Patent Grant
7786257
A printed Sequence Listing accompanies this application, and has also been submitted with identical contents in the form of a computer-readable ASCII file on a floppy diskette and a CDROM.
1. Field of the Invention
The present invention concerns immune responses initiated by the recognition of a peptide:MHC complex on the surface of antigen presenting cells by T-cells. The present invention also concerns immune responses initiated by the binding of a Signal-2 moiety to its complement protein on the surface of an antigen presenting cell. More particularly, the present invention concerns the immune responses initiated by the recognition of the peptide:MHC by the T-cell and by the binding of a Signal-2 moiety to its complement protein. Still more particularly, the present invention concerns the modification of the typical immune response generated by a particular individual in response to this binding. Most particularly, the present invention concerns the conjugation of peptides derived from the peptide portion of the peptide:MHC complex to the preferred Signal-2 moiety in order to modify or shift a given immune response from type-1 to type-2 or from type-2 to type-1. This may include specific phenotypes of regulatory T-cells including suppressor T-cells.
2. Description of the Prior Art
Autoimmune diseases are characterized by the activation of T-cells against self-antigens. These T-cells then destroy cells presenting these antigens. For example, insulin-dependent diabetes mellitus (IDDM, also called Type-I diabetes) is characterized by the activation of T-cells against the insulin-producing cells of the pancreas and their subsequent destruction by these T-cells. The diseases and conditions associated with autoimmune responses are strongly associated with specific subtypes (alleles) of cell surface proteins called major histocompatability complex (MHC) class II molecules. MHC molecules bind fragments (peptides) of proteins from infectious agents, allergens, and selfproteins, and this MHC:peptide complex is the structure that T-cells recognize with their receptor (called the T-cell receptor, or TCR). The MHC:peptide complex is displayed on the surfaces of other cells of the immune system (i.e., B cells, dendritic cells and macrophages) which are called antigen presenting cells (APC). In order for an immune response to ensue, the major regulatory cell of the immune system, the undifferentiated T-cell, must be presented with small breakdown products (peptides) of the foreign invader. This presentation occurs on the surface of the APC. The T-cell must then interact with the APC, and this interaction stimulates the T-cell to divide and differentiate to produce molecules that attack, either directly or indirectly, cells displaying the same or highly similar MHC:peptide complex. It is well known that the genes that encode the MHC molecules are extremely variable within the species, and the different MHC alleles prefer to bind some peptides over others. Along with other genetic and environmental factors, the existence of different MHC alleles helps to explain why some members of a species develop conditions such as autoimmune diseases, allergies, asthma, and even certain infectious diseases, while others remain seemingly unaffected, or immune, to the same substances. Other differences arise because cell surface proteins distinct from the peptide:MHC complex must also bind to specific receptors on the T-cell. These other protein:protein pairs at the interface of the T-cell and APC membranes provide a costimulatory signal, known as Signal-2 which, along with the signal generated by the TCR recognition of the MHC:peptide complex (known as Signal-1), initiates an immune response.
A defining stage of the immune response is the differentiation of CD4+ T-cells into either type-1 helper T-cells (TH1 cells) or type-2 helper T-cells (TH2 cells) as a result of the two signals. These two subtypes of TH cells and the regulatory network of cells that they selectively activate are well-known correlates of human health conditions and disease states. Differentiation into TH1 cells results in predominantly cell-mediated immunity while differentiation into TH2 cells results in predominantly humoral immunity. Each of these immunity types helps to protect the body against different types of invasion. Type-1 immunity protects the body against intracellular pathogens such as bacteria, but is also implicated in organ-specific autoimmune diseases. Type-2 immunity is important for protection against extracellular parasites, but is associated with allergic reactions as well. Development of TH1 cells is driven by a cytokine called interleukin-12, which is produced by immune cells known as macrophages and dendritic cells. Interleukin-12 induces or stimulates the naive T-cell (CD4+ T-cells) to produce interferon-γ (IFN-γ) and interleukin-2 (IL-2). These two cytokines (IL-2 and IFN-γ) are involved in classic cell-mediated functions such as clonal expansion of cytotoxic T-lymphocytes (CTLs), macrophage activation, and class switching to IgG isotypes that mediate complement lysis of sensitized cells. Commitment to a TH1 immune response is enhanced by the presence of IFN-γ which up-regulates expression of the interleukin-12 (IL-12) receptor while inhibiting the development of TH2 cells. TH2 immunity results from the production of interleukin-4 (IL-4) by the naive T-cell. IL-4 induces TH2 development and the subsequent production of interleukins-4 (IL-4), -5 (IL-5), -10 (IL-10), and -13 (IL-13). IL-4 also operates to down-regulate expression of the IL-12 receptor on developing cells, thereby inhibiting TH1 development and helping undifferentiated T-cells to commit to TH2 cell development. Additionally, IL-4 and IL-5 are known to activate B cells and switch to neutralizing antibody (IgG1 in the mouse) and IgE, the initiator of immediate hypersensitivity.
In order for either of these immune pathways to be activated, a two-signal mechanism is required to fully activate the T-cell. Signal-1 (S-1) occurs when the T-cell antigen receptor (TCR) recognizes the peptide:MHC-II complex on the surface of an antigen presenting cell (APC). This first signal passes through the T-cell receptor and initiates a cascade of tyrosine phosphorylation/dephosphorylation events mediated by kinases and phosphatases and leads to the activation of Ca++ flux, nuclear factor of activated T cells (NF-AT) and NFκB transcription factors. These factors enter the nucleus of the T-cell and bind to promoters of genes responsible for effector functions. Signal-2 (S-2) arises from the binding of Signal-2 receptors to their ligands on the surface of an APC. Signal-2 receptors include CD28 and its ligand B7 as well as LFA-1 and its ligand ICAM-1. When a Signal-2 receptor and its ligand form a complex at the interface between the T-cell and APC receptor membranes, a series of signaling events occur. These events include serine/threonine phosphorylation/dephosphory-lation and activation of guanine nucleotide exchange factors that activate adapter proteins with GTPase activity. These signaling events activate a separate set of transcription factors. The signal delivered through the CD28:B7 complex is distinct from that delivered from the ICAM-1:LFA-1 complex, particularly with respect to the differentiation of CD4+ T-cells into TH1 versus TH2 effector populations. When the predominant binding occurs between LFA-1 and ICAM-1, the CD4+ T-cell differentiation favors TH1 cells which are abundant producers of IL-2 and IFNγ, the preeminent initiators of inflammatory immune responses including delayed-type hypersensitivity (DTH), immunity to intracellular pathogens, and several autoimmune diseases. When the predominant binding occurs between CD28 and B7, the CD4+ T-cells differentiate into TH2 cells. In contrast to TH1 cells, TH2 cells do not produce abundant IL-2 or IFNγ cytokines, but instead release the mediators of immediate-type hypersensitivity such as allergy and asthma, i.e., IL-4, IL-5, IL-10, and IL-13. Thus, the ability to manipulate the relative contribution of the complex providing the second signal has a profound effect on the type of immune response that is elicited against a given self-tissue antigen.
