an influenza vaccine for more than one sub-type and/or strain of type A influenza virus contains from about 1 to about 10 or less peptide conjugates containing an immune cell binding ligand (ICBL) as described herein. In other embodiments, a composition for use as a treatment or as an influenza vaccine for more than one sub-type and/or strain of type A influenza virus contains from about 3 to about 5 or less peptide conjugates containing an ICBL as described herein. In further embodiments, a composition for use as a treatment or as an influenza vaccine for more than one sub-type and/or strain of type A influenza virus contains from about 3 to about 10 or less peptide conjugates containing an ICBL as described herein.
In certain embodiments, peptide heteroconjugates having an immunomodulatory effect for influenza infection include the peptide heteroconjugate DLLKNGERIEKVEGGGNDATYQRTRALVRTG (SEQ ID No. 1), containing two elements of the LEAPS™ heteroconjugate construct, namely an immune cell binding ligand (ICBL) Peptide J, DLLKNGERIEKVE (SEQ ID No. 3) linked to a peptide derived from the nucleoprotein (NP) of the Type A influenza virus NDATYQRTRALVRTG (SEQ ID No. 7). Another LEAPS™ peptide heteroconjugate having immunomodulatory effect for influenza infection is the peptide construct DLLKNGERIEKVEGGGSLLTEVETPIRNEWGCRCNDSSD (SEQ ID No. 2), containing two elements of the LEAPS™ heteroconjugate construct, namely a ICBL Peptide J, DLLKNGERIEKVE (SEQ ID No. 3) linked to a peptide derived from the matrix 2 ectodomain (M2e) of the A virus SLLTEVETPIRNEWGCRCNDSSD (SEQ ID No. 8). Additional peptide constructs having immunomodulatory effects are disclosed.
In certain embodiments, a peptide construct for directing an immune response against various infectious disease causing agents or for maturing dendritic cells from immature dendritic cells to a more matured and now activated dendritic cell are provided. The peptide has the formula P1-x-P2 or P2-x-P1, where P2 represents a specific antigenic peptide, P1 represents an immunomodulatory peptide, which is a portion of an immunoprotein capable of promoting binding to a class or subclass of immune cells, and x represents a covalent bond or a divalent linking group.
In certain embodiments, a composition having a population of matured dendritic cells is provided. The population of matured dendritic cells is formed by treating immature dendritic cells or monocytes with an effective amount of a peptide construct having the formula P1-x-P2 or P2-x-P1 under conditions suitable for maturation of the cells to form the matured or effective dendritic cells which interacts with T cells, where P2 represents a specific antigenic peptide, P1 represents an immunomodulatory peptide which is a portion of an immunoprotein capable of promoting binding to a class or subclass of DC or T cells, and -x- represents a covalent bond or a divalent linking group.
In certain embodiments, a peptide for directing an immune response against Type A influenza virus or for maturing dendritic cells from immature dendritic cells to a more matured and now activated dendritic cell is provided. The peptide has the formula P1-x-P2 or P2-x-P1 where P2 represents a specific antigenic peptide derived from a Type A influenza virus, P1 represents an immunomodulatory peptide which is a portion of an immunoprotein capable of promoting binding to a class or subclass of immune cells, and x represents a covalent bond or a divalent linking group.
In certain embodiments, a composition comprising a population of matured dendritic cells is provided. The population of matured dendritic cells is formed by treating immature dendritic cells or monocytes with an effective amount of a peptide construct having the formula P1-x-P2 or P2-x-P1 under conditions suitable for maturation of the cells to form the matured or effective dendritic cells which interacts with T cells, where P2 represents a specific antigenic peptide derived from an influenza virus, P1 represents an immunomodulatory peptide which is a portion of an immunoprotein capable of promoting binding to a class or subclass of DC or T cells, and -x- represents a covalent bond or a divalent linking group.
In certain embodiments, a method for targeting matured or effective dendritic cells to an infection site in a subject is provided. Immature dendritic cells or monocytes are treated with a peptide construct ex vivo under conditions suitable for maturation of the cells to form more matured or activated dendritic cells, and an effective amount of these matured dendritic cells are administered to the subject, wherein a majority of the dendritic cells administered to the subject locate to the infection site.
In certain embodiments, a therapeutic method of inducing an immune response in an animal subject infected with an infectious agent is provided by maturing dendritic cells (DC) by ex vivo treatment with an effective amount of a LEAPS™ heteroconjugate construct having immunomodulatory effect for infections or a mixture of LEAPS™ heteroconjugates having immunomodulatory effect when administered to a subject. The matured dendritic cells can then be transferred to an animal subject to confer immunity to an infectious agent or to treat an on-going viral, bacterial, parasitic or rickettsial infection. In certain embodiments, the LEAPS™ heteroconjugate can be administered to a subject to confer active immunity to infections in a manner similar to traditional vaccines containing attenuated virus, live virus, or viral proteins or protein fragments, wherein the LEAPS™ heteroconjugate is administered direct to a subject with or without an optional adjuvant.
In certain embodiments, therapeutic method of inducing an immune response in an animal subject infected with Type A influenza virus is provided by maturing dendritic cells (DC) by ex vivo treatment with an effective amount of a LEAPS™ heteroconjugate construct having immunomodulatory effect for influenza infection or a mixture of LEAPS™ heteroconjugates having immunomodulatory effect for influenza infection. The matured dendritic cells can then be transferred to an animal subject to confer immunity to Type A influenza virus or to treat an ongoing influenza infection. In certain embodiments, the LEAPS™ heteroconjugate can be administered to a subject to confer active immunity to influenza infections in a manner similar to traditional influenza vaccines containing attenuated virus, live virus, or viral proteins or protein fragments, wherein the LEAPS™ heteroconjugate is administered direct to a subject with or without an optional adjuvant.
In certain embodiments, a method for modulating a response to an infection in a subject in need thereof is provided by combining dendritic cells (DCs) and/or monocytes, that can be derived from an infected subject or a suitably matched donor, with a LEAPS™ heteroconjugate having immunomodulatory effect for the infection ex vivo to form a mixture, and administering the mixture to the subject. A method for modulating a response to an infection in a subject is provided by treating isolating DCs and/or monocytes from blood derived monocytes and/or bone marrow taken from the subject with a LEAPS™ heteroconjugate to induce maturation of the DCs and/or monocytes into matured DCs and administering, optionally without any supplementary immunomodulators, an effective amount of the treated matured DCs back into the subject.
In certain embodiments, method for modulating a response to Type A influenza virus in a subject in need thereof is provided by combining dendritic cells (DCs) and/or monocytes, that can be derived from an influenza infected subject or a suitably matched donor, with a heteroconjugate having immunomodulator effect for influenza virus ex vivo to form a mixture and administering the mixture to the subject. A method for modulating a response to Type A influenza virus in an infected subject is provided by treating isolated DCs and/or monocytes from blood derived monocytes and/or bone marrow taken from the subject with a heteroconjugate to induce maturation of the DCs and/or monocytes into matured DCs and administering, optionally without any supplementary immunomodulators, an effective amount of the treated matured DCs back into the subject.
In certain embodiments, additional heteroconjugates having immunomodulatory effect for influenza virus include, in addition to the heteroconjugates SEQ ID No. 1 or SEQ ID No. 2, heteroconjugates including the Peptide J (SEQ ID No. 3), Peptide G (SEQ ID No. 6) or CEL-1000 (SEQ ID No. 4) sequences conjugated with HA2 core 1, GLFGAIAGFIEGG (SEQ ID No. 10) or HA2 core 2, LKSTQNAIDEITNKVN (SEQ ID No. 9). A heteroconjugate of Peptide J (SEQ ID No. 3) and HA2 core 1, GLFGAIAGFIEGG (SEQ ID No. 10), with a spacer GGG, is DLLKNGERIEKVEGGGGLFGAIAGFIEGG (SEQ ID No. 12). A heteroconjugate of Peptide J (SEQ ID No. 3) and HA2 core 2, LKSTQNAIDEITNKVN (SEQ ID No. 9), with a spacer GGG, is DLLKNGERIEKVEGGGLKSTQNAIDEITNKVN (SEQ ID No. 11). Any of the heteroconjugate can optionally be combined with an adjuvant for in vivo use by administration into a living animal or human.
In certain embodiments, a tracking marker or a therapeutic agent is conjugated to an antibody having affinity for any one of MHC II, CD11c, DEC-205, Dectin-1, DC-SIGN, and DC-LAMP.
In certain embodiments, a peptide conjugate or an dendritic cell is conjugated to a therapeutic agent selected from the group consisting of reverse transcriptase inhibitors, portmanteau inhibitors, integrase inhibitors, protease inhibitors, entry inhibitors, CCR5 receptor antagonists, maturation inhibitors, agents from the ARV NRTI group, penicillins, cephalosporins, vancomycin, tetracyclines, macrolides, chloramphenicol, clindamycin, spectinomycin, sulfonamides, DNA-gyrase inhibitors, antimycobacterial agents, protein synthesis inhibitors, mefloquine, doxycycline, atovaquone, proguanil hydrochloride, proguanil, quinacrine, chloroquine, primaquine, amoxicillin, penicillin G, and clarithromycin.
In certain embodiments, matured dendritic cells exhibit an upregulation of one or more of CD80, CD86 and Major Histocompatibility Complex II relative to immature dendritic cells or monocytes not contacted with the peptide construct.
In certain embodiments, matured dendritic cells are isolated away from bone marrow or blood tissues.
In certain embodiments, matured dendritic cells produce an increased amount of Interleukin 12p70 (IL-12p70) compared to immature dendritic cells or monocytes not contacted with the peptide construct.
In certain embodiments, a therapeutic agent or a tracking marker is conjugated to a peptide conjugate the peptide construct is conjugated to the therapeutic agent by a cathepsin cleavable valine-citrulline dipeptide linker or by linking with a cysteine or lysine residue of the peptide construct by conjugation to a group selected from OH groups, COOH groups, amine groups, and amide groups of the peptide construct.
In certain embodiments, a peptide construct is conjugated to a lysosmatropic agent.
In certain embodiments, a peptide for directing an immune response against an infection or for maturing dendritic cells is a peptide construct selected from the group consisting of SEQ ID No.'s 140-189 and 209-218 or a variant thereof.
In certain embodiments, a composition containing matured dendritic cells is provided. The matured dendritic cells are formed by contacting immature dendritic cells or monocytes with an effective amount of a peptide construct having the formula P1-x-P2 or P2-x-P1 under conditions suitable for maturation of the immature dendritic cells to form the matured dendritic cells, wherein P2 represents a specific antigenic peptide derived from infectious, viral, bacterial, parasitic disease causing agent; P1 represents an immunomodulatory peptide which is a portion of an immunoprotein capable of promoting binding to a class or subclass of dendritic cells; and x represents a covalent bond or a divalent linking group.
In certain embodiments, a composition containing a population of matured dendritic cells is provided. The matured dendritic cells formed are by contacting immature dendritic cells or monocytes with an effective amount of a peptide construct selected from the group consisting of SEQ ID No.'s 140-189 and 209-218 or a variant thereof under conditions suitable for maturation of the dendritic cells or monocytes.
In certain embodiments, a method for inducing a systemic immune response to an infection includes administering an immunologically effective amount of a peptide construct selected from the group consisting of SEQ ID No.'s 140-189 and 209-218 or a variant thereof to a subject.
In certain embodiments, a method for producing a matured dendritic cell population is performed by contacting or treating immature dendritic cells or monocytes with an effective amount of a peptide construct having the formula P1-X-P2 or P2-X-P1 under conditions suitable for maturation of dendritic cells or monocytes to form matured dendritic cells, wherein P2 represents a specific antigenic peptide derived from infectious, viral, bacterial, parasitic disease causing agent; P1 represents an immunomodulatory peptide which is a portion of an immunoprotein capable of promoting binding to a class or subclass of dendritic cells; and x represents a covalent bond or a divalent peptide linking group.
In certain embodiments, an immune response in a subject is modulated or a subject is vaccinated by contacting immature dendritic cells or monocytes with a peptide construct having the formula P1-x-P2 or P2-x-P1 under conditions suitable for maturation of the cells to form matured dendritic cells and administering an effective amount of the matured dendritic cells to the subject. In the peptide construct, P2 represents a specific antigenic peptide derived from infectious, viral, bacterial, parasitic disease causing agent; P1 represents an immunomodulatory peptide which is a portion of an immunoprotein capable of promoting binding to a class or subclass of dendritic cells; and x represents a covalent bond or a divalent peptide linking group.
In certain embodiments, the peptide construct having the formula P1-x-P2 or P2-x-P1 has a peptide P1 selected from the group consisting of SEQ ID No.'s 3-6 and 40 or variants thereof.