The associations between the TCR and APC occur at a specialized junction or interface between the TCR and the APC called the immunological synapse. An immune synapse is depicted schematically in
In more detail, to activate the pathway leading to TH1 dominance, the TCR recognizes the peptide:MHC-II complex and sends Signal-1 to the T-cell. Additionally, LFA-1 binds to ICAM-1, and these molecules, along with the peptide:MHC-II complex, translocate to form the end-stage immune synapse. This leads to the effective expression of the CD40 ligand (CD154) by the uncommited TH cell. CD40 interaction (expressed on the antigen presenting cell) with its ligand generates NFκB up-regulation of the inflammatory cytokine, IL-12. IL-12 then binds to its receptor on the undifferentiated TH cell and initiates the TH1 program, including the up-regulation of the transcription regulators, Stat4 and Tbet. This leads to TH1 dominance against the autoantigen (e.g., glutamic acid decarboxylase, GAD65), which was initiated by the GAD65 peptide component of the TCR:peptide:MHC-II complex. For the pathway leading to TH2 dominance, the TCR can recognize the same peptide:MHC-II complex, thereby sending Signal-1. However, in this case, a weaker strength of Signal-1 and/or altered or blocked binding between Signal-2 moieties leads to an altered form of the end-stage immune synapse. Likely, this lower strength of Signal-1 or distinct participation of the LFA-1 second signal leads to this different result, i.e., dominant TH2 differentiation. For example, the altered immune synapse can dictate that the CD40 ligand is not expressed and IL-12 is therefore not released by the APC. This pathway is schematically represented in
A major goal of modern applied immunology is to be able to switch from TH1-dominant immunity (e.g., as seen in autoimmune diseases and transplant rejection) to TH2 responses against these same tissue antigens. In other cases, it would be extremely valuable to replace weak TH2 immunity with TH1 dominance leading to strong T-cell proliferation and the effective generation of cytotoxic T-cells (CTL). These cases may include chronic viral illnesses, like hepatitis-C and AIDS; and could include certain cancers like melanoma. Accordingly, what is needed in the art is modifiers of these immune responses so that type-2 immunity can be replaced with type-1 immunity or type-1 immunity can be replaced with type-2 immunity, as desired in order to combat different human disease states or health conditions.
The present invention solves the problems found in the prior art and provides a distinct advance in the state of the art. Briefly, the present invention embraces a peptide which includes a portion of a Signal-1 moiety at one end and a portion of a Signal-2 moiety at the other end. These two ends can be directly connected to each other or connected via a flexible, non-substrate linker. This conjugation of the peptide portions directly and via a linker into a continuous peptide chain produces a new class of immunotherapeutic peptides termed bifunctional peptide inhibitors (BPI). These BPI are based upon the two signal mechanism of T-cell activation and link Signal-1 and Signal-2 moieties in order to alter T-cell activation. In other words, the present invention provides a method of modulating T-cells and subsequent immunity in a very specified manner such that only specific disease-associated populations of these cells are targeted by the products of the present invention. Thus, the present invention leaves necessary components of the intact immune system to operate in their nominal protective manner.
In more detail, the present invention describes constructing a peptide sequence having a TCR epitope of interest (a Signal-1 moiety) at one end and a peptide derived from the protein:protein interaction (the Signal-2 moiety) which generates Signal-2. These two peptide sequences can be connected via a flexible linker which couples the Signal-1 moiety to the Signal-2 moiety or can be directly linked together. In some cases, the linkage between the two peptides sequences may include flanking residues from each portion. The combination of the Signal-1 moiety coupled with the Signal-2 moiety constitutes a BPI. Accordingly, once a TCR epitope of interest is identified and the desired immune response (type-1 or type-2) determined, a BPI according to the present invention, can be generated.
As noted above, an important stage of the immune response is the differentiation of CD4+ T-cells into either type-1 helper T-cells (TH1 cells) or type-2 helper T-cells (TH2 cells). Differentiation into TH1 cells results in predominantly cell-mediated immunity while differentiation into TH2 cells results in predominantly humoral immunity. Each of these immunity types help to protect the body against different types of invasion. TH1 cells protect the body against intracellular pathogens such as bacteria, and are also implicated in organ-specific autoimmune diseases. TH2 cells are important for protection against extracellular parasites as well as allergic reactions. Development of TH1 cells is driven by a cytokine called interleukin-12, which is produced by immune cells known as macrophages and dendritic cells. Interleukin-12 induces or stimulates the naive T-cell to produce interferon-γ (IFN-γ) and interleukin-2 (IL-2). These two cytokines (IL-2 and IFN-γ) are involved in classic cell-mediated functions such as clonal expansion of cytotoxic T-lymphocytes (CTLs), macrophage activation, and class switching to IgG isotypes that mediate complement lysis of sensitized cells. Commitment to a TH1 immune response is enhanced by the presence of IFN-γ which up-regulates expression of the interleukin-12 (IL-12) receptor while inhibiting the development of TH2 cells. This pathway is shown schematically in
TH2 immunity results from the production of interleukin-4 (IL-4) by the naive T-cell. IL-4 induces TH2 development and the subsequent production of interleukins 4 (IL-4), 5 (IL-5) and 13 (IL-13), through activation of the transcription regulator Stat6. IL-4 also operates to down-regulate expression of the IL-12 receptor on developing cells, thereby inhibiting TH1 development and helping undifferentiated T-cells to commit to TH2 cell development. Additionally, IL-4 and IL-5 are known to activate B cells and switch to neutralizing antibody (IgG1 in the mouse) and IgE, the initiator of immediate hypersensitivity. Again, a schematic representation of this process is depicted in
As noted above, a two-signal mechanism is required to fully activate the TH cell. Signal-1 occurs when the T-cell antigen receptor (TCR) recognizes or engages the peptide:MHC-II complex on the surface of an antigen presenting cell (APC). This first signal is transmitted through the T-cell receptor and initiates a cascade of tyrosine phosphorylation/dephosphorylation events mediated by kinases and phosphatases and leads to the activation of Ca++ flux, NF-AT and NFκB transcription factors. These factors enter the nucleus of the T-cell and bind to promoters of genes responsible for effector functions. Signal-2 arises from the binding of a Signal-2 receptor on the T-cell to its protein ligand on the APC. Signal-2 receptors include CD28 and its ligand B7 as well as LFA-1 and its ligand ICAM-1. When a Signal-2 receptor and its ligand form a complex at the interface between the T-cell and APC membranes, a series of signaling events occurs including serine/threonine phosphorylation/dephosphorylation along with actuation of guanine nucleotide exchange factors that activate adapter proteins with GTPase activity. These signaling events activate a separate set of transcription factors. The signal delivered through the CD28:B7 complex is distinct from that delivered from the ICAM-1:LFA-1 complex, particularly with respect to the differentiation of CD4+ T-cells into TH1 versus TH2 effector populations. A schematic representation of this signaling is provided herein as
The associations between the TCR and APC occur at a specialized junction called the immunological synapse (shown in
In one aspect of the present invention, known TCR epitopes are used as the first peptide portion of the BPI. In this manner, minimal peptide sequences that are potent immunogens are utilized. These minimal peptide sequences (e.g. antigenic peptides) effectively engage the TCR involved in immune responses of interest (i.e. autoimmune diseases, infectious diseases, allergies, cancers, etc). There are already many known TCR epitopes of interest (Signal-1 moieties) and their sequences have been defined in the literature. A partial list of some representative Signal-1 moieties is provided in Table 1. This list is by no means exhaustive as there are potentially thousands of Signal-1 moieties.