In certain embodiments, the peptide construct having the formula P1-x-P2 has a peptide P2 selected from one of the following groups: the group consisting of SEQ ID No.'s 7-10 and 41-46; the group consisting of SEQ ID No.'s 53-56; the group consisting of SEQ ID No.'s 57-60; the group consisting of SEQ ID No.'s 61-64; the group consisting of SEQ ID No.'s 65-66; the group consisting of SEQ ID No.'s 67-68; the group consisting of SEQ ID No.'s 69-70; the group consisting of SEQ ID No.'s 71-72; the group consisting of SEQ ID No.'s 73-74; the group consisting of SEQ ID No.'s 75-80; the group consisting of SEQ ID No.'s 81-82; the group consisting of SEQ ID No.'s 83-86; the group consisting of SEQ ID No.'s 87-90; the group consisting of SEQ ID No.'s 91-99; the group consisting of SEQ ID No.'s 100-114; the group consisting of SEQ ID No.'s 115-120; the group consisting of SEQ ID No.'s 121-124; the group consisting of SEQ ID No.'s 125-126; the group consisting of SEQ ID No.'s 128-129; SEQ ID No. 127; the group consisting of SEQ ID No.'s 131-133; the group consisting of SEQ ID No.'s 194-195; the group consisting of SEQ ID No.'s 135-136; the group consisting of SEQ ID No.'s 137-138; SEQ ID No. 139; the group consisting of SEQ ID No.'s 196-204; and the group consisting of SEQ ID No.'s 205-208, or variants of any of the foregoing sequences.
In certain embodiments, the peptide construct is selected from one of the following groups: the group consisting of SEQ ID No.'s 1-2, 11-36, 47-52; the group consisting of SEQ ID No.'s 140-141; the group consisting of SEQ ID No.'s 142-143; the group consisting of SEQ ID No.'s 144-146; SEQ ID No. 147; SEQ ID No. 148; SEQ ID No. 149; SEQ ID No. 150; SEQ ID No. 151; the group consisting of SEQ ID No.'s 152-154; SEQ ID No. 155; the group consisting of SEQ ID No.'s 156-157; the group consisting of SEQ ID No.'s 158-159; the group consisting of SEQ ID No.'s 160-165; the group consisting of SEQ ID No.'s 166-173; the group consisting of SEQ ID No.'s 174-176; the group consisting of SEQ ID No.'s 177-178; SEQ ID No. 179; SEQ ID No. 180; the group consisting of SEQ ID No.'s 181-182; SEQ ID No. 183; SEQ ID No. 184-185 and 209-210; and SEQ ID No. 186 and 211; SEQ ID No. 187; SEQ ID No. 188; SEQ ID No. 189; the group consisting of SEQ ID No.'s 212-216; and the group consisting of SEQ ID No.'s 217-217, or variants of any of the foregoing sequences.
One of ordinary skill in the art will appreciate that other aspects of this invention will become apparent upon reference to the following detailed description.
The present invention provides LEAPS™ peptide conjugates useful for treatment or prevention of various infections. The novel heteroconjugates disclosed herein are based upon conserved, non-changing epitope sequences common to various strains of viruses, bacteria, rickettsia or parasites, as applicable, in order to minimize the chance of insufficient immunity due to mutation and minor strain variations. The use of LEAPS™ vaccine technology for immunization in animal models has been shown to provide protection from viral diseases without causing an immune response associated with the deadly “cytokine-storm” seen in some of the victims of viral infections in particular. The present invention also provides new approaches for detection and/or treatment that are suitable for use in treatment as well as in research, diagnostics, etc. Numerous other benefits will become apparent upon review of the following.
The present invention provides LEAPS™ peptide conjugates useful for treatment of Type A influenza. The novel heteroconjugates disclosed herein are based upon conserved, non-changing epitope sequences common to various strains of Type A Influenza viruses (H1N1, H5N1, H3N2, etc.), including “swine,” “avian” or “bird,” and “Spanish Influenza,” in order to minimize the chance of insufficient immunity due to mutation. The use of LEAPS™ vaccine technology for immunization in animal models has been shown to provide protection from viral diseases without causing an immune response associated with the deadly “cytokine-storm” seen in some of the victims of influenza. The present invention also provides new and/or newly isolated influenza hemagglutinin and neuraminidase fragments that are capable of use in production of numerous types of vaccines as well as in research, diagnostics, etc. Numerous other benefits will become apparent upon review of the following.
The present invention provides LEAPS™ peptide heteroconjugates useful for treatment of diseases such as influenza, HSV, other viruses, bacteria, rickettsia or parasitic infections and localization of these LEAPS™ heteroconjugate-activated DCs at the site of the ongoing disease or infection whether visualized by labeling with CFSE, a radioactive label such as 131I or 125I, some other visualization means or as unlabeled DCs. The novel heteroconjugates disclosed herein are based upon conserved, non-changing epitope sequences common to the disease causing organism or virus (pathogen) or protein (or peptide) and are often or usually essential for its existence.
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the relevant art.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “adjuvant” refers to substance that accelerates, prolongs or enhances antigen-specific immune responses when used in combination with vaccine antigens.
The terms “administering,” “administer,” “delivering,” “deliver,” “introducing,” and “introduce” can be used interchangeably to indicate the introduction of a therapeutic or diagnostic agent into the body of a patient in need thereof to treat a disease or condition, and can further mean the introduction of any agent into the body for any purpose.
The term “antigen” refers to a substance or molecule that generates an immune response when introduced to the body or any molecule or fragment thereof now also refers to any molecule or molecular fragment that can be bound by a major histocompatibility complex (MHC).
The term “blood tissue” refers to cells suspended in or in contact with plasma.
The term “bone marrow cell” refers to any cell originating from the interior of bones.
The terms “CD80,” “CD86,” “CD11c, “CD85” and similar terms refer to cell surface molecules present on leukocyte cells through a nomenclature protocol maintained by Human Cell Differentiation Molecules (www.hcdm.org; Paris, France).
The term “comprising” includes the recited steps, elements, structures or compositions of matter and does not exclude any un-recited elements, structures or compositions of matter.
The term “consisting of” includes and is limited to whatever follows the phrase the phrase “consisting of.” Thus, the phrase indicates that the limited elements are required or mandatory and that no other elements may be present.
The phrase “consisting essentially of” includes any elements listed after the phrase and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase indicates that the listed elements are required or mandatory but that other elements are optional and may or may not be present, depending upon whether or not they affect the activity or action of the listed elements.
A “dendritic cell” or “DC” refers to an antigen-presenting leukocyte that is found in the skin, mucosa, and lymphoid tissues and having a capability under appropriate conditions to initiate a primary immune response by activating T cells, lymphocytes and/or secreting cytokines.
The term “diagnostic” refers to any technique for determining the presence of any influenza viral infection or antigen in a subject.
The term “divalent linker” refers to any moiety having a structure forming a peptide bond to a first peptide moiety and forming a second bond to a second peptide moiety.
The term “effective amount” is an amount of a therapeutic which produces a therapeutic response, including an immune response, in the subject to which the therapeutic is administered.
The term “ex vivo” refers to an operation or procedure that is performed outside of the body of a patient or subject to be treated for an influenza viral disease. For example, an ex vivo procedure can be performed on living cells originating from the patient, subject or donor removed from the body.
The term “autologous” refers to a situation where the donor and recipient of cells, fluids or other biological sample or material is the same individual.
The term “homologous” refers to a situation where the donor are recipient of cells, fluids or other biological sample or material are not the same individual.
The term “Herpes simplex virus 1 and 2” (HSV-1 and HSV-2) refers to members of the family Herpesviridae of double-stranded DNA viruses. HSV-1 is associated with producing cold sores in humans and HSV-2 is associated with producing genital herpes.
The term “human T-lymphotropic virus Type I” (HTLV-1) refers to a human RNA retrovirus that causes T-cell leukemia and T-cell lymphoma in humans and other primates.
The terms “conjugate,” “conjugation” and similar terms refer to two species being spatially associated with each other by covalent linkage, non-covalent binding or by a combination of covalent linkage and non-covalent binding. For example, an antibody can be conjugated to an epitope through non-covalent binding to the epitope as well as the antibody serving to conjugate the epitope (such as a cell surface marker) to a compound that is linked to the antibody.
An “immature dendritic cell” is a “dendritic cell” in a state characteristic of immune cells prior to contact with an antigen and having a limited present ability to activate T cells, lymphocytes and/or to secrete cytokines; however, “immature dendritic cells” may acquire the ability to activate T cells, lymphocytes and secrete cytokines upon contact with an antigen.
The terms “immunomodulatory” and “immunoprotein” refer to a protein, peptide or cell having the ability to bind or interact with an immune cell to alter or to regulate one or more immune functions.
The term “infection” refers to the colonization in a host organism by a pathogenic influenza virus.
The term “Influenza virus” refers to an RNA virus from the Orthomyxoviridae family.
The term “influenza subtype” means an influenza virus having a specific sub-type of hemagglutinin protein (H) on the viral envelope and a specific sub-type of neuraminidase (N) as classified by the Centers for Disease Control and Prevention (Atlanta, Ga.).
The term “strain” as it relates to influenza virus refers to an influenza virus having a specific strain number as classified by the Centers for Disease Control and Prevention (Atlanta, Ga.), wherein the strain number can include an identifier that incorporates information or identification of the site or location where the specific strain was found, features of the sub-types of the hemagglutinin (H) and neuraminidase (N) proteins and the year of isolation or passage.
The term “multi-strain influenza vaccine” means a vaccine that is active in conferring immunity to multiple (usually only 3 to 4 on 1-9 different H and 1-9 different N proteins) strains of type A influenza virus that have accumulated genetic drift between strains. For example, a multi-strain influenza vaccine may, in some embodiments, confer immunity to influenza strains having 3 or 4 or more different hemagglutinin (H) and neuraminidase (N) protein sub-types as classified by the Centers for Disease Control and Prevention (Atlanta, Ga.).
The term “multi-subtype influenza vaccine” means a vaccine that is active in conferring immunity to multiple (usually only 3 to 4 on 1-9 different H and 1-9 different N proteins) strains of type A influenza virus that have accumulated genetic drift between strains. For example, a multi-strain influenza vaccine may, in some embodiments, confer immunity to influenza strains having 3 or 4 or more different hemagglutinin (H) and neuraminidase (N) protein sub-types as classified by the Centers for Disease Control and Prevention (Atlanta, Ga.).
The term “Interleukin 12p70” refers to a cytokine produced by dendritic cells capable of directing the development of lymphocytes in a Th1 immune response, and possessing two peptides of approximately 40 kd and 35 kd in size.
The terms “isolated matured dendritic cells” or “isolated dendritic cells” refer to dendritic cells suspended in a liquid medium, a cell culture or a composition wherein at least 50% of the viable cells present in the liquid medium, the cell culture or the composition are dendritic cells or monocytes.
An “isotype control” is an antibody having the same serological structure and can have a fluorescent conjugate dye as an antibody conjugate having affinity for a cellular surface or cytokine marker, except the isotype control does not have affinity for the cellular surface or cytokine marker.
A “heteroconjugate” refers to a protein or peptide containing at least two amino acid sequences covalently linked to form a single molecule, wherein two sequences originate or are homologous to proteins expressed by different genes.
The term “maturation” refers to a process for generating a “matured dendritic cell.”
The terms “matured dendritic cell,” “maturated dendritic cell,” “activated dendritic cell” or “effective dendritic cell” refer to a “dendritic cell” in a state characteristic of cells after contact with an antigen and having a present ability to initiate a primary immune response by activating T cells, lymphocytes and/or secreting cytokines.
The term “MDCK” refers to the Madin-Darby canine kidney epithelial cell line.
The term “monocyte” refers to immune cells produced by bone marrow and haematopoietic stem cell having the ability to differentiate into macrophages or dendritic cells.
The term “magnetic resonance imaging” refers to any technique where information is collected from the exposure of a subject or sample to a magnetic field.
The terms “H1N1,” “H5N1,” “H7N3,” “H9N2,” and similar terms refer to specific subtypes of influenza Type A virus, where the numeral after “H” designates a type of hemagglutinin protein on the viral envelope and the numeral after “N” designates a type of neuraminidase as classified by the Centers for Disease Control and Prevention (Atlanta, Ga.).
The terms “originating” and “derived” as related to a peptide sequence refers to an organism or cell type that produces a protein containing the peptide sequence.
The term “TCID50” refers to the median tissue culture infective dose of a pathogenic agent that produces pathological change in 50% of cell cultures inoculated.
The terms “peptide” and “peptide construct” refer to a molecule including two or more amino acid residues linked by a peptide bond. The term “peptide” includes molecular species where only part of the molecule has peptide character and/or where two parts of the molecular species formed of peptide bonds are covalently linked by a divalent linker.
The term “phenotype” as relating to the phenotype of immune cells refers to any observable characteristic or trait of a cell such as its morphology, development, biochemical or physiological properties including the expression or presence of specific cell surface proteins or markers.
The term “poliovirus” refers to a human enterovirus and member of the family Picornaviridae with a single-stranded, positive-sense RNA genome. Poliovirus is associated with causing poliomyelitis also known as infantile paralysis.
The term “prophylactic” or “prophylactically” refers to a method or use of a peptide, cells or biological matter in a manner to prevent the onset or occurrence of a disease or infection including use as a vaccine.
The term “red blood cells” refers to erythrocytes having an intact phospholipid bilayer membrane.
The term “rickettsia” refers to bacteria that are obligate intracellular parasites that have a cell well and are typically gram-negative.
The term “subject” or “patient” refers to an animal, including mice and humans, to which a therapeutic agent is administered.
The term “systemic immune response” refers to an immune response where antibodies, cytokines or immune cells generated by the immune response are detectable throughout the circulatory and lymph systems of the body.
The term “T cell” refers to a lymphocyte having a T cell receptor protein on the surface of the cell.
“Type A influenza virus” refers to an RNA virus from the Orthomyxoviridae family characterized by the presence of at least three membrane proteins on the viral envelope: hemagglutinin, Neuraminidase and M2 proton-selective ion channel protein.
The terms “treating” and “treatment” as related to treating or treatment of immune cells refers to bringing an immune cell into contact with a substance or composition for a time period sufficient to cause a change in phenotype. The term “vaccine” refers to composition containing one or more antigens that stimulates an immune response when administered to an organism in vivo.
The term “virus” refers to a small infectious agent that can replicate only inside the living cells of another organism or host through the use of some of the host's own cellular machinery (e.g. ribosomes) for growth and replication. Viruses outside of the host cells are formed from a nucleic acid with an associated protein coat. Structure of Immunomodulatory LEAPS™ Heteroconjugates
The peptide constructs disclosed herein are based on LEAPS™ technology and are conjugates of two peptides which are linked together covalently. The peptide constructs can be synthesized artificially using solid-phase synthesis or other synthetic technique or expressed using recombinant DNA technology. The two peptides can be synthesized separately and joined covalently or can be synthesized or expressed as a single construct. The LEAPS™ heteroconjugates are formed by joining a Peptide P1 and a Peptide P2 originating from different species by a linker “-x-,” such that the heteroconjugate has the structure P1-x-P2 or A first peptide (hereinafter may be referred to as Peptide P1) of the conjugate is a portion of an immunoprotein capable of promoting binding to a class or subclass of dendritic cells (DCs) or T cells and is referred to as an immune cell binding ligand (ICBL). Without wishing to be bound by any particular theory, it is believed that Peptide P1 has a structure for promoting interaction and/or binding with specific surface receptors present on DCs. Peptide P1 can be a peptide sequence derived from Major Histocompatibility Complex (MHC) I or II. A more detailed discussion of the Peptide P1 and peptide conjugates involved with LEAPS™ technology can be found in U.S. Pat. No. 5,652,342, which is incorporated herein by reference.
A second peptide (hereinafter may be referred to as Peptide P2) is a specific antigen peptide derived from a virus or infecting agent such as Type A influenza or other virus, parasite, bacteria or infectious agent. As described, the sequence of Peptide P2 is associated with sequences with a low-degree of variability between strains of viruses and can be identical to sequences present across several strains of the particular virus, bacteria, rickettsia, parasite, etc. In particular for Type A influenza virus, the sequence of Peptide P2 can be identical to sequences present across several strains of influenza virus, e.g. H1N1, H5N1, H7N3, H9N2, etc. Without wishing to be bound by any particular theory, it is believed that the antigen Peptide P2 being covalently bound to the ICBL Peptide P1 allows for a more effective recognition of the antigen Peptide P2 by the immune system and specific immune cells. Peptide epitopes having a limited number of amino acid residues have sufficient structure to be bound by an antibody or an MHC molecule with a high degree of specificity. However, peptide epitopes of limited size are less competent to cross-link immunoglobulins to cause lymphocyte activation and/or to be effectively displayed to T cells to stimulate cellular or humoral immune response. As such, small peptide epitopes introduced into a subject may produce a poor immune response. In the LEAPS™ heteroconjugates disclosed herein, the antigen Peptide P2 is covalently bound to ICBL Peptide P1 or other immunomodulatory peptide having the capability to bind to molecules present on the surface of dendritic cells or monocytes. Once bound to the surface of a dendritic cell, the antigen Peptide P2 can then be recognized by local T cell receptor (TCR) or Major Histocompatibility Complex (MHC) molecules to trigger a corresponding immune response and immune recognition of the antigen Peptide P2. Through such a mechanism, a latter challenge with a competent Type A influenza virus will generate a secondary immune response in a subject previously administered one or more of the peptide constructs disclosed herein. Further, the LEAPS™ heteroconjugates described herein can be used to stimulate an immune response and increase survivability in subjects having an active infection such as with influenza vim s.
In certain embodiments the Peptide P2 can be derived from disease causing organism or agents, for example, viruses, bacteria, rickettsia or parasites. Example disease causing organisms, viruses, parasites, etc. include adeno-virus, hepatitis C virus (HCV), hepatitis B virus (HBV), human papilloma virus (HPV), human T-lymphotropic virus 1 (HTLV-1), respiratory syncytial virus (RSV), vaccinia virus, West Nile virus (WNV), polyomavirus, human T-lymphotropic virus 2 (HTLV-2), cytomegalovirus (CVM), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (HSV-VIII), varicella zoster virus (VZV), herpes simplex 1 virus (HSV-1), herpes simplex 2 virus (HSV-2), poliovirus type 3 included strains P3/LEON/37 and PS/LEON/12A[1]B, human polio virus 1 Mahoney, tuberculosis, Lyme disease (caused by bacteria Borrelia burgdorferi), stomach diseases caused by bacteria Helicobacter pylori (H. pylori), Chlamydia, malaria, and Treponema pallidum.
In the LEAPS™ heteroconjugates disclosed herein, the antigen Peptide P2 is covalently bound to ICBL Peptide P1 or other immunomodulatory peptide having the capability to bind to molecules present on the surface of dendritic cells or monocytes. Once bound to the surface of a dendritic cell, the antigen Peptide P2 can then be recognized by local T cell receptor (TCR) or Major Histocompatibility Complex (MHC) molecules to trigger a corresponding immune response and immune recognition of the antigen Peptide P2. Through such a mechanism, a latter challenge with a disease-causing agent will generate a secondary immune response in a subject previously administered one or more of the peptide constructs disclosed herein. Further, the LEAPS™ heteroconjugates described herein can be used to stimulate an immune response and increase survivability in subjects having an active infection.
A further aspect of the LEAPS™ heteroconjugates disclosed herein is that the extent of pro-inflammatory or inflammatory cytokines produced during the immune response to the peptide constructs is reduced relative to levels typically associated with larger antigen proteins containing many different epitope sequences. Further, a Th1 type of immune response or a Th2 type of immune response may be promoted based upon the identity of the ICBL Peptide P1 conjugated with the antigen Peptide P2.
A further aspect of the LEAPS™ heteroconjugates disclosed herein is that the heteroconjugates can be treated or contacted with dendritic cells isolated from a subject or donor under conditions where the dendritic cells differentiate into more matured immune cells capable of directing immunity toward influenza virus. The matured dendritic cells increase resistance against influenza infection when administered to the subject. LEAPS™ Heteroconjugates
Specifically, the novel peptides of this invention include peptide constructs of the following Formulae (I) and (11):
P1-x-P2 (I)
P2-x-P1 (II)
where P2 is a peptide derived with Type A influenza, which will bind to an antigen receptor on a set or subset of dendritic cells or T cells; P2 is an immune response modifying peptide, which will cause a directed immune response by said set or subset of T cells or dendritic cells to which the peptide P1 is attached and initiate an immune response focused on IL-12 without or with low levels of pro-inflammatory or inflammatory cytokines (Patricia R Taylor; Christopher A Paustian, Gary K Koski, Daniel H Zimmerman, K S Rosenthal, Maturation of dendritic cell precursors into IL12 producing DCs by J-LEAPS, Cellular Immunology, 2010; 262:1-5; Taylor P R, G K Koski, C C Paustian, P A Cohen, F B-G Moore, D H Zimmerman, K S Rosenthal, J-L.E.A.P.S.™ Vaccines Initiate Murine Th1 Responses By Activating Dendritic Cells, Vaccine 2010; 28:5533-4, both of which are incorporated herein by reference). As shown in Formulae (I) and (II), the Peptide P1 can be N-terminal or C-terminal to the Peptide P2.
In certain embodiments, the Peptide P1 contains an ICBL termed “J” or “Peptide J.” Peptide J is derived from amino acids 38-50 from the β-2-microglobulin chain of the MHC I molecule (DLLKNGERIEKVE) (SEQ ID No. 3). ICBL Peptide J is believed to promote Th1-type immune responses to the coupled antigen P2 peptide, but is not limited to such activity.
In certain embodiments, the Peptide P1 of the peptide constructs contains an ICBL termed “CEL-1000” (DGQEEKAGVVSTGLI) (SEQ ID No. 4). The CEL-1000 peptide is derived from the β-chain of MHC II (MHC II (3134-148) and binds to murine as well as human CD4+ cells. The chemical structure of conjugated peptides containing CEL-1000 can have an amidated carboxyl terminal, (amino)-DGQEEKAGVVSTGLI-(amide) (SEQ ID No. 5). CEL-1000 can be prepared by F-MOC chemistry and purified by Reverse Phase (RP)-HPLC, analyzed by another RP-PLC system, ion exchange chromatography (IEC)-HPLC as well as mass spectroscopy. Based on site directed mutagenesis studies of MHC II β-chain and/or peptide competition studies, peptides such as CEL-1000, were shown to bind to CD4, a T cell co-stimulator molecule (Charoenvit et al., A small peptide derived from human MHC β2 chain induces complete protection against malaria in an antigen-independent manner, Antimicrobial Agents and Chemotherapy, July 2004; 48(7):2455-63; Cammarota et al., Identification of a CD4 binding site on the beta 2 domain of HLA-DR molecules, Nature, 1992; 356:799-801) and cell surface protein on some Dendritic Cell (DCs) (Konig, et al., MHC class II interaction with CD4 medicated by a region analogous to the MHC class I binding site for CD8, Nature, 1992; 356:796-798; Shen X. and Konig R., “Regulation of T cell immunity and tolerance in vivo by CD4”, Int. Immunol., 1998 10:247-57; Shen X. et al., Peptides corresponding to CD4-interacting regions of murine MHC class II molecules modulate immune responses of CD4+ T lymphocytes in vitro and in vivo, J Immunol., 1996; 157:87-100, all of which are incorporated herein by reference).
In certain embodiments, the Peptide P1 contains an ICBL termed “G” or “Peptide G.” Peptide G has the sequence NGQEEKAGVVSTGLI (SEQ ID No. 6) derived from the MHC-II beta 2 chain (Zimmerman et al., A new approach to T cell activation: natural and synthetic conjugates capable of activating T cells, 1996, Vacc. Res., 1996; 5:91, 5:102; Rosenthal et al., Immunization with a LEAPS™ heteroconjugate containing a CTL epitope and a peptide from beta-2-microglobulin elicits a protective and DTH response to herpes simplex virus type 1,1999, Vaccine, 1999; 17(6):535-542, both of which are incorporated herein by reference).
In certain embodiments, the Peptide P1 contains an ICBL termed “IL-1β” or “Peptide IL-1β.” Peptide IL-1β has the sequence VQGEESNDK (SEQ ID No. 40) derived from the human interleukin-1β chain (e.g., Bajpai et al., Immunomodulating activity of analogs of noninflammatory fragment 163-171 of human interleukin-lbeta 1998 Immunopharmacology, 38:237, incorporated herein by reference).
Novel epitope sequences that can serve as the antigen P2 peptide and conjugated with the ICBL Peptide P1 to form a LEAPS™ heteroconjugate will now be described. In certain embodiment, the Peptide P2 is derived from nucleoprotein of the Type A influenza virus (NP-A), NDATYQRTRALVRTG (SEQ ID No. 7). In certain embodiments, the Peptide P2 is derived from the matrix 2 ectodomain (M2e) of the A virus, SLLTEVETPIRNEWGCRCNDSSD (SEQ ID No. 8). In certain embodiments, the Peptide P2 is derived from the hemagglutinin monomer 2 core 1 protein (HA2 core 1), GLFGAIAGFIEGG (SEQ ID No. 10). In certain embodiments, the Peptide P2 is derived from the HA2 domain of the hemagglutinin protein (HA2 core 2), LKSTQNAIDEITNKVN (SEQ ID No. 9).
In certain embodiments, the novel epitope sequence is selected from one of SEQ ID No.'s 7-10. In some embodiments, the novel epitope sequences that can serve as the antigen P2 peptide and conjugated with the ICBL Peptide P1 to form a LEAPS™ heteroconjugate can be selected from YLEEHPSAGKDPKKTGGPIY (SEQ ID No. 41) from influenza nucleoprotein and TGTFEFTSFFYRYGFVANF (SEQ ID No. 43) from influenza polymerase protein 1 (PB1). In further embodiments, the novel epitope sequences that can serve as the antigen P2 peptide can be selected from AQNAISTTFPYTGDPPY (SEQ ID No. 42), VERLKHGTFGPVHFRNQVKI RR (SEQ ID No. 44), RNDDVDQSLI IAARNIVRRA (SEQ ID No. 45), HQLLRHFQKD AKVLF (SEQ ID No. 46). See Reference number 5 (Tan et al.) below.