In another aspect of the present invention, TCR epitopes of interest are identified so that the first portion of the BPI can be synthesized. In this aspect, these dominant TCR epitopes have been so determined by previous art and the sequences are available in the literature. The peptide to which a given T-cell response is focused upon, (e.g., the response against the diabetes-associated antigen GAD65) is identified by the fact that most effector T-cells respond to this portion of the antigen and not other portions. In mouse model systems, animals are immunized with the whole protein antigen. Next, T-cells are removed after the antigen has primed the immune system. These T-cells are placed separately in cultures with short overlapping peptides of the antigen. Most of the response will be to a single peptide and this is the dominant TCR epitope. In humans, T-cells are first cloned from patients. These cloned T-cells are placed separately in cultures with overlapping peptides (again, representing individual portions of the antigen involved, e.g., HIV-1, p24 (SEQ ID No. 8)). Again, the peptide to which most T-cell clones respond is the dominant TCR epitope. The foregoing is described by Schountz et al., MHC Genotype Controls the Capacity of Ligand Density to Switch T Helper (Th)-1/Th-2 Priming In Vivo, 157 The Journal of Immunology 3893-3901 (1996), the teachings and content of which are hereby incorporated by reference herein.
In another aspect of the present invention, peptides derived from Signal-2 receptors are used to alter interactions between the nominal receptors on T-cells and their complementary ligands on the APC surface. Table 3 includes a representative list of some known Signal-2 receptor moieties. Of course, those of ordinary skill in the art will be able to identify other Signal-2 moieties not listed therein, as this list is representative and not all-inclusive.
Another aspect of the present invention is the linking of the TCR epitope (i.e. the Signal-1 moiety) to a Signal-2 receptor peptide mimic (i.e., the Signal-2 moiety) in order to modify the resultant immune response. This linkage can be between the Signal-1 moiety and the Signal-2 moiety directly, or through flanking residues. Alternatively, this linking can be done via a linker which is positioned between the Signal-1 moiety and the Signal-2 moiety. The linker could be any amino acid including naturally occurring or chemically synthesized amino acids. Preferably, non-substrate amino acids will be used due to their resistance to protease attack. Still more preferably, the linker will comprise a non-substrate amino acid alternating with a small or hydrophilic amino acid. Even more preferably, the linker is synthesizable as one continuous sequence along with the Signal-1 and Signal-2 moieties, which flank the linker at each respective end. Still more preferably, the linker has the general formula (A,B)X, wherein A and B are amino acid residues, and the A amino acid residue is individually and respectively selected from the group consisting of aminocaproic acid, aminohexanoic acid, aminododecanoic acid, and β-alanine, and the B amino acid residue is a small or hydrophilic amino acid. In this formula, X can range from 1 to 100. A particularly representative B residue is glycine. In this embodiment, a linker could potentially have aminocaproic acid (Ac), aminohexanoic acid (Ahx), aminododecanoic acid (Ado), and β-alanine (βA) alternating with glycine residues (G) (e.g., Ac-G-Ahx-G-Ado-G-βA). The choice of the residues used to construct the linker can be based upon the desired length of the linker as well as steric hindrance considerations. One preferred linker comprises alternating Ac and G residues. This linker can be lengthened or shortened by the inclusion of the other amino acid residue choices (Ahx, Ado, βA). Some representative linkers are included in Table 2 as SEQ ID Nos. 26-29.
Approximately 109 different TCR account for protective immunity to the universe of infectious agents and contain the repertoire of TCR that may turn against self in autoimmune diseases. Moreover, the TCR are also specific for potential tumor antigens and the myriad of allergenic substances in the environment. By changing the TCR epitope of a given BPI we direct the immunomodulating capacity of the BPI to a select group of TCR. In other words, the selection of a TCR epitope to incorporate into the BPI targets T-cells that are involved in a particular human disease in a highly specific fashion. For example, incorporating the GAD65 epitope into a BPI targets autoaggressive T-cells involved in the induction of type-1 diabetes. This targeting to specific TCR allows that T-cells necessary for immunity to infectious agents or cancers will not be significantly compromised. Thus, BPI offer the possibility to specifically modulate T-cell immunity to one antigen while leaving intact the T-cell repertoire necessary for protective immunity to infectious agents and developing cancers.
As noted above, the Signal-1 moieties of the present invention are preferably derived from TCR epitopes and a list of representative known epitopes is provided in Table 1 wherein these known epitopes are presented as SEQ ID Nos. 1-25. When a derivative of a TCR epitope is used to construct the BPI, preferably, the TCR epitope selected will be correlated with a known health condition or disease state. When using one of the representative peptides shown in Table 1 to construct the BPI, it is preferred that the peptides include a sequence having at least about 10% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 1-25. More preferably, the peptide will have at least 30% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 1-25. More preferably, the peptide will have at least 50% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 1-25. Even more preferably, the peptide will have at least 70% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 1-25. Most preferably, the peptide will have at least about 95% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 1-25. Of course, it is also well known in the art to use peptidomimetics to construct moieties having similar functions as the peptides derived from the TCR epitopes. In this respect, the teachings of Falcioni et al. in Peptidomimetic Compounds That Inhibit Antigen Presentation by Autoimmune Disease-Associated Class II Major Histocompatability Molecules, 17 Nature Biotechnology, 562-567 (1999), are incorporated by reference herein. Accordingly, all or part of this Signal-1 moiety portion of the BPI can include such peptidomimetics. Preferably, the peptidomimetic will be a mimetic of a peptide selected from the group consisting of SEQ ID Nos. 1-25. Alternatively, the Signal-1 moiety will be a derivative of a TCR epitope or a peptide selected from the group consisting of SEQ ID Nos. 1-25. At any rate, it is desired that this first portion of the BPI (or the portion responsible for initiating the first signal) be capable of binding with a major histocompatability complex (MHC) on an antigen presenting cell (APC). Furthermore, it is preferred that this resulting peptide:MHC complex be capable of engaging important TCR and initiating some form of the signal to the T-cell.
As noted above, the peptides used on the side of the linker opposite the Signal-1 moiety are preferably derived from Signal-2 receptors. This second portion of the BPI is connected to the first portion either directly or via the linker. In preferred forms, the second portion includes a sequence having at least about 10% sequence homology with a sequence selected from a group consisting of SEQ ID Nos. 30-41. More preferably, the second portion peptide has at least about 30% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 30-41. Still more preferably, the second portion peptide has at least about 50% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 30-41. Even more preferably, the second portion peptide has at least about 70% sequence homology with a sequence selected from the group consisting of SEQ ID Nos. 30-41. Most preferably, the second portion peptide includes a sequence having at least about 95% sequence homology with a sequence selected from the group of SEQ ID Nos. 30-41. As with the first portion, peptidomimetics can be used in place of all or some of the amino acid residues of the second portion. In preferred forms the peptidomimetic of the second portion will be a mimic of a peptide selected from the group consisting of SEQ ID Nos. 30-41. Alternatively, the second portion of the BPI will comprise a derivative of a peptide selected from the group consisting of SEQ ID Nos. 30-41. Similar to the first portion, it is preferred that the second portion be capable of binding with a complementary ligand (e.g. the Signal-2 ligand) on an antigen presenting cell. For example, when a peptide derived from LFA-1 is used as this Signal-2 moiety of the BPI, it should bind to ICAM-1 on the surface of the APC. Additionally, it is preferred that this binding with the Signal-2 ligand on the APC inhibits or alters the binding of the moiety's parent receptor (on the T-cell) to this same APC ligand.