A LEAPS™ heteroconjugate having immunomodulatory effects toward an infectious or other disease causing agent such as influenza virus is contemplated containing any combination of sequences selected from embodiments of Peptide P1 and Peptide P2 having the structure of one of Formulae (I) and (II), as described above. In Formulae (I) and (II), -x- represents a covalent bond or a divalent peptide linking group providing a covalent linkage between Peptide P1 and Peptide P2. In certain embodiments, -x- is a divalent peptide linking group having one or more glycine residues, such as the divalent linking group -GGG- or -GG-. In order to avoid synthesis of peptides having four glycine residues in a row, which may be difficult to synthesize, a linking group of -GG- can be used.
In certain embodiments, the divalent linking group is not limited to any particular identity so long as the linking group -x- serves to covalently attach the Peptide P1 and Peptide P2 as shown in Formulae (I) and (II). The linking group -x- can contain one or more amino acid residues or a bifunctional chemical linking group, such as, for example, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), m-maleimidobenzoyl-N-hydroxy-succimide ester (MBS), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). In certain embodiments, the linking group -x- can be a direct peptide or other covalent bond directly coupling Peptide P1 and Peptide P2. In certain embodiments where the linking group -x- contains amino acid residues, the linking group -x- can contain from 1 to about 5 amino acid residues or from 1 to about 3 amino residues. In certain embodiments, the linking group -x- can be cleavable or non-cleavable under physiological conditions.
The LEAPS™ heteroconjugates of Formulae (I) and (II) can be modified including modifications to the N- or C-terminal of the heteroconjugates. The LEAPS™ heteroconjugates described by Formulae (I) and (II) contain a sequence of amino acid residues consistent with the described Peptide P1 and Peptide P2. However, the N- or C-terminal of the described LEAPS™ heteroconjugates can be modified by any one or more of amidation or acylation, including myristoylation.
In certain embodiments, Peptides P1 and P2 include variants of any sequence disclosed herein. A variant is herein defined as a sequence wherein 1, 2, 3, 4 or 5 amino acid residues of sequence disclosed herein are replaced with a different amino acid residue without affecting the ability of the LEAPS™ heteroconjugates to stimulate an immune response against an infectious agent. In certain embodiment, variants have amino acid residues substituted in a conserved manner. In certain other embodiments, variants have amino acid residues substituted in a non-conserved manner. Variants include amino acid sequences where 1, 2, 3, 4 or 5 amino acid residues are deleted from the sequences and/or 1, 2, 3, 4 or 5 amino acid residues are added to the sequences. Variants include embodiments where combinations of conserved or non-conserved substitutions, additions and/or deletions are made to a sequence. In certain embodiments, Peptides P1 and P2 include variants of SEQ ID No.'s 1-36 and 41-52. A variant is herein defined as a sequence wherein 1, 2, 3, 4 or 5 amino acid residues of any of SEQ ID No.'s 1-36 and 41-52 or any other sequence disclosed herein are replaced with a different amino acid residue without affecting the ability of the LEAPS™ heteroconjugates to stimulate an immune response against Type A influenza virus. In certain embodiments, variants to SEQ ID No.'s 1-36 and 41-52 have amino acid residues substituted in a conserved manner. In certain other embodiments, variants to SEQ ID No.'s 1-36 and 41-52 or any other sequence disclosed herein have amino acid residues substituted in a non-conserved manner. Variants to SEQ ID No.'s 1-36 and 41-52 or any other sequence disclosed herein include amino acid sequences where 1, 2, 3, 4 or 5 amino acid residues are deleted from the sequences and/or 1, 2, 3, 4 or 5 amino acid residues are added to the sequences. Variants include embodiments where combinations of conserved or non-conserved substitutions, additions and/or deletions are made to a sequence.
A conserved substitution is a substitution where an amino acid residue is replaced with another amino acid residue having similar charge, polarity, hydrophobicity, chemical functionality, size and/or shape. Substitution of an amino acid residue in any of the following groups with an amino acid residue from the same group is considered to be a conserved substitution: 1) Ala and Gly; 2) Asp and Glu; 3) Ile, Leu, Val and Ala; 4) Lys, Arg and His; 5) Cys and Ser; 6) Phe, Trp and Tyr; 7) Phe and Pro; 8) Met and Nle (norleucine); 9) Asn and Gln; and 10) Thr and Ser.
Table 1 shows exemplary LEAPS™ heteroconjugates consistent with Formulae (I) and (II), including permutations of constructs of CEL-1000, Peptide J and Peptide G (Peptides P1) with the NP and M2e, HA2 core 1 and HA2 core 2 epitope peptides (Peptides P2), as described above. Those skilled in the art will recognize that other constructs can be formed substituting for Peptide P1 and Peptide P2 where the examples on Table 1 are merely illustrative and are not limiting.
Table 1 presents exemplary LEAPS™ heteroconjugates using antigens (Peptide P2) derived from Type A influenza virus. Table 2 presents antigen sequences, either as a core epitope or as an extended region, useful for making LEAPS™ heteroconjugates, along with other information and references about these epitopes. Further, LEAPS™ heteroconjugates incorporating an antigen sequence from Table 2, including antigens derived from Type A influenza, can be used to treat DCs to form matured DCs ex vivo, as will be described below. Such LEAPS™ heteroconjugate-treated DCs can be transferred to a subject to confer resistance, immunity, or to treat an active or acute infection caused by an influenza virus. Such LEAPS™ heteroconjugate-treated DCs also have the unexpected property to localize in a region of the body where a source of antigen originating from an influenza virus can be found.
Table 2 shows exemplary antigens described that can be employed as peptide P2 in certain embodiments. LEAPS™ heteroconjugates consistent with Formulae (I) and (II) can be formed by combining any permutation of ICBL peptide including, but not limited to, CEL-1000, Peptide J and/or Peptide G (Peptides P1) with an antigen peptide (Peptide P2) as presented in Table 2. Specifically, the first column of Table 2 lists the SEQ ID No. for the sequence presented in each row. The second column specifies the protein from which the individual amino acid sequences are derived. The third column gives the abbreviation for which the sequence presented in each row can be referred to. Also provided on Table 2 are example LEAPS™ heteroconjugates where Peptide P1 is Peptide J (SEQ ID No. 3) combined with an antigen Peptide P2. The fourth column specifies the core epitope sequence, if any, for the protein described in each row, and the fifth column specifies an extended epitope sequence associated with the protein described in each row. The sixth column indicates the range of amino acids from the described protein corresponding to the epitope sequence. The seventh column presents a non-limiting example LEAPS™ heteroconjugate containing Peptide J. The eighth column lists any know references describing the extended or core epitope sequences, if known. References are specified by a number corresponding to the list of references found at the end of this disclosure.
Table 3 shows exemplary LEAPS™ heteroconjugates consistent with Formulae (I) and (II), including exemplary LEAPS™ heteroconjugates formed by linking Peptide J to various infectious disease antigens in addition to Influenza virus. Those skilled in the art will recognize that other constructs can be formed substituting for Peptide P1 and Peptide P2 where the examples on Table 3 are merely illustrative and are not limiting.
Table 3 present antigen sequences, either as a core epitope or as an extended region, useful for making LEAPS™ heteroconjugates. The antigen sequences useful as serving as Peptide P2 in Formulae (I) and (II), described above, are derived from several viruses, bacteria and parasites including adeno-virus, hepatitis C virus (HCV), hepatitis B virus (HBV), human papilloma virus (HPV), human T-lymphotropic virus 1 (HTLV-1), respiratory syncytial virus (RSV), vaccinia virus, West Nile virus (WNV), polyomavirus, human T-lymphotropic virus 2 (HTLV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpes virus (HSV-VIII), varicella zoster virus (VZV), herpes simplex 1 virus (HSV-1), herpes simplex 2 virus (HSV-2), poliovirus type 3 included strains P3/LEOM/37 and PS/LEON/12A[1]B, human polio virus 1 Mahoney, tuberculosis, Lyme disease, Chlamydia, malaria, Helicobacter pylori (H. pylori) for stomach diseases and ulcers, and Borrelia burgdorferi (B. burgdorferi) causing Lyme disease and Treponema pallidum. The antigen sequences described on Table 3 are useful for forming LEAPS™ heteroconjugates that can confer increase resistance or immunity to the viruses, bacteria or parasites from which the specific antigen sequences originate, or can be used to treat an active or acute infection in a subject.
Further, LEAPS™ heteroconjugates incorporating an antigen sequence from Tables 1 and 3, including antigens can be used to treat DCs to form matured DCs ex vivo, as will be described below. Such LEAPS™ heteroconjugate-treated DCs can be transferred to a subject to confer resistance, immunity or to treat an active or acute infection caused by a virus, bacteria or parasite. Such LEAPS™ heteroconjugate-treated DCs also have the unexpected property to localize in a region of the body where a source of antigen originating from a virus, bacteria or parasite can be found. Many disease causing substances and organisms, including bacteria, can be present in the body in a quiescent phase or condition that does not cause any active symptoms. In particular, certain viruses can even be incorporated into the host genome with a period of years or decades between expression or have very low levels of antigen expression. For example, HSV-I, HSV-II, EBV, CMV, VZV, HSV-VI, HSV-VIII, polio, HTLV-I, HTLV-II, Human immunodeficiency virus (HIV), mumps, RSV and perhaps H. pylori and B. burgdorferi are suspected to have the capability to enter a long-term quiescent phase with the possibility of later emergence of symptoms or lower level of activity. As will be described in the Examples, LEAPS™ heteroconjugate-treated DCs have the capability to localize to location where sources of antigen can be found. As such, LEAPS™ heteroconjugate-treated DCs can be used to diagnose the presence of viruses that are in a quiescent phase and otherwise difficult to detect.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 53-56 to form a heteroconjugate capable of modulating an immune response to Adeno-virus or to mature DCs having the capability to modulate an immune response to Adeno-virus. Exemplary LEAPS™ heteroconjugates specific to Adeno-virus include, but are not limited to, SEQ ID No.'s 140-141.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 57-60 to form a heteroconjugate capable of modulating an immune response to hepatitis C virus or to mature DCs having the capability to modulate an immune response to hepatitis C virus. Exemplary LEAPS™ heteroconjugates specific to hepatitis C virus include, but are not limited to, SEQ ID No.'s 142-143.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 61-64 to form a heteroconjugate capable of modulating an immune response to hepatitis B virus or to mature DCs having the capability to modulate an immune response to hepatitis B virus. Exemplary LEAPS™ heteroconjugates specific to hepatitis B virus include, but are not limited to, SEQ ID No.'s 144-146.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 65-66 to form a heteroconjugate capable of modulating an immune response to human papilloma virus or to mature DCs having the capability to modulate an immune response to human papilloma virus. An exemplary LEAPS™ heteroconjugates specific to human papilloma virus includes, but is not limited to, SEQ ID No. 147.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 67-68 to form a heteroconjugate capable of modulating an immune response to HTLV-1 or to mature DCs having the capability to modulate an immune response to HTLV-1 virus. An Exemplary LEAPS™ heteroconjugates specific to HTLV-1 includes, but is not limited to, SEQ ID No. 148.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 69-70 to form a heteroconjugate capable of modulating an immune response to RSV or to mature DCs having the capability to modulate an immune response to RSV. An exemplary LEAPS™ heteroconjugate specific to RSV includes, but is not limited to, SEQ ID No. 149.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 71-72 to form a heteroconjugate capable of modulating an immune response to vaccinia virus or to mature DCs having the capability to modulate an immune response to vaccinia virus. An exemplary LEAPS™ heteroconjugate specific to vaccinia virus includes, but is not limited to, SEQ ID No. 150.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 73-74 to form a heteroconjugate capable of modulating an immune response to West Nile virus or to mature DCs having the capability to modulate an immune response to West Nile virus. An exemplary LEAPS™ heteroconjugate specific to West Nile virus includes, but is not limited to, SEQ ID No. 151.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 75-80 to form a heteroconjugate capable of modulating an immune response to polyomavirus or to mature DCs having the capability to modulate an immune response to polyomavirus. Exemplary LEAPS™ heteroconjugates specific to polyomavirus include, but are not limited to, SEQ ID No.'s 152-154.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 81-82 to form a heteroconjugate capable of modulating an immune response to HTLV-2 or to mature DCs having the capability to modulate an immune response to HTLV-2. An exemplary LEAPS™ heteroconjugate specific to HTLV-2 includes, but is not limited to, SEQ ID No. 155.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 83-86 to form a heteroconjugate capable of modulating an immune response to cytomegalovirus or to mature DCs having the capability to modulate an immune response to cytomegalovirus. Exemplary LEAPS™ heteroconjugates specific to cytomegalovirus include, but are not limited to, SEQ ID No.'s 156-157.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 87-90 to form a heteroconjugate capable of modulating an immune response to EBV or to mature DCs having the capability to modulate an immune response to EBV. Exemplary LEAPS™ heteroconjugates specific to EBV include, but are not limited to, SEQ ID No.'s 158-159.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 91-99 to form a heteroconjugate capable of modulating an immune response to HSV-VIII or to mature DCs having the capability to modulate an immune response to HSV-VIII. Exemplary LEAPS™ heteroconjugates specific to HSV-VIII include, but are not limited to, SEQ ID No.'s 160-165.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 100-114 to form a heteroconjugate capable of modulating an immune response to VZV or to mature DCs having the capability to modulate an immune response to VZV. Exemplary LEAPS™ heteroconjugates specific to VZV include, but are not limited to, SEQ ID No.'s 166-173.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 115-120 to form a heteroconjugate capable of modulating an immune response to HSV-1 or to mature DCs having the capability to modulate an immune response to HSV-1. Exemplary LEAPS™ heteroconjugates specific to HSV-1 include, but are not limited to, SEQ ID No.'s 174-176.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 121-124 to form a heteroconjugate capable of modulating an immune response to HSV-2 or to mature DCs having the capability to modulate an immune response to HSV-2. Exemplary LEAPS™ heteroconjugates specific to HSV-2 include, but are not limited to, SEQ ID No.'s 177-178.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 125-126 to form a heteroconjugate capable of modulating an immune response to herpes simplex virus or to mature DCs having the capability to modulate an immune response to herpes simplex virus. An exemplary LEAPS™ heteroconjugate specific to herpes simplex virus includes, but is not limited to, SEQ ID No. 179.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates SEQ ID No. 127 to form a heteroconjugate capable of modulating an immune response to EBV (Epstein-Barr virus) or to mature DCs having the capability to modulate an immune response to EBV. An Exemplary LEAPS™ heteroconjugate specific to poliovirus type 3 include, but are not limited to, SEQ ID No. 180.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 128-129 to form a heteroconjugate capable of modulating an immune response to polio virus type 3 or to mature DCs having the capability to modulate an immune response to polio virus type 3. Exemplary LEAPS™ heteroconjugates specific to poliovirus type 3 include, but are not limited to, SEQ ID No.'s 181-182.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates SEQ ID No. 130 to form a heteroconjugate capable of modulating an immune response to human poliovirus 1 Mahoney or to mature DCs having the capability to modulate an immune response to human polio virus 1 Mahoney. An exemplary LEAPS™ heteroconjugate specific to human polio virus 1 Mahoney includes, but is not limited to, SEQ ID No. 183.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 131-133 and 190-193 to form a heteroconjugate capable of modulating an immune response to tuberculosis or to mature DCs having the capability to modulate an immune response to tuberculosis. Exemplary LEAPS™ heteroconjugates specific to tuberculosis include, but are not limited to, SEQ ID No.'s 184-185 and 209-210.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 134 and 194-195 to form a heteroconjugate capable of modulating an immune response to Lyme disease or to mature DCs having the capability to modulate an immune response to Lyme disease. Exemplary LEAPS™ heteroconjugates specific to Lyme disease includes, but is not limited to, SEQ ID No.'s 186 and 211.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 135-136 to form a heteroconjugate capable of modulating an immune response to Chlamydia or to mature DCs having the capability to modulate an immune response to Chlamydia. An exemplary LEAPS™ heteroconjugate specific to Chlamydia virus includes, but is not limited to, SEQ ID No. 187.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 137-138 to form a heteroconjugate capable of modulating an immune response to malaria or to mature DCs having the capability to modulate an immune response to malaria. An exemplary LEAPS™ heteroconjugate specific to malaria virus includes, but is not limited to, SEQ ID No. 188.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No. 139 to form a heteroconjugate capable of modulating an immune response to Treponema pallidum or to mature DCs having the capability to modulate an immune response to Treponema pallidum. An exemplary LEAPS™ heteroconjugate specific to Treponema pallidum includes, but is not limited to, SEQ ID No. 189.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 205-208 to form a heteroconjugate capable of modulating an immune response to H. pylori or to mature DCs having the capability to modulate an immune response to H. pylori. An exemplary LEAPS™ heteroconjugate specific to H. pylori includes, but is not limited to, SEQ ID No.'s 217-218.