As explained above, the immune response involves a two signal mechanism and the purpose of the present invention is to modify a given immune response, e.g., from type-1 immunity to type-2 immunity or from type-2 immunity to type-1 immunity. This modification or shifting of immune response phenotype is brought about by BPI according to the present invention. It is preferred in some cases for the BPI to modify an immune response from a TH1 dominated or cytolytic immune response to a TH2 dominated response; and, in other cases, it is preferred for the BPI to modify an immune response from a TH2 dominated response to a TH1 or cytolytic dominated response. In some cases, BPI may operate via the activation of very specific T-cell phenotypes, e.g., peptide-specific suppressor T-cells. In contrast to the nominal situation where an antigen stimulates the system toward a TH1 response (depicted in
Thus, an important aspect of the present invention is that tethering a specific TCR epitope to a Signal-2 receptor peptide mimic leads to alteration of T-cell differentiation involving T-cells bearing only these receptors and/or T-cell populations indirectly linked to these peptide specific subsets. The ability to block or alter T-cell responses to a given immunodominant peptide antigen would offer extremely precise treatments for immunopathological conditions. A major drawback to current immunotherapies is that broad specificities of T-cells are affected leaving the host more susceptible to infections and cancers. The BPI of the present invention should block and/or alter only the desired T-cell population and subsequent responses that depend on these initial T-cells. Also, BPI will target a specific TCR-bearing population for activation toward a desired effector function.
In another aspect of this invention, the relative strength of signal generated by the T-cell-APC interaction has an affect on whether the ultimate immune response is a type-1 or a type-2 response. In this regard, the teachings of Murray in How the MHC Selects TH1/TH2 Immunity, 19 Immunology Today 157-163 (1998) are hereby incorporated by reference.
In another aspect of the present invention, an immune response is modified by contacting an APC with a peptide capable of binding to an MHC and to a Signal-2 ligand on the APC and causing an altered signal to be transmitted to the T-cell. Thus, the immune response is deviated from the immune response generally associated with the immunogenic peptide and its corresponding antigen (i.e., infectious agent, self protein, or allergen).
In another aspect of the present invention, a peptide having the general formula AXB is provided. The A, X, and B represent a chain of amino acid residues wherein the A chain has at least about five residues and at least about 10% sequence homology with a TCR epitope, the B chain has at least four residues and at least about 10% sequence homology with a peptide derived from a Signal-2 moiety, and the X chain is a linker. The linker could be any amino acid including naturally occurring or chemically synthesized amino acids. Additionally, the X chain has at least one residue. It is possible to link A to B directly without X as well, although a linker of some size is preferred in order to span the distance between the MHC-II and second signal ligands on the APC surface. As noted above, preferably, non-substrate amino acids will be used due to their resistance to protease attack. Still more preferably, the linker will comprise a non-substrate amino acid alternating with a small or hydrophilic amino acid. Even more preferably, the linker is synthesizable as one continuous sequence along with the Signal-1 and Signal-2 moieties, which flank the linker at each respective end. Still more preferably, the linker has the general formula (A,B)X, wherein A and B are amino acid residues, and the A amino acid residue is individually and respectively selected from the group consisting of aminocaproic acid, aminohexanoic acid, aminododecanoic acid, and β-alanine, and the B amino acid residue is a small or hydrophilic amino acid. In this formula, X can range from 1 to 100. A particularly representative B residue is glycine. In this embodiment, a linker could potentially have aminocaproic acid (Ac), aminohexanoic acid (Ahx), aminododecanoic acid (Ado), and β-alanine (βA) alternating with glycine residues (G) (e.g., Ac-G-Ahx-G-Ado-G-βA). The choice of the residues used to construct the linker can be based upon the desired length of the linker as well as steric hindrance considerations, hydrophobicity, charge, etc. One preferred linker comprises alternating Ac and G residues. This linker can be lengthened or shortened by the inclusion of the other amino acid residue choices (Ahx, Ado, βA). Some representative linkers are included in Table 2 as SEQ ID Nos. 26-29. Additionally, the X chain is positioned between the A chain and the B chain and the entire peptide can be synthesized as one continuous sequence. Some preferred sequences will have an A chain having at least about 10% sequence homology with any one of SEQ ID Nos. 1-25, an X chain having at least about 2% sequence homology with any one of SEQ ID Nos. 26-29, and a B chain having at least about 10% sequence homology with any one of SEQ ID Nos. 30-41. Preferably, the peptide is capable of shifting a type-1 response to a type-2 response, or vice versa. Of course, peptidomimetics may be synthesized to mimic any part of the BPI, including the linker. Preferably, the A chain binds to the MHC on an APC to form a peptide:MHC complex. This complex is capable of engaging the TCR on critical T-cell populations. Still more preferably, the B chain is capable of binding to a Signal-2 ligand on the APC at the same time as the formation of the peptide:MHC complex. This combined binding to the APC should be capable of altering the signal delivered to the T-cell. The combination of the first signal and the second signal are capable of fully activating a T-cell and by selecting the peptide used for the A chain and the peptide used for the B chain, the immune response can be deviated from its normal progression. In the case of a normally activated type-1 response leading to the up-regulation of TH1 cells, the response can be altered to give a type-2 response leading to the up-regulation of TH2 cells. In the case of a normally activated type-2 response leading to the up-regulation of TH2 cells, the response can be altered to give a type-1 response leading to the up-regulation of TH1 cells. Again, the A chain can be chosen based on the health condition normally associated with the sequence (for example, see Table 4).
In another aspect of the present invention, a method is provided for preparing a peptide for modulating immune responses. This method comprises the steps of selecting a first peptide sequence which has at least about 10% sequence homology with a sequence derived from a TCR epitope, selecting a second peptide sequence which has at least about 10% sequence homology with a sequence derived from a Signal-2 receptor moiety, selecting a third peptide sequence which is a flexible, non-substrate linker, and synthesizing the peptides as a continuous peptide chain. Preferably, the linker is flanked on one end with the peptide derived from the TCR epitope and flanked on the other end with the peptide derived from the Signal-2 moiety. Preferably, the first peptide sequence should be associated with a known health condition and be capable of binding with an MHC on an APC. Similarly, it is preferred that the second peptide sequence be capable of binding with a Signal-2 ligand moiety on the APC. The method can further comprise the step of contacting the nominal peptide immunogen with the TCR, thereby binding the first peptide sequence to the MHC and the second peptide sequence to the Signal-2 ligand, thereby generating APC bearing potent first and altered/blocked second signal ligands which activate a desired immune response.
Inherent in the BPI design is the antigen-specific moiety that a given T-cell population is activated to respond against (i.e., the TCR epitope), ultimately leading to the cascade of immune reactions that generate protective or in some cases pathologic immune responses. These epitopes when bound to major histocompatibility complex (MHC) molecules on the antigen presenting cell (APC) surface provide the Signal-1 of the two-signal mechanism of T-cell activation. Thus, an important consideration is the affinity of a given peptide for MHC molecules. This affinity was directly tested by the binding of biotinylated peptide to the cells that present the peptide:MHC complex to the T-cells in mouse and man. T-cell clones to possible peptide epitopes were generated and tested for binding to the immunodominant TCR of a response and specifically stimulate T-cell functions in vitro by the ELISPOT assay. Because modifications in the peptide residues that actually contact the TCR are part of the BPI development, it is also preferable that known crystallographic structures of the epitope bound to MHC molecules are available. This allows for precise three-dimensional predictions of how a particular amino acid substitution or mimetic will affect the actual structure encountered by the developing T-cells. However, in the absence of known crystal structures, it is possible to predict the shape of a hypothetical peptide:MHC structure based upon the available coordinates of other peptide:MHC structures. This predictability is partially due to established peptide binding motifs, that allow for prediction of which residues of the epitope fit most readily into the particular binding pockets of a given MHC allele. These predictions have been accomplished using two different alleles (I-As and I-Ab) binding a collagen peptide wherein each polymorphic position of the I-A molecules were substituted with the known amino acids at these positions. The overall structure was based upon the published coordinates of a reference allele I-Ak binding a different peptide. Thus, in cases where the 3D structure of a particular disease-associated epitope is not known, a structure for predicting where the TCR contacts the peptide by a similar allelic-substitution and modeling approach can still be generated. It is important to identify (or at least predict) these TCR contact positions. It is well-known that certain alterations to TCR-contact positions can change the functional differentiation of T-cells into the TH1 or TH2 types that can determine the course of immunity (see Murray, et al., Major Histocompatibility Complex (MHC) Class II Molecules Direct TCR-Specificity for Opposite Ends of the Same Immunogenic Peptide in TH1 or TH2 responses (unpublished manuscript, 2000); and Murray; 19 Immunology Today 157-163 (1998), the teachings of which are hereby incorporated by reference.
Specifically, it was verified that the peptide binds to MHC molecules on live APC. Briefly, spleen cell density-gradient fractions (from mice), or PBL, or APC lines (from humans) were incubated in round bottom 96-well plates with increasing concentrations of individual biotinylated peptides at 37° C., 5% CO2 for 16 hours. Following binding of the BPI to the APC, Avidin-FITC was incubated with the cells on ice for 30 minutes, followed by biotinylated anti-Avidin for 1 hour, then again with Avidin-FITC. For BPI titrations, increasing concentrations (0.1-100 μM) of the biotinylated derivatives in sterile 0.5% BSA-PBS were incubated with the APC for 16 hours as above. As shown in
Next, T-cell clones were generated for determination of TCR epitopes for later use in BPI. These experiments utilized CD4+ or CD8+ T-cell clones from humans or mice immunized against predicted TCR epitopes using previously described methods (Murray et al., 24 Eur. J. Immunol. 2337-2344 (1994); Murray; 19 Immunology Today 157-163; and Schountz et al., 157 The Journal of Immunology 3893-3901 (1996)). These clones were maintained by biweekly restimulation with irradiated histocompatible lymphocytes, the peptide, and recombinant IL2. To determine if a given TCR-epitope is effective for the activation to cytokine synthesis, an ELISPOT assay was used. Of course, other cytokine assays could also be used. For analysis, BPI that have been substituted at predicted TCR-contact positions will be used to determine which of these BPI variants are most effective in the inhibition of proliferation and cytokine release from individual clones as analyzed above. Predicted positions will be scanned with different amino acids or mimetics to alter the interaction with the TCR in the structures generated by molecular modeling.
Next, Molecular Dynamics/Energy Minimizations (MDEM) will be used to study the peptide conformations. MD simulations will be used in conjunction with crystallographic data to predict the solution conformations of the BPI. MDEM simulations can predict flexibility and conformational changes during binding of BPI to MHC-II and ICAM-1. Amino acid substitutions can also be introduced into the Signal-1 and/or Signal-2 moieties in order to favor simultaneous binding to both receptors, as first predicted by these model studies. The I-Ag7:BPI:D1 structure (see
The second stage in the BPI process is selection of peptide mimics of established second signal receptor molecules involved in the functional differentiation of T-cells. To determine these structures, the crystallographic structures and available models of the second signal receptors bound to their physiological ligands will be used to predict the regions of the receptors that make contact with the ligand. This approach was used to design the EGAD-BPI which can be depicted as
These interactions can also be determined empirically by making site-directed changes in the second signal molecules and determining whether such mutant molecules still bind to the ligand in question. However, many of these studies have been performed with known second signal receptors, including LFA-1, CD28/CTLA4, CD40L, and CD95L. Molecular modeling studies will be used as above to model the interactions of these second signal moieties with their ligands separately and when the moiety is part of the BPI structure.
Once candidate BPI moieties that bind effectively to the MHC and second signal ligands and that alter T-cell functions in vitro were identified, these moieties were synthesized at opposite ends of relatively short synthetic peptides, connecting them by a non-proteolytic substrate linker of variable length. As described by the prototypical EGAD-BPI, the Signal-1 moiety (i.e., the disease-associated TCR epitope) was linked by this flexible linker to the Signal-2 receptor mimic peptide (i.e., a second signal receptor thought to be involved in TH1/TH2 differentiation. These synthetic peptides were generated by conventional methods of peptide synthesis. In some cases, BPI are tested for binding to isolated MHC and second signal ligands, and NMR, molecular modeling and crystallography are used to determine their exact 3D structures. Finally, it was determined whether a given BPI was biologically active in vivo. Mice were treated with the BPI and immune cells isolated and tested for cytokine production by ELISPOT (see
To synthesize the BPI, Fmoc chemistry on chlorotrityl resins was used. Protected amino acids were double coupled at 8-fold excess for 1 hour. Resins were DMF and MeOH washed and cleaved in Reagent R: TFA, EDTA, Thioanisole, Anisole. The TFA mixture containing the peptide in solution was precipitated in ether and washed extensively. Preparative HPLC of peptides was accomplished by a gradient of 0-80% acetonitrile in 0.1% TFA. Lyophilization of the various fractions and verification by MALDI-TOF using a Voyager mass spec (PerSeptive, Foster City, Calif.) yielded the synthetic peptide as a TFA salt. Modeling, crystallography and binding studies, as described above, were used to generate the predicted BPI complex structure.