In certain embodiments, a LEAPS™ heteroconjugate in accordance to Formulae (I) or (II) incorporates any of SEQ ID No.'s 196-204 to form a heteroconjugate capable of modulating an immune response to HIV or to mature DCs having the capability to modulate an immune response to HIV. An exemplary LEAPS™ heteroconjugate specific to HIV, includes, but is not limited to, SEQ ID No.'s 212-216.
Table 3 below shows exemplary antigens described that can be employed as peptide P2 in certain embodiments. LEAPS™ heteroconjugates consistent with Formulae (I) and (II) can be formed by combining any permutation of ICBL peptide including, but not limited to, CEL-1000, Peptide J and/or Peptide G (Peptides P1) with an antigen peptide (Peptide P2) as presented in Table 3. Table 3 lists antigen sequences grouped by disease causing organism or agent associated with such sequences. Specifically, the first column of Table 3 lists the SEQ ID No. for the sequence presented in each row. The second column lists the disease, agent or organism for which the sequences presented in each row relate. The third column specifies the protein from which individual amino acid sequences are derived. The fourth column gives the abbreviation for which the sequence presented in each row can be referred to. For example, AH51ext stands for adenovirus 5 hexon protein (extended sequence), whereas J-AH51ext indicates a LEAPS™ heteroconjugate having the ICBL Peptide J linked to AH51ext. Also provided on Table 3 are example LEAPS™ heteroconjugates where Peptide P1 is Peptide J (SEQ ID No. 3) combined with an antigen Peptide P2. The fifth column specifies the core epitope sequence, if any, for the protein described in each row, and the sixth column specifies an extended epitope sequence associated with the protein described in each row. The seventh column indicates the range of amino acids from the described protein corresponding to the epitope sequence. The eighth column presents a non-limiting example LEAPS™ heteroconjugate containing Peptide J. The ninth column lists any know references describing the extended or core epitope sequences, if known. References are specified by a number corresponding to the list of references found at the end of this disclosure.
Embodiments also contemplate reversal sequences where the order of amino acids in Peptides P1 and/or P2 is reversed from N-term to C-terminus. For example peptide J has the sequence DLLKNGERIEKVE (SEQ ID No. 3). The reversal sequence for any ICBL disclosed herein is envisioned for inclusion in a LEAPS™ heteroconjugate as described herein. Further, the non-reversal sequence for an ICBL can be conjugated with an antigen sequence from Tables 1, 2 or 3 or with a reversal antigen sequence from Tables 1, 2 or 3. The reversal sequence for SEQ ID No. 7 is GTRVLARTRQYTADN, such that LEAPS™ heteroconjugates contemplated in certain embodiments include DLLKNGERIEKVEGGG GTRVLARTRQYTADN and DLLKNGERIEKVEGGGNDATYQRTRALVRTG, where Peptide J (SEQ ID No. 3) is conjugated with the reversal sequence of SEQ ID No. 7 or the non-reversal sequence of SEQ ID No. 7, respectively. Further, the reversal sequence for an ICBL can be conjugated with an antigen sequence from Tables 1, 2 and 3 or with a reversal antigen sequence from Tables 1, 2 and 3. Such LEAPS™ heteroconjugates contemplated in certain embodiments include EVKEIREGNKLLDGGGGTRVLARTRQYTADN and EVKEIREGNKLLDGGGNDATYQRTRALVRTG, where the reversal sequence for Peptide J (SEQ ID No. 3) is conjugated with the reversal sequence of SEQ ID No. 7 or the non-reversal sequence of SEQ ID No. 7, respectively.
Alternatively, the invention contemplates a variable immunomodulatory peptide construct having the Formula (III)
P3-x-P4 (III)
where P3 is a peptide construct comprised of X1 to X14 said peptide P3 being associated with Type A influenza or another infectious agent. For example, the immunomodulatory peptide can contain a highly conserved protein such as but not limited to the M2e or other matrix protein, or NP1 nucleoprotein, and P4 is a peptide construct comprised of X1 to X14 causing a Th1 directed immune response by said set or subset of T cells to which the peptide P3 is attached or which binds to a dendritic cell or T cell receptor causing said set or subset of DC or T cells to which the peptide P3 is attached to initiate and complete, an immune response.
Alternatively, the invention contemplates a variable immunomodulatory peptide construct having the formula (IV)
P5-x-P6 (IV)
where P5 is a peptide construct comprised of X1 to X14 said peptide P5 being associated with Type A influenza or another infectious agent, and P6 is a peptide construct comprised of X1 to X14 causing a Th2 directed immune response by said set or subset of T cells to which the peptide P5 is attached or which binds to a T cell receptor causing said set or subset of T cells to which the peptide P5 is attached to initiate an immune response, such that X1 to X10 and X14 describe a group of amino acids based on their features and X11 to X13 describe modifications to the peptide construct, wherein
X1 is selected from the group consisting of Ala and Gly,
X2 is selected from the group consisting of Asp and Glu,
X3 is selected from the group consisting of Ile, Leu and Val,
X4 is selected from the group consisting of Lys, Arg and His,
X5 is selected from the group consisting of Cys and Ser,
X6 is selected from the group consisting of Phe, Trp and Tyr,
X7 is selected from the group consisting of Phe and Pro,
X8 is selected from the group consisting of Met and Nle,
X9 is selected from the group consisting of Asn and Gln,
X10 is selected from the group consisting of Thr and Ser,
X11 is Gabax where X2X3, X3X2, X2X3, X3X2, X3X3, or X2X2 can be substituted with
X11;
x is a direct bond or linker for covalently bonding P5 and P6. For example, a variable immunomodulatory peptide construct of formulae (III)-(IV) can contain a peptide causing a Th2 directed immune response related to peptide J (SEQ ID No. 3), such X2X3X3X4X9X1X2X4X3X2X4X3X2 as (SEQ ID No. 225). One having skill in the art would recognize that each of X1 to X14 represents a group of amino acids having similar charge, polarity, hydrophobicity, chemical functionality, size and/or shape. As such, one having skill in the art will recognize that a variable immunomodulatory peptide can be identified by substituting a specific amino acid residue in any sequence disclosed herein with the corresponding group X1 to X14 including and representing the properties of that specific residue. For example, one having skill in the art will recognized that a Gly residue can be represented by group X1, and Trp residue can be represented by X6 and an Arg residue can be represented by group X4. As such, one having skill in the art will be able to unambiguously assign the tripeptide GWR as X1X6X4. Similarly, one having skill in the art will be able to unambiguously assign any of the sequences disclosed herein to a variable immunomodulatory peptide construct having residue represented by X1 to X14. For example, SEQ ID No. 7 derived from influenza virus can be converted to the variable immunomodulatory peptide X9X2X1X10X6X9X4X10X4X1X3X3X4X10X1 (SEQ ID No. 226) that can be incorporated into an immunomodulatory peptide construct.
Methods of Treating a Subject with LEAPS™ Heteroconjugates
Any of the LEAPS™ heteroconjugates in accordance with Formulae (I)-(IV) described above can be combined with an appropriate pharmaceutically suitable carrier with one or more optional adjuvants for administration to a subject. Such combination of the LEAPS™ heteroconjugates and a pharmaceutically suitable carrier can function as a vaccine to confer immune resistance to a broad spectrum of Type A influenza viruses or any of the infections agents described in Table 3. For example, an immunomodulatory peptide construct having any of SEQ ID No.'s 1, 2, 11-36, 47-52, 140-189 and 209-218 individually or as a mixture thereof, can be combined with a pharmaceutically suitable carrier to form a vaccine composition.
These LEAPS™ heteroconjugate peptide-based vaccines can provide prophylactic protection and also have the potential for therapeutic treatment of recurrent disease. The LEAPS™ technology is a T cell modulation platform technology that can be used to design and synthesize proprietary epitope sequences compatible with LEAPS™ technology. Each LEAPS™ heteroconjugate is composed of an ICBL which has the ability to induce and elicit protective immunity and antigen specific response in animal models.
LEAPS™ technology directly mimics cell to cell interactions on the dendritic and T-cell surfaces using synthetic peptides. The LEAPS™ heteroconjugates containing the antigenic disease epitope linked to an ICBL can be manufactured by peptide synthesis or by covalently linking two peptides. Depending on the type of LEAPS™ heteroconjugates and ICBL used, the peptide construct is able to direct the outcome of the immune response towards the development of T-cell function with primary effector T-cell functions: T Lymphocyte; helper/effector T Lymphocyte, type 1 or 2 (Th1 or Th2), cytotoxic (Tc) or suppressor (Ts) without excessive amounts of proinflammatory and inflammatory cytokines.
The type of the immune response elicited against an immunogen or a natural infection can be classified as Th1/Tc1, Th2/Tc2 or Th3 based on the predominant IgG subtype, the cytokines that are induced, or the presence or absence of delayed type hypersensitivity (DTH) response. A Th0 response is an earlier response that can mature into either a Th1 or a Th2 response and has features of both. The Th1 (CD4)/Tc1 (CD8) response is characterized by activation of CD4+ and CD8+ T cells to produce IL-2, TNF-β, and IFN-γ and to promote the production of IgM and specific IgG antibody subtypes and cell-mediated immune responses including delayed-type hypersensitivity (DTH). These responses reinforce early, local and inflammatory responses. Th2 responses promote different IgG subclasses, IgE and IgA responses but not cell mediated responses to antigen (Ag). Th2 responses prevent the onset of protective Th1 cell mediated responses important for infection control, which may exacerbate disease. Initiation of Th1 and Th2 responses has important implications in terms of resistance and susceptibility to disease. Th1-dominated responses are potentially effective in eradicating influenza viral infections, and are important for the induction of cytotoxic T lymphocytes (CTL). Most importantly, for many vaccines it is thought that initiation of immunity with a Th1 response and then progression to a Th2 response promotes better immune memory.