In another example which tested the diabetes BPI in vivo, 15 million disease-linked lymphocytes (i.e., patient T-cells, or T-cell populations linked to the disease process) were injected with or without T-cells that received the BPI compound (in vivo or in vitro) and were expanded for 24 hours in recombinant IL-2. Some experiments will deplete specific subsets of the T-cells using mAb to CD154, CD25, CD62L, CD152, etc. and magnetic particles prior to adoptive transfer. T-cells (from mice or humans) treated with the individual moieties of the BPI will be used as negative controls along with CD4+ cells from mice treated with saline alone (
To evaluate the disease process by immunohistology (see, e.g.,
A few representative assembled BPI consisting of a Signal-1 moiety and a Signal-2 receptor moiety joined together via a linker are provided in Table 4 as SEQ ID Nos. 42-46. These representative BPI are operable for shifting specific immune responses from a type-1 to a type-2 response and vice-versa. Advantageously, other immune responses to other antigenic peptides will be preferably unaffected.
a is a graph representing the results of a flow cytometry analysis comparing the binding of a representative BPI to different mouse strains;
b is a graph representing the results of a flow cytometry analysis comparing the binding of a representative BPI portion to different mouse strains;
c is a graph representing the results of a flow cytometry analysis comparing the binding of a representative BPI portion to different mouse strains;
a is a graph illustrating the results of a flow cytometry analysis of a representative BPI binding to the APC of a mouse strain, without antibodies to MHC-II or ICAM-1;
b is a graph illustrating the results of a flow cytometry analysis of a representative BPI binding to the APC of a mouse strain, when antibodies to ICAM-1 are present;
c is a graph illustrating the results of a flow cytometry analysis of a representative BPI binding to the APC of a mouse strain, when antibodies to MHC-II are present;
a is a graph representing the ELISPOT analysis of IL-4 cytokine release by T-cells taken from NOD mice treated with the EGAD-BPI or the saline control;
b is a graph representing the ELISPOT analysis of IFN-γ cytokine release by T-cells taken from NOD mice treated with the EGAD-BPI or the saline control;
c is a graph representing the ELISPOT analysis of IL-4 cytokine release by T-cells taken from NOD mice treated with the AGAD-BPI or the saline control;
d is a graph representing the ELISPOT analysis of IFN-γ cytokine release by T-cells taken from NOD mice treated with the AGAD-BPI or the saline control;
a is a representative color photograph of the histological analysis of pancreata infiltration by mononuclear cells in NOD mice treated with the saline control;
b is a representative color photograph of the histological analysis of pancreata infiltration by mononuclear cells in NOD mice treated with the GAD peptide;
c is a representative color photograph of the histological analysis of pancreata infiltration by mononuclear cells in NOD mice treated with the EGAD-BPI;
d is a representative color photograph of the histological analysis of pancreata infiltration by mononuclear cells in NOD mice treated with the CD11a peptide; and
As used herein, the following definitions will apply: “Sequence Identity” as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are “identical” at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. et al., eds., M. Stockton Press, New York (1991); and Carillo, H., et al. Applied Math., 48:1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLMNIH Bethesda, MD 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% “sequence identity” to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 95% identity relative to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 95% sequence identity with a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.
Similarly, “sequence homology”, as used herein, also refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned as described above, and gaps are introduced if necessary. However, in contrast to “sequence identity”, conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence.
A “conservative substitution” refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, charge, hydrophobicity, etc., such that the overall functionality does not change significantly.
“Isolated” means altered “by the hand of man” from its natural state., i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein. Finally, all references and teachings cited herein which have not been expressly incorporated by reference are hereby incorporated by reference.
Sequences including or having a sequence which has at least about 10% sequence identity with any one of SEQ ID Nos. 1-46 and which exhibit similar binding properties to APC or linking properties between two peptide sequences are within the scope of the present invention. Preferably, such sequences will have at least about 30% sequence identity with any one of SEQ ID Nos. 1-46, still more preferably at least about 50% sequence identity, even more preferably, at least about 70% sequence identity, and most preferably at least about 95% sequence identity. Alternatively, sequences including or having a sequence which has at least about 10% sequence homology with any one of SEQ ID Nos. 1-46 and which exhibit similar binding properties to APC or linking properties between the two adjacent peptide sequences are embraced in the present invention. More preferably, such sequences will have at least about 30% sequence homology with any one of SEQ ID Nos. 1-46, still more preferably at least about 50% sequence homology, even more preferably at least about 70% sequence homology, and most preferably at least about 95% sequence homology. Additionally, sequences which differ from any one of SEQ ID Nos. 1-46 due to a mutation event, a series of mutation events, or chemical derivatization but which still exhibit desired properties are also embraced in the present invention. Such mutation events or derivatizations include but are not limited to point mutations, deletions, insertions, rearrangements, peptidomimetics, and other chemical modifications.
A “linker” is defined as any amino acid including naturally occurring or chemically synthesized amino acids. Preferably, a “linker” is a flexible, non-substrate sequence of amino acid residues resistant to proteolytic degradation which can be used to conjugate and/or couple a Signal-1 moiety to a Signal-2 moiety.
A “Signal-1 moiety” is defined as a peptide epitope, i.e., the peptide portion of an antigen and/or mimetics of these antigenic peptides to which important TCRs bind.
A “Signal-2 moiety” or a “Signal-2 receptor moiety” is defined as a peptide portion of a second signal receptor known to bind to and/or affect binding of the receptor to its complimentary ligand on the APC. This can include peptide mimics and mimetics of the receptor/ligand structure of interest.
A “Signal-2 ligand” is the complementary protein of the Signal-2 receptor moiety on the APC to which the receptor portion and/or the Signal-2 receptor moiety has significant affinity and binds.
As used herein “derivative” with respect to peptides refers to changes produced by amino acid addition, deletion, replacement, substitution, and/or modification; mutants produced by recombinant and/or DNA shuffling; and salts, solvates, and other chemically synthesized/modified forms of the peptide that retain in part the activity of the isolated native peptide.
BPI were generated using automated peptide synthesis by a robotic multiple peptide synthesizer employing Fmoc amino acid chemistry by standard methods. Wang resin (p-benzyloxybenzyl alcohol polystyrene) was used as the solid support. Peptides were characterized by reversed-phase HPLC and electrospraymass-spectrometry. This synthesis, referred to as Merrifield peptide synthesis, utilizes traditional organic chemical reactions carried out on a solid material so that the peptide chain is lengthened while attached to the support structure. The peptides will be cleaved from the resin using TFA, and purified by reverse-phase HPLC and analyzed by mass spectroscopy. Alternatively, these reactions can be carried out in solution when larger amounts of the peptides are desired. Of course, the peptides of the invention may be synthesized or prepared by a number of techniques which are well known in the art. See, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W. H. Freeman and Co., New York, which is incorporated herein by reference in its entirety. Short peptides, for example, can be synthesized on a solid support or in solution. Longer peptides maybe made using recombinant DNA techniques. Here, the nucleotide sequences encoding the peptides of the invention may be synthesized, and/or cloned, and expressed according to techniques well known to those of ordinary skill in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning, A is Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, New York.