Many suitable pharmaceutical carriers are known to persons skilled in the art. The primary function of the pharmaceutical carrier is to assist in the delivery and/or administration the immunomodulatory peptide construct to a subject. The pharmaceutical carrier can be as simple as sterilized water. In certain embodiments, the pharmaceutical suitable carrier is a sterile pyrogen-free formulation containing from about 0.2 mg/mL to about 10 mg/mL of the immunomodulatory peptide construct in phosphate-buffered saline (PBS) and trehalose or other sugar that has been lyophilized to remove water and reconstituted prior to use with sterilized water for injection to a subject.
Optional adjuvants include products such as GMP products including Montanide™ ISA-51 (Seppic, Fairfield, N.J.), Depovax™, a liposomal adjuvant currently in phase I trials by Immunovaccine Technologies, and MAS1™, a proprietary water-in-oil GMP adjuvant from MerciaPharma currently in phase II clinical studies. Alum is currently the only FDA licensed adjuvant of the group. In certain embodiments, the composition administered to a subject containing a LEAPS™ heteroconjugate, as described herein, has mixture of an aqueous phase and an adjuvant oil phase from about 1:4 to about 4:1.
A composition having the LEAPS™ heteroconjugate and a suitable pharmaceutical carrier with or without an optional adjuvant can be administered to a subject by subcutaneous or intramuscular injection in a therapeutically effective amount. The subject can be a mammal subject including a human subject.
Freund's Incomplete Adjuvant is also contemplated (Sigma Corp., St. Louis, Mo.). For Product Number F5506, the Storage Temperature is 2-8° C. where F5881 is a clear, amber liquid containing particulate matter (dried cells). F5506 is a clear amber liquid. Freund's incomplete Adjuvant is one of the most commonly used adjuvants in research. It is used as a water-in-oil emulsion. It is prepared from non-metabolizable oils (paraffin oil and mannide monooleate). First developed by Jules Freund in the 1940's, Freund's Adjuvant is designed to provide continuous release of antigens necessary for stimulating a strong, persistent immune response. The main disadvantage of Freund's Adjuvant is that it can cause granulomas, inflammation at the inoculation site and lesions. To minimize side-effects, Incomplete Freund's Adjuvant is used for the boosts. (Freund, J. and McDermott, K., Proc. Soc. Exp. Biol. Med., 1942; 49:548-553; Freund, J., Ann. Rev. Microbiol., 1947; 1:291; Freund, J., Adv. Tuberc. Res., 1956; 7:130; Bennett, B. et al., J. Immuno. Meth., 1992; 153:31-40; Deeb, B. J. et al., J. Immuno. Meth., 1992; 152:105-113; Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
Maturation of Dendritic Cells with LEAPS™ Heteroconjugates
In certain embodiments, a subject is stimulated to have an immune response to Type A influenza by being administered DCs matured and activated in the presence of a LEAPS™ heteroconjugate consistent with Formulae (I) or (II). Induction of an optimal immune response requires mimicking nature's approach to immunization. DCs play a major role in initiating and directing the immune response to a vaccine. The initial host response to an antigen requires internalization of the antigen into the DC followed by processing and presentation by the MHC I or II proteins for T cell recognition. DCs, macrophages and B cells are capable of presenting antigens to CD4+ helper T cells and CD8+ cytotoxic T cells as peptides held within grooves of the class II and I MHC proteins, respectively. These cells can be functionally divided into DC1 and DC2 cell types based on the means of their activation, their cytokine output and the nature of their influence on T cells. DC1 cells produce IL12 and promote Th1-type responses whereas DC2 cells promote Th2-type responses.
As described above, the LEAPS™ heteroconjugates described herein can be administered to a subject to generate an immune response in vivo. The immunomodulatory peptide constructs can also modulate the properties of immune cells, including DCs, ex vivo, in order to activate, mature, and direct the character and phenotype of immune cells (e.g. DCs) contacted with the LEAPS™ heteroconjugate. Such treated immune cells, which can include DCs and/or monocytes, can then be introduced into a subject for the purpose of increasing immunity against Type A influenza virus. DCs treated with a LEAPS™ heteroconjugate ex vivo and transferred to a subject can confer acquired immunity to the subject.
In certain embodiments, DCs and/or monocytes are extracted from a subject or donor away from other tissues of the body. The dendritic cells and/or monocytes are then contacted with one or more LEAPS™ heteroconjugates. DCs and/or monocytes isolated from a subject or donor can be in an immature state characteristic of immune cells prior to contact with an antigen. Such cells are herein referred to as immature DCs (iDCs). The DCs and/or monocytes can be isolated from blood derived monocytes and/or bone marrow taken from a subject or donor. DCs and/or monocytes expressly include monocyte cells capable of differentiating into macrophages and/or dendritic cells that can function to present antigens to T cells under appropriate conditions. Upon contact or treatment of the isolated dendritic cells and/or monocytes with the LEAPS™ heteroconjugates, the dendritic cells and/or monocytes undergo a maturation to a state that directs immune response.
As defined herein, the term “immature DCs” (iDCs) refers to cells derived from a donor or subject that are not competent to induce T cell activation upon interaction with T cells. Such iDCs are also known in the art as naïve DCs. Such iDCs can have certain physical characteristics such as a reduced level of expression of CD80 and/or CD86, MHC molecules (class I and/or class II), other surface markers and a reduced appearance of dendrites. Immature DCs, as defined herein, expressly includes monocytes that can be stimulated to form dendritic cells. As defined herein, terms “matured DCs” and “more matured DCs” refer to DCs after contact with any of the LEAPS™ heteroconjugates described herein. Such matured DCs can have certain physical characteristics including upregulation of CD80 and/or CD86, MHC I or II molecules, an increased appearance of dendrites and secretion of IL-12p70.
In certain embodiments, contacting monocyte cells with one or more LEAPS™ heteroconjugates can induce the development of the monocyte cells or iDCs to DC1 (Th1-inducing dendritic cells) and/or DC2 cells (Th2-inducing dendritic cells) or other cell type allowing for acquired immunity to influenza when transferred to a subject. In certain other embodiments, iDCs and/or monocytes isolated from a subject or donor are contacted with a media containing granulocyte-macrophage colony stimulating factor (GM-CSF) to stimulate the expression of CD11c on the surface of the iDCs or monocytes. The iDCs and/or monocytes after exposure to GM-CSF are contacted with one or more LEAPS™ heteroconjugates to induce the maturation of the monocyte cells or iDCs to DC1 (Th1-inducing dendritic cells) and/or DC2 cells (Th2-inducing dendritic cells) or other cell type allowing for acquired immunity to influenza when transferred to a subject.
In certain embodiments, DCs and/or monocytes are extracted from a subject or donor away from other tissues of the body in a composition of isolated iDCs and/or monocytes. As described herein, a composition of isolated DCs and/or monocytes is a composition in which the DCs and/or monocytes are present away from other body tissue including blood or bone marrow. In certain embodiments, a composition of isolated DCs and/or monocytes contains at least 50% of the viable cells present in the composition being DCs and/or monocytes. In certain embodiments, the composition of DCs and/or monocytes is substantially free from whole red blood cells.
In certain embodiments, the iDCs and/or monocytes are contacted or treated with one or more LEAPS™ heteroconjugates for about 6 hours to about 96 hours or from about 12 hours to about 72 hours. In certain other embodiments, the iDCs and/or monocytes are contacted or treated with one or more LEAPS™ heteroconjugates for a period of time longer than about 6 hours. In additional embodiments, the iDCs and/or monocytes are contacted or treated with one or more LEAPS™ heteroconjugates for a period of time longer than about 12 hours. In certain additional embodiments, the iDCs and/or monocytes are contacted or treated with one or more LEAPS™ heteroconjugates for a period of time longer than about 24 hours. In certain embodiments, the iDCs and/or monocytes are contacted or treated with one or more LEAPS™ heteroconjugates at a ratio from about 5 to about 50 micromoles of one or more LEAPS™ heteroconjugates per 106 iDCs and/or monocytes. In certain other embodiments, the iDCs and/or monocytes are contacted or treated with one or more LEAPS™ heteroconjugates at a ratio greater than about 5 micromoles of one or more LEAPS™ heteroconjugates per 106 iDCs and/or monocytes.
In certain embodiments, the iDCs and/or monocytes are contacted with GM-CSF for a period of about 1 day to about 10 days or from about 3 days to about 10 days. In certain additional embodiments, the iDCs and/or monocytes are contacted with GM-CSF for a period greater than about 5 days. In certain embodiments, the iDCs and/or monocytes are contacted with a media having a concentration of from about 5 to about 200 ng/mL of GM-CSF or from about 10 to about 150 ng/mL of GM-CSF. In other embodiments, the DCs and/or monocytes are contacted with a media having a concentration of GM-CSF greater than about 5 ng/mL. In certain embodiments, the DCs and/or monocytes are contacted with a media having a concentration of GM-CSF greater than about 15 ng/mL of GM-CSF.
Upon contact of iDCs and/or monocytes with the LEAPS™ heteroconjugate, an increased expression level of interleukin-12p70 (IL-12p70) can be observed relative to iDCs and/or monocytes not contacted with the LEAPS™ heteroconjugate. In certain embodiments, iDCs and/or monocytes contacted with the LEAPS™ heteroconjugate exhibit an up-regulation of at least one of the following: CD80, CD86, MHC class I, or MHC class II cell surface markers relative to iDCs and/or monocytes not contacted with the LEAPS™ heteroconjugate.
Immature dendritic cells and/or monocytes after contact with an immunomodulatory LEAPS™ heteroconjugate can be referred to as matured dendritic cells. The matured dendritic cells can modulate an immune response when administered or introduced into a subject. An immune response can be induced in a subject under situations where matured dendritic cells are washed free of LEAPS™ heteroconjugate that is unbound from the surface of a dendritic cell. As such, the amount of any antigen, including the antigen peptide P2 forming part of the LEAPS™ heteroconjugate, introduced into a subject is limited. As such, introduction of the matured DCs described herein minimizes the production of a “cytokine storm” or other inflammatory responses in a treated subject. Matured DCs created through contact with a LEAPS™ heteroconjugate do not produce acute phase cytokines that can trigger a cytokine storm. See Taylor et al., Vaccine 28 (2010) 5533-5542.
Without wishing to be bound by any one particular theory, it is believed that the LEAPS™ heteroconjugate is retained on the surface of DCs in manner allowing for the interaction of the LEAPS™ heteroconjugate with T cell receptor present on the surface of T cells. As such, DCs matured in the manner described above can be introduced or administered to a subject such that the LEAPS™ heteroconjugate present of the surface of the introduced or administered DCs can interact with the subject's in situ T cells to direct an antigen specific immune response. More specifically, activation of T cell-mediated immune response requires multiple stimulator interactions, including interaction with T cell receptor (TCR) present on the surface of T cells. It is believed that these more matured DCs having the LEAPS™ heteroconjugate present of the surface can provide the necessary interaction to activate T cells and direct an immune response to the Peptide P2 antigen of the LEAPS™ heteroconjugate. Further, it is believed that these more matured DCs formed using the methods described herein have an advantageous profile of secreted cytokines that do not stimulate a cytokine storm or other deleterious inflammation response in a subject.
The more matured dendritic cells and/or T cells can be used in an autologous fashion. In certain embodiments, iDCs and/or monocytes are isolated from a subject to be treated, such isolated cells can be blood derived monocytes and/or bone marrow cells taken from the subject. The isolated iDCs and/or monocytes are contacted with one or more LEAPS™ heteroconjugates having the structure P1-x-P2 or P2-x-P1 to induce maturation to more matured dendritic cells. An effective amount of the LEAPS activated DCs are administered to the same subject from which the matured cells were originally isolated. The subject can be a mammal, including a human. As such, the method for modulating an immune response to Type A influenza virus is autologous and minimizes exposure to any potential non-self antigen that can trigger a “cytokine storm” or other undesirable inflammatory response.
In certain other embodiments, iDCs and/or monocytes can be isolated from a compatible donor, treated with a heteroconjugate peptide having the structure P1-x-P2 or P2-x-P1 to induce maturation to form matured dendritic cells, and an effective amount of the matured dendritic cells and/or T cells administered to a subject having compatibility with the donor.
Development of DC1 or DC2 cells is determined by environmental factors, including dose and form of the antigen, but mostly by stimulation of Toll-Like Receptors (TLR) and other receptors for microbial pathogen associated molecular patterns, artificial ligands of these receptors and other stimuli. Many of these TLR molecules are triggered by adjuvants made from the TLR ligands such as Lipid A, MPL, CpG, LPS, etc. Other receptors on DCs known as LIR (leukocyte immunoglobulin-like receptors or also known as CD85) are known to recognize self-epitopes found on various MHC molecules. Both CEL-1000 and Peptide J are derived from MHC molecules and are likely ligands for LIRs. Many of these receptors' responses are also triggered by their own adjuvants. However, the identity of the receptor for LEAPS™ heteroconjugate on DCs is not definitively known. (Annunziato F. et al., Expression and release of LAG-3-encoded protein by human CD4+ T cells are associated with IFN-gamma production, FASEB J., 1996 May; 10(7):769-76; Anderson K J, Allen R L., Regulation of T-cell immunity by leucocyte immunoglobulin-like receptors: innate immune receptors for self on antigen-presenting cells, Immunology, 2009 May; 127(1):8-17; Sloane D E et al., Leukocyte immunoglobulin-like receptors: novel innate receptors for human basophil activation and inhibition, Blood, 2004 Nov. 1; 104(9):2832-9; Shiroishi M et al., Efficient leukocyte Ig-like receptor signaling and crystal structure of disulfide-linked HLA-G dimer, J Biol. Chem., 2006 Apr. 14; 281(15):10439-47; Shiroishi M et al., Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G, Proc Natl Acad Sci USA. 2003 Jul. 22; 100(15):8856-61; Colonna M et al., A novel family of Ig-like receptors for HLA class I molecules that modulate function of lymphoid and myeloid cells, J Leukoc. Biol., 1999 September; 66(3):3; 75-81; Borges L, et al., A family of human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I molecules, J Immunol., 1997 Dec. 1; 159(11):5192-6; Shiroishi M et al., Structural basis for recognition of the non-classical MHC molecule HLA-G by the leukocyte Ig-like receptor B2 (LILRB2/LIR2/ILT4/CD85d), Proc. Natl. Acad. Sci. U.S.A., 2006 Oct. 31; 103(44):16412-7; all of which are incorporated herein by reference).
The matured DCs having been treated with the LEAPS™ heteroconjugate having the structure P1-x-P2 or P2-x-P1 can be administered to a subject with an active influenza virus infection. A significant reduction in influenza virus titer can be observed within about 3 days of the administration of the matured LEAPS™ heteroconjugate-activated DCs to a subject having an active influenza infection. In certain embodiments, influenza virus titer can be reduced by 20% or more within 3 days post administration of the matured LEAPS™ heteroconjugate-activated DCs. In certain embodiments, virus titer can be reduced by 50% or more three days post administration of the matured LEAPS™ heteroconjugate-activated DCs.
Alternatively, the matured DCs having been contacted by the LEAPS™ heteroconjugate having the structure P1-x-P2 or P2-x-P1 can be administered as a prophylactic against future influenza virus exposure. Specifically, the matured DCs produce an immune response to the antigen of the Peptide P2 that can lead to long-term immune memory in a similar fashion as direct vaccination with LEAPS™ heteroconjugates.
In a surprising observation, the matured DCs having been contacted by the LEAPS™ heteroconjugate having the structure P1-x-P2 or P2-x-P1 have a capability to be recruited directly to a site of an infection. That is, the matured DCs can be recruited to an infection site involving an organism or virus expressing the epitope of the antigenic Peptide P2.
DCs can be tagged or tracked using several known techniques. For example, cells can be labeled or stained with carboxy fluorescein succinimidyl ester (CSFE), Cy5.5 or Alexa Fluor® which have fluorescent properties. The distribution of CSFE-dyed dendritic cells throughout different tissues can be determined through fluorescent microscopy or flow cytometry techniques. Those skilled in the art will recognize that the technique for tracking the distribution of DCs is not limited. For example, radio labeling techniques can be employed to track the distribution of DCs. In certain embodiments, at least a majority of the matured DCs contacted with the peptide construct having the structure P1-x-P2 or P2-x-P1 are found in the lung tissue of an influenza virus infected mouse at a time point 8 hours post administration of the matured DCs. The lungs are the primary location of influenza infection in mice. In other species, the primary location of influenza infection can be different, such as the upper respiratory tract for humans infected with influenza virus. In certain embodiments, a majority of the LEAPS™ heteroconjugate-activated DCs locate to the site of an infection within 8 hours of administration of the DCs to a patient, where the heteroconjugate contains an antigen derived from the virus causing the infection.
LEAPS™ heteroconjugate-activated matured dendritic cells have been previously reported to be useful for prophylactic use providing immunity against a later challenge with herpes simplex virus 1 (HSV-1) virus, Taylor et al., Vaccine 28 (2010), 5533-43. In the Taylor et al. study, mice were not exposed to nor suffering from an active HSV-1 infection at the time of treatment with LEAPS™ heteroconjugate-activated more matured DCs. As such, the DCs administered in the Taylor et al. study could not migrate to a site of infection for HSV-1, since there was no active HSV-1 infection in the subject mice at the time of administration. As demonstrated in the Examples below, the ability of LEAPS™ heteroconjugate-activated DCs to migrate or accumulate at the site of infection for an organism or virus is unexpected.
The matured DCs contacted with the LEAPS™ heteroconjugate having the structure P1-x-P2 or P2-x-P1 were found to have a beneficial effect in ameliorating influenza infection without the use of additional immunomodulators.
Diagnostic Application of Dendritic Cells with LEAPS™ Heteroconjugates
Matured DCs treated with a LEAPS™ heteroconjugate in vitro have a property allowing for the localization of such matured DCs to the site of an infection within the body of a subject. The LEAPS™ heteroconjugate has a P2 peptide sequence originating or derived from an antigen, as described above. Maturation of DCs through treatment with a LEAPS™ heteroconjugate allows for the matured DCs to collect or to locate in an area of a subject's body where a source of the antigen from which the P2 is derived can be found. As such, an infection by a pathogen, virus, rickettsia, bacteria or parasite can be detected by observing matured dendritic cells administered to a subject collecting, locating or concentrating at a site within the subject's body where the virus can be found.
In certain embodiments, matured DCs can be labelled with a tracking marker to allow for their location within a subject's body to be tracked after administration to the subject. For example, in the matured DC's can be labelled with radionuclides to allow for the location of the labeled, matured DCs to be detected using appropriate equipment. Appropriate radionuclides include radioactive isotopes of iodine such as 131I or 125I. The location of radionuclides can be determined using a radiation detector or photographic film sensitive to radiation.
As the Examples below demonstrate, DCs matured by treatment with a LEAPS™ heteroconjugate, as describe herein, have the ability to concentrate in a region of a subject's body hosting or similar antigen source. Specifically, the matured DCs obtained by treatment with a LEAPS™ heteroconjugate are sensitized to sources of the antigen from which the peptide P2 forming the specific LEAPS™ heteroconjugate used to mature the matured DCs is derived. For example, DCs matured with a LEAPS™ heteroconjugate containing a peptide P2 derived from an infectious agent will accumulate in area of the subject's body where antigens originating from the virus can be found.
Early detection of specific conditions is often crucial to successful treatment. Unfortunately, many traditional techniques for verifying the presence of an infection, pathogen, virus, bacteria, rickettsia or parasite are not effective until the condition has progressed to a significant degree such that symptoms are manifest Likewise, the presence of antibodies in the blood serum having affinity for a particular antigen is often not discernible until a substantial time has past since infection and an immune response is well on the way.
The immunomodulatory LEAPS™ heteroconjugates can be used to modulate a subject's immune system to detect the presence of an infection at an early stage. The LEAPS™ heteroconjugates can be used to mature immature DCs or monocytes isolated from the subject or a compatible donor to be sensitive to a desired antigen originating or derived from an infectious agent whose detection is desired. Since the DCs can be manipulated outside of the body, the matured DCs can be labelled with a tracking marker in a manner allowing for sensitive detection. In particular, labelling with radionuclides can allow for detection down to very low levels. Similarly, the presence of a fluorescent dye can be discerned at very low levels.
In certain embodiments, matured DCs can be labelled with a tracking marker to allow for location within a subject's body to be tracked after administration to the subject. For example, matured DC's can be labelled with radionuclides (radioisotopes) to allow for the location of the labeled, matured DCs to be detected using appropriate equipment. Appropriate radionuclides include radioactive isotopes of iodine such as 131I or 125I as well as other radionuclides including 18F, 32P, 64Cu, 90Y, 99mTc, 124I, 89Zr, 11In, 188Re, or 177Lu. The location of radionuclides can be determined using a radiation detector, single-photon tomography/computed tomography (SPECT/CT), scintillation camera, positron emission tomography or photographic film sensitive to radiation. In certain further embodiments, matured DC's can be labeled with a fluorescent dye, such as carboxyfluorescein succinimidyl ester (CSFE), Cy 5.5 or Alexa Fluor® where the presence of such dye-labelled DCs can be detected in tissues taken by biopsy from a patient administered the matured DCs. In certain embodiments, the tracing marker is a luminescence dye. In certain embodiments, the presence of fluorescence in a tissue sample taken from a subject's body is determined by flow cytometry.
In certain embodiments, immature DCs and/or monocytes are collected from a subject or a compatible donor and matured by treatment or contact with a LEAPS™ heteroconjugates having a structure of Formulae (I) or (II) and incorporating an antigen peptide (P2) sequence derived from an infection agent such as an infection pathogen virus, rickettsia, bacteria or parasite. The matured DCs are administered to the subject through an intravenous route or another appropriate route and a period of time is allowed to elapse. A diagnostic determination of the presence of the desired infection, pathogen virus, rickettsia, bacteria or parasite can be made by observing the location of the administered matured dendritic cells and/or tracking marker. The tracking marker can be conjugated to the LEAPS™ heteroconjugate used to treat the DCs or can be conjugated to an antibody (mAb) having affinity for a cell surface marker or other protein present on the DC. Example cell surface markers to which such an antibody can have affinity include MHC II, CD11c, DEC-205, Dectin-1, DC-SIGN, and DC-LAMP. When the desired infection, pathogen virus, rickettsia, bacteria or parasite is present in the body of the subject, the matured DCs and/or tracking marker will concentrate at the location, tissue type or organ structure where the infection is present within the subject's body. When the targeted infection is not present in the body of the subject, the matured DCs and/or tracking marker is expected to be diffused in different locations of the subject's body and not concentrated in any particular location, tissue type or organ structure.
The diagnostic determination can be made by only observing the location, tissue type or organ structure where the infection, pathogen virus, rickettsia, bacteria or parasite is expected to be found. For example, if matured DCs are made with a LEAPS™ heteroconjugates containing an antigen sequence derived from a virus known to cause respiratory infections, then only observation of the presence of the matured DCs and/or tracking marker in the subject's lungs or other respiratory organs needs to be performed. Since the amount of tracking marker administered to the subject is known, a determination of a concentration of the matured DCs and/or tracking marker in a specific location, tissue type or organ structure can be made without the need for a direct comparison with other body tissues.
In certain embodiments, a majority of the matured DCs and/or tracking marker is present in a specific location, tissue type or organ structure of the subject indicating the presence of the targeted infection, pathogen virus, rickettsia, bacteria or parasite. In certain other embodiments, at least about 75% of the matured DCs and/or tracking marker are present in a specific location, tissue type or organ structure of the subject indicating the presence of an influenza infection. In certain other embodiments, less than a majority of the matured DCs and/or tracking marker present in a specific location, tissue type or organ structure can indicate the presence of an infection, pathogen virus, rickettsia, bacteria or parasite, where the amount of the DCs and/or tracking marker in the specific location, tissue type or organ structure is higher than in surrounding areas.
The ability of LEAPS™ heteroconjugate-treated DCs to localize to the site of an infection can be utilized to deliver therapeutic agents directly to the site of the infection in addition to the diagnostic applications discussed above. The therapeutic agent or radioisotope conjugated to a LEAPS™ heteroconjugate or to a monoclonal antibody (mAb) can by conjugated or linked to a lysosomatropic agent. A lysosomatropic agent is a weak organic base that can diffuse through membranes but will become protonated in the lysosome of a cell, where the protonated lysosomatropic agent is unable to diffuse through membranes and will, therefore, be trapped within the cell. Hydrophobic amines, including butylamine, spermidine, spermine, methylamine, and cyanine dyes (including those used for studying membrane potential or that are used as tracers in neurobiology) are examples of lysosomatropic agents. These lysosomatropic agents can be modified to be conjugated to a radioisotope or to a therapeutic compound (e.g. cytokines, staphylococcal enterotoxin A superantigen, staphylococcal enterotoxin B superantigen, or other molecules) by a cleavable linkage to the radioisotope or compound.
Antiviral therapeutic agent, especially anti-influenza drugs, can be delivered by conjugation to a LEAPS™ heteroconjugate or to an mAb. Alternatively, antiviral therapeutic agents can be delivered by lysosomal means. The two main classes of antiviral therapeutic agents used against influenza are neuraminidase inhibitors, such as zanamivir (RELENZA™) and oseltamivir (TAMIFLU™), or inhibitors of the viral M2 protein, such as amantadine (SYMMETREL™) and rimantadine (FLUMADINE™). Some interferons either normal (non-pegylated) or pegylated and especially forms that can be conjugated or delivered by lysosomal encapsulated methods may also be useful in severe cases.