Alternatively, the peptides of the invention may be synthesized such that one or more of the bonds which link the amino acid residues of the peptides are non-peptide bonds. These alternative non-peptide bonds may be formed by utilizing reactions well known to those in the art, and may include, but are not limited to amino, ester, hydrazide, semicarbazide, and azo bonds, to name but a few. In yet another embodiment of the invention, peptides comprising the sequences described above may be synthesized with additional chemical groups present at their amino and/or carboxy termini, such that, for example, the stability, bioavailability, and/or inhibitory activity of the peptides is enhanced. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups, may be added to the peptides' amino termini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonyl group may be placed at the peptides' amino termini. Additionally, the hydrophobic group, t-butyloxycarbonyl, or an amido group may be added to the peptides' carboxy termini.
Purchasing preformed peptides provides another alternative source of peptides having 25 amino acids or less as these are easily purchased from commercial peptide synthesis laboratories. In later synthesis schemes, peptide mimetic compounds may be synthesized in place of the peptide moieties and linked by the same chemistry. The design of peptidomimetics is an established technique and known correlates of key amino acids of the peptide can be synthesized by previously published methods. Furthermore, as it is well known in the art, peptidomimetics may be developed which have the same modulation properties as the preferred peptides detailed herein. As these peptidomimetics require no more than routine skill in the art to produce, such peptidomimetics are embraced within the present application. Notably, the side chains of these peptidomimetics will be very similar in structure to the side chains of the preferred peptides herein, however, their peptide backbone may be very different or even entirely dissimilar. If resistance to degradation in vivo or greater conformational stability were desired, the peptides of the present invention could be cyclized by any well known method. One such method adds Penicillamine (Pen) and cysteine (Cys) residues to the N- and C-termini to form cyclic peptides via a disulfide bond between the Pen and Cys residues. The formation of this cyclic peptide restricts the peptide conformation to produce a conformational stability, thereby providing better selectivity for cell surface receptors than its linear counterpart.
The portion of the BPI which spans between the Signal-1 moiety and the Signal-2 moiety is referred to as a linker. As noted above, the linker is not essential in forming a BPI. However, when a linker is used, the linker can be any naturally occurring or chemically synthesized amino acid. Preferably, the linker is a non-substrate amino acid residue chain which helps to prevent protease attack. A particularly preferred linker is a repeating chain of the non-natural amino acid, aminocaproic acid (Ac), and the amino acid glycine (G) (e.g. Ac-G-Ac-G-Ac). If a shorter length was needed for the linker, beta-alanine residues (βAla) could be substituted for one or more of the Ac residues. If a longer chain was needed for the linker, amino-dodecanoic acid residues (Adod) could be substituted for one or more of the Ac residues. As is well known in the art, peptide mimetics of these linker amino acids may also be synthesized and inserted into the BPI structure.
The following examples set forth preferred embodiments of the present invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.
This example describes the methods used to generate the BPI.
Materials and Methods:
Synthesis of peptides was via Fmoc on chlorotrity resins. Protected amino acids were double coupled at 8-fold excess for one hour. Resins were dimethylforamide (DMF) and methanol (MeOH) washed and cleaved in Reagent R: trifluorolacetic acid (TFA), ethylene diamine tetraacetic acid (EDTA), Thioanisole, Anisole. The TFA mixture containing the peptide in solution is precipitated in ether and washed extensively. Preparative HPLC of peptides was accomplished by a gradient of 0-80% acetonitrile in 0.1% TFA. Lyophilization of the various fractions and verification by mass spectroscopy yielded the synthetic peptides as a TFA salt. Modeling, crystallography and binding studies are as described above.
Results:
The peptides produced in this example are provided in Table 1 and are also listed as SEQ ID Nos. 1-46. These peptides include the Signal-1 moiety, the Signal-2 moiety and the non-substrate linker between the two moieties. To produce the BPI, any Signal-1 moiety could be linked with any Signal-2 moiety via any linker using the peptide synthesis described above. In other words, the BPI are generated as one continuous peptide chain comprising a Signal-1 peptide sequence followed by a linker sequence followed by a Signal-2 peptide sequence. Additionally, some representative BPI were generated for later use in the experiments. These BPI are included herein in Table 4. However, it is important to note that these BPI are representative (as are each of the BPI portions listed in Tables 1-4) and not all inclusive.
Homo sapiens
Mus musculus
Homo sapiens
Yersinia HSP (322-333)
Yersinia enterocolitica
Homo sapiens
Homo sapiens
Homo sapiens
Felis domesticus
Felis domesticus
Ambrosia artenisiifdia
Ambrosia trifida
Cryptomeria japonica
Cryptomeria japonica
Apis mellifera
Hevia brasiliensis
Dermatophagoides
pteronyssinus
Homo sapiens
Homo sapiens
Homo sapiens
Mus musculus
Homo sapiens
Mus musculus
Mus musculus
Homo sapiens
Mus musculus
Homo sapiens
Mus musculus
Mus musculus
Discussion:
Once the linker, the Signal-1 moiety, and the Signal-2 moiety are chosen, the entire BPI can be synthesized using the above-described methods. There are thousands of potential Signal-1 moieties which could be used in connection with the present invention. Each of these moieties maybe associated with a distinct immunological response or disease state. Once the epitope structure and sequence are determined, an appropriate linker could be selected and the other portion of the BPI, i.e., the second signal moiety, can be chosen. Once all of these sequences have been determined, BPI can be designed using the peptide sequences themselves, peptidomimetics, or combinations of the two. Construction of appropriate peptidomimetics is detailed by Falcioni, et al, 17 Nature Biotechnology, 562-567 (1999), the content and teachings of which are hereby incorporated by reference herein.
These structures are illustrated by the preliminary mechanism depicted schematically (
Of course, it is possible that one of ordinary skill in the art could produce any number of peptidomimetics or derivatives which would have similar activity to the BPI, and such modifications are encompassed by the present invention as described in more detail above.
This example uses biotinylated BPI to test for competitive inhibition of BPI binding by unlabeled peptides or monoclonal antibodies to MHC-II and ICAM-1 on live APC, and to verify antigenic peptide binding to live APC. Additionally, it was shown that monoclonal antibodies to MHC-II or ICAM-1 effectively block binding of the diabetes BPI (GAD65(208-217)-[Ac-G-Ac-G-Ac]-CD11a (237-247)) (hereinafter referred to as EGAD-BPI) to NOD spleenocytes.
Materials and Methods:
To obtain biotinylated BPI, the synthesized EGAD BPI was biotinylated with NHS-Biotin as described in Murray et al., 24 Eur. J. Immunol. 2337-2344 (1994). Spleen cell density-gradient fractions from normal (unimmunized) NOD, BALB/c and other MHC congenic strains were incubated in round bottom 96-well plates with increasing concentrations of individual biotinylated peptides at 37° C., 5% CO2 for 16 hours. Following binding of the BPI to the APC, Avidin-FITC was incubated with the cells on ice for 30 minutes, followed bybiotinylated anti-Avidin for 1 hour, then again with Avidin-FITC. For BPI titrations, increasing concentrations (0.1-100 μM) of the biotinylated derivatives in sterile 0.5% BSA-PBS were incubated with the APC for 16 hours as above. Three-color analyses used Cy-Chrome or PE-conjugated anti-B220 (mAb RA3-6B2 (CD45R B-cell marker), anti-MHC class II (KH74 or 10-3.62 mAb), or anti-ICAM-1 (3E2 mAb); (all purchased from PharMingen, San Diego, Calif.). Bound peptide was detected with avidin-FITC/biotinylated anti-avidin/avidin-FITC on live cells gated by forward/side scatter analysis. Controls contained all detecting reagents in absence of the biotinylated peptide; 20,000 events were analyzed for each histogram with a FACScan (Becton-Dickinson) flow cytometer.