Conjugation of an antiviral therapeutic agent or any other therapeutic agent or a dye can be done through the selective use or engineering of an amino acid residue to conjugate to the antiviral therapeutic agent. For example, a cysteine or lysine residue can be engineered into the LEAPS™ heteroconjugates to serve as a conjugation site for a therapeutic compound. Other active sites for attachment on other amino acids include OH groups on serine or threonine residues, COOH groups on aspartic or glutamic acid residues, the carboxyl terminal COOH, amide groups on glutamine or asparagine residues, NH2 on amino terminal amino acid or lysine residues, and —SCH3 groups on methionine residues. A therapeutic agent or a dye conjugated to a LEAPS™ heteroconjugates or to an mAb that can in turn be conjugated to DCs via the LEAPS™ heteroconjugates or the mAb.
In a further embodiment, the LEAPS™ heteroconjugate or an mAb can be conjugated to a fluorescent dye. Suitable dyes include but are not limited to N,N′di-carboxypentyl-indodicarbocyanine-5,5′-disulfonic acid (Cy5.5), Alexa Fluor® probes, carboxyfluorescein succinimidyl ester (CFSE), 4-N(S-glutathionylacetylaminophenyl)arsenoxide-Cy5.5, 2,3-dicyanonaphthalene-Cy5.5 or Alexa Fluor, or CFSE and other near-infrared probes. Additional NIR probes include Cy 5.5 covalently linked to 4-N(S-glutathionylacetylaminophenyl)arsenoxide-Cy5.5 and 2,3-dicyanonaphthalene-Cy5.5. Additionally, DCs can be directly stained by carboxyfluorescein succinimidyl ester (CFSE). In particular, Cy 5.5, Alexa Flour® and other NIR dyes exhibit low absorption of the NIR signal in tissue at operating wavelengths and may be quenched by conjugation of two or more NIR probe molecules together. Cleavage of the conjugation bonds results in fluorescence dequenching and generation of a signal that is suitable for imaging. A fluorescent image can be made by endoscopy or by taking a tissue biopsy. A tissue biopsy can be examined by flow cytometry to identify the presence of fluorescent cells.
Bone marrow (BM) cells were extracted from the femurs and tibias of BALB/c mice using a sterile disposable 27g needle. The ends of the femurs and tibias were removed to expose the bone marrow, and the BM cells were flushed out with Hanks Balanced Salt Solution (HBSS). BM cells were washed with HBSS, passed through a Nytex filter, and red blood cells were lysed with ACK buffer (NH4C1, KHCO3, EDTA, neutral pH 6-8-7.4). The BM cells were suspended in culture medium containing RPMI at a concentration of approximately 5×106 cells/mL.
The remaining BM cells were decanted from the plastic tissue culture flasks and further washed and resuspended in RPMI medium with 10% fetal calf serum (FCS) containing 20 ng/mL murine recombinant (GM-CSF) at a concentration of approximately 1 to 1.5×106 cells/mL. BM cells were incubated in RPMI medium with 10% FCS containing 20 ng/mL GM-CSF at 37° C. by seeding approximately 1×106 BM cells per well in well culture plates or approximately 50×106 BM cells in tissue culture flasks. Following 2 days and 4 days of incubation, the culture media was replaced with fresh RPMI medium with 10% FCS containing 20 ng/mL GM-CSF. BM cells were harvested after 8 days.
The BM cells after incubation with GM-CSF were analyzed for expression of CD3, CD19, CD11c, CD86, MHC II and F4/80 (CD80). At least 106 cells were analyzed by flow cytometry (Altra FACS, Beckman Coulter) using forward and side scatter parameters to limit the immunofluorescence analysis to cells having the size and granularity anticipated for monocytes and dendritic cells. In
The BM cells were labeled with an appropriate antibody-fluorescent conjugate for each cell surface marker analyzed. The antibody-fluorescent conjugates used are indicated in Table 4. All antibody-fluorescent conjugates are commercially available and have affinity to the mouse (anti-mouse) cell surface markers as indicated. Flow cytometry analysis was also performed using an appropriate isotype control, as indicated in Table 4.
1BD Bioscience, 2350 Qume Dr., San Jose, CA 95131
2eBioscience, 10255 Science Center Dr., San Diego, CA 92121
The results of flow cytometry analysis of the BM cells are presented in
As described above, flow cytometry analysis for the BM cells incubated with GM-CSF indicated that the BM cells display a phenotype consistent with the presence of iDCs and/or monocytes. These cells were incubated with LEAPS™ heteroconjugate or an appropriate control as described below.
BM cells isolated from mice, as described above, were seeded into a 24-well culture plate at about 106 cells per well in RPMI media with 10% FCS and 20 ng/mL GM-CSF. The BM cells were incubated with GM-CSF media over the course of 8 days and replenished with fresh media after 2 days and 4 days. After 8 days, a specific LEAPS™ heteroconjugate, a control composition or a combination of LEAPS™ heteroconjugates were added to individual wells.
LEAPS™ heteroconjugates were added to individual wells at an amount of 14.5 micromoles. Four different LEAPS™ heteroconjugates were utilized having an antigen peptide derived from influenza virus: J-NP (SEQ ID No. 1), J-M2e (SEQ ID No. 2), J-HA1 (SEQ ID No. 12) and J-HA2 (SEQ ID No. 11). In addition, a LEAPS™ heteroconjugate having an antigen portion derived from p17 gag protein from HIV was used as a control, J-H (DLLKNGERIEKVEGGGYSVHQRIDVKDTKEALEKIEEEQNKSKKKA) (SEQ ID No. 37). LEAPS™ heteroconjugate J-H is conjugate of Peptide J with and a peptide isolated from the p17 gag protein of HIV, “Protein H” (YSVHQRIDVKDTKEALEKIEEEQNKSKKKA) (SEQ ID No. 38), through a -GGG- linker. Additional wells were incubated with an approximately equimolar mixture of J-NP, J-M2e, J-HA1 and J-HA2 at a total combined amount of 14.5 micromoles per well. An additional non-LEAPS™ heteroconjugate control was used, lipopolysaccharide (LPS), which was expected to produce a significant immune response.
BM cells were incubated at 37° C. with the appropriate LEAPS™ heteroconjugate or LPS for a period of 24 hours and harvested. Trials of incubation for periods of 24 hours, 48 hours and 72 hours were also explored; however, 24 hours of incubation was found to yield the most satisfactory maturation signals. The BM cells were analyzed by flow cytometry, in the same manner described above, after incubation with the LEAPS™ heteroconjugate or LPS for a period of 24 hours, 48 hours or 72 hours. Results of flow cytometry are presented in
No significant levels for CD3, CD19, or F4/80 were observed. Therefore, flow cytometry data for those cell surface markers are not shown in
BM cells are known to be good source of stem cells for monocytes and naïve myeloid DCs with few or no T cells. Stem cells/monocytes can be stimulated to take on a DC phenotype by exposure to GM-CSF, which is indicated by expression of CD11c. CD11c is a type I transmembrane protein found on DCs. In
For clarity,
CD80 and CD86 are cell markers for matured DCs competent for signaling and activating T cells. The change in phenotype observed between the non-LEAPS™-activated DCs and the LEAPS™ heteroconjugate-treated DCs is a directed indication that the LEAPS™ heteroconjugates described herein are capable of directly developing iDCs into matured DCs expressing cell surface markers needed for antigen presentment and T cell activation.
Treatment of Mice with LEAPS Heteroconjugate-Treated DCs
45 mice were infected with 106 TCID50 of mouse-adapted PR8 influenza A virus. The effectiveness of DCs treated with a mixture of conjugates J-NP (SEQ ID No. 1), J-M2e (SEQ ID No. 2), J-HA1 (SEQ ID No. 12) and J-HA2 (SEQ ID No. 11) for mitigating influenza infections was evaluated. The LEAPS™ heteroconjugate-activated DCs are the same as those presented in
The statistical significance between the observed survivability between the non-LEAPS™-activated DC group and the LEAPS™ heteroconjugate-activated DCs group was evaluated by Mantel-Cox test and Gerhan-Breslow-Wilcoxon test. The Mantel-Cox test yielded a p-value of 0.0035 and the Gerhan-Breslow-Wilcoxon test yielded a p-value of 0.0046. As such, a null hypothesis that there is no statistical difference in survivability between the two groups is rejected.
Treatment of Mice with LEAPS Heteroconjugate Treated-DCs/Time from Infection
An additional survival study for treating influenza-infected mice with DCs was performed to determine any effect based on time between infection and treatment with DC cells. Mice were infected with 105 TCID50 PR8 strain Type A influenza virus. 45 mice were treated by IV at 8 hours post infection and 30 mice were treated by IV at 24 hours post infections with either 107 cells/mouse of not treated with LEAPS™-heteroconjugate DCs or 107 cells/mouse of LEAPS™ heteroconjugate-treated DCs. A total of four groups of mice were monitored as follows: 15 mice treated with non-LEAPS™-activated DCs (“non-pulsed DCs”) at 8 hours post-infection; 15 mice treated with LEAPS™ heteroconjugate-activated DCs at 8 hours post-infection; 15 mice treated with non-LEAPS™-activated DCs (“non-pulsed DCs”) at 24 hours post-infection; and 15 mice treated with LEAPS™ heteroconjugate-activated DCs at 24 hours post-infection. From each group, 5 individuals were sacrificed at 3 days post IV treatment and 10 individuals were observed for 14 days post IV treatment.
Virus titers found in lung tissue of the sacrificed mice 3 days post IV were measured using a standard assay in MDCK cells. The mean virus titers (log10 TCID50/g) for the non-LEAPS™-activated DC groups treated at 8 and 24 hours post infection were about 7.3 TCID50 g−1 and 7.6 TCID50 g−1, respectively. The mean virus titers for the LEAPS™ heteroconjugate-activated DC groups treated at 8 and 24 hours were both about 6.1 TCID50 g1. The virus titers for the 8-hour non-LEAPS™-activated DC group and the 8-hour LEAPS™ heteroconjugate-activated DC group were compared by paired t test. A similar comparison by t test was also performed for the 24-hour non-LEAPS™-activated DC group and the 24-hour LEAPS™ heteroconjugate-activated DC group. The paired t test yielded p-values of 0.01 and 0.04, respectively. As such, there is a statistical basis to reject a null hypothesis of no difference in viral load between compared groups. Almost no difference was seen in mean virus titers between the 8- and 24-hour LEAPS™ heteroconjugate-activated DC groups. As such, acquired immunity obtainable by treatment with LEAPS™ heteroconjugate-activated DCs does not appear to be time dependent at least within the 24-hour period following infection.
The results presented herein appear to be the first time that DCs matured with a designed, artificial peptide are reported to be sufficient to provide protection from a lethal infection. That is, the results presented here demonstrate the usefulness of LEAPS™ heteroconjugate-activated DCs for combating an active infection and not only for prophylactic use. Taylor et al., Vaccine 28 (2010), 5533-43, cited above, reports the use of DCs treated with an artificial peptide for prophylactic use against challenge with HSV-1 virus; however, no indication is made for effectiveness against an active infection. As discussed in Taylor et al., the J Peptide alone does not induce protection against infection nor a change in phenotype of iDCs exposed to Peptide J.
DCs were administered to individual mice after inoculation with competent Type A influenza virus. Even if symptoms are not actively present in the mice, it is presumed that an active infection is on-going prior to treatment with DC cells. The course of influenza infection can be monitored through observation of weight of infected mice.
As seen in
Surprisingly, observations indicate that LEAPS™ heteroconjugate-activated DCs administered to influenza-infected mice by IV were not evenly distributed in the tissue of infected mice. The above-described iDCs were incubated with the combined LEAPS™ heteroconjugates for 24 hours or left in a non-LEAPS™-activated or iDC state. The DCs were then labeled by incubation with 5 μM carboxyfluorescein succinimidyl ester (CSFE) for 30 minutes at 37° C. to fluorescently label the DCs. Those skilled in the art will recognize that the manner for labeling DCs for later detection is not limited to any particular method. For example, DCs can be labeled for later detection by other methods including labeling with other cell surface labeled dyes, or radionuclides. The CSFE-labeled DCs were administered IV into 18 individual mice in two groups: 9 mice were administered 107 non-LEAPS™-activated DCs cells and 9 mice were administered 107 LEAPS™ heteroconjugate-activated DCs, as described-above. 3 individuals in each group were sacrificed at 8, 24 and 48 hours post IV and tissue collected from lung, spleen and lymph nodes.
The two-dimensional data presented represents CFSE fluorescence intensity and side scattering. The outlined boxes in
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| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/039473 | 5/24/2012 | WO | 00 | 2/2/2015 |
| Number | Date | Country | |
|---|---|---|---|
| 61490050 | May 2011 | US | |
| 61538427 | Sep 2011 | US | |
| 61490056 | May 2011 | US |