To test for competitive inhibition of BPI binding with unlabeled peptides or monoclonal antibodies to MHC-II and ICAM-1 on live APC, freshly isolated fractions of spleen cells were incubated with the previously biotinylated BPI. However, for this portion of the experiment, the experimental wells contained various unlabeled peptides (e.g., antigenic peptides or LFA-1 peptides), and/or monoclonal antibody (e.g., anti-MHC-II or anti-ICAM-1 mAb) inhibitors. Negative selection methods with monoclonal antibodies conjugated to magnetic particles were used to enrich the spleen cell fractions for B cells, macrophages, or dendritic cells as well as to examine differences in BPI binding to these different populations. These methods are detailed in Schountz et al., 157 The Journal of Immunology 3893-3901 (1996), the teachings and content of which were incorporated by reference above.
To verify antigenic peptide binding to live APC, initial EGAD-BPI were screened for selective binding to NOD (I-Ag7) APC and assayed for simultaneous binding using monoclonal antibodies against either MHC-II or ICAM-1 by flow cytometry methods using live APC (Murray et al., 24 Eur. J. Immunol. 2337-2344 (1994); Murray; 19 Immunology Today 157-163; and Schountz et al., 157 The Journal of Immunology 3893-3901 (1996)). In this assay, increasing concentrations of the biotinylated-BPI, -CD11a(237-247) or -GAD65(208-217) peptide were incubated overnight with spleenocytes from each inbred strain. Bound peptide was detected with amplification of avidin-FITC fluorescence by the use of a biotinylated anti-avidin reagent, followed by a second round of avidin-FITC binding. Biotinylated peptide was incubated with 106 viable cells at a peptide concentrations of 50 μM. Bound peptide was detected with avidin-FITC/biotinylated anti-avidin-FITC. For analysis, a forward/side scatter gate was set on live lymphocytes and 20,000 events were collected in this gate. Background fluorescence (detection reagents only) is shown in each panel of
To demonstrate that monoclonal antibodies to MHC-II or ICAM-1 effectively block binding of the diabetes BPI (EGAD-BPI) to NOD spleenocytes, an assay identical to that used for
Results and Discussion:
As illustrated by the data provided in
Additionally, as illustrated in
This example utilizes co-capping experiments to demonstrate simultaneous binding of the BPI to MHC-II and ICAM-1 molecules.
Materials and Methods:
Further support for simultaneous binding of the BPI to MHC-II and ICAM-1 molecules has been observed in co-capping experiments using biotinylated mAb 10-3.62 and streptavidin to cap MHC-II in the presence or absence of the BPI. To test the ability of the BPI peptide to link MHC-II and ICAM-1 molecules on the APC surface, we used a modification of a co-capping experiment originally described for monoclonal antibodies. Briefly, biotinylated monoclonal antibody to MHC-II (10-3.62) is incubated with freshly-isolated APC from NOD mice previously treated by intravenous (i.v.) injection of a given BPI variant or saline. Antibody-bound cells are then incubated with streptavidin (37 C×15 min.) to cap the MHC-II molecules on the APC surface. The cells were transferred to ice and labeled with a fluorescent (PE) monoclonal antibody to ICAM-1 (3E2). The cells are plated and observed for evidence that a given BPI links ICAM-1 into the MHC-II cap, i.e., by standard fluorescence microscopy and image analysis. In one experiment, T-depleted spleenocytes from mice treated 16 hours previously with EGAD-BPI (i.v.) exhibited co-capping of ICAM-1 with MUC-II in the presence of bio-10-3.62/streptavidin. In the other experiment, T-depleted spleenocytes from mice treated 16 hours previously with saline only did not exhibit co-capping. The results for these experiments are given in
Results:
The top panels of
Therefore, it appears evident that BPI have the capacity to bind simultaneously to MHC-II and ICAM-1 structures on the surface of live APC and therefore may provide signal alterations involving pathways necessary for TH1/TH2 differentiation. To directly examine the effects of BPI on TH1/TH2 immune deviation, T-cells from mice injected with EGAD-BPI were examined for cytokine analysis.
This example used an ELISPOT to determine TH1/TH2 frequency as altered by BPI injection.
Materials and Methods:
Groups of 3-5 NOD mice were immunized subcutaneously (s.c.) with the GAD 65 peptide in CFA (40 nanomoles/mouse) at the tail base. Different groups received either the EGAD-BPI, its single TCR epitope (Signal-1 moiety), or its CD11a peptide (Signal-2 moiety) i.v. (all 40 nanomoles/mouse). After 6-8 days, another identical 40 nanomole injection was given to each mouse, and the next day lymph nodes draining the site of the s.c. injection were made into single cell suspensions for culture. Identical primary cultures were incubated for 96 hours; then, viable T-cells were recovered by density gradient centrifugation. One million of these cells were combined in nitrocellulose-bottomed 96-well plates (Millititer-HA, Millipore, Bedford, Mass.), previously coated (50 μl/well) with mAb to either mouse IFNγ (clone R4-6A2), or mouse IL-4 (clone BVD4-1D11) at a concentration of 10 μg/ml in PBS. Groups of triplicate cultures were incubated with either Concanavalin-A (2 μg/ml), or the Signal-1 peptide moiety plus 20 U/ml recombinant IL2 (R & D Systems). After 72-96 hours of culture at 37° C. and 5% CO2, plates were washed three times with PBS-0.05% Tween-20. In appropriate wells are added biotinylated anti-IFNγ (clone XMG1.2) or biotinylated anti-IL-4 (clone BVD6-24G2) at a concentration of 1 μg/ml and incubated for 1 hour at room temperature. Positive control wells receive known TH1 or TH2 clones in place of normal T-cells. MAb pairs to IL12, IL10, and IL2 are also available and will be used to test for these cytokines in the same assay. All mAb and recombinant controls are purchased from PharMingen (San Diego, Calif.). Finally, plates were washed three times with PBS-Tween, and then exposed to 100 μl of a 1:2000 dilution of streptavidin alkaline phosphatase (Jackson ImmunoResearch, West Grove, Pa.) for 1 h and washed as before. Cytokine-producing cells were enumerated by development of the membrane with BCIP/NBT substrate kit (BioRad Labs, Richmond, Calif.), followed by image capture and analysis using a standard stereomicroscope connected with a digital camera and NIH image software (Murray et al., 24 Eur. J. Immunol. 2337-2344 (1994); Murray; 19 Immunology Today 157-163; and Schountz et al., 157 The Journal of Immunology 3893-3901 (1996)). In separate experiments, CD4+ T-cell clones from NOD mice immunized with the GAD65(208-217) peptide by our previously described methods can be used in the same assay. The clones were generated using the methods described in Murray et al., 24 Eur. J. Inmunol. 2337-2344 (1994), the teachings of which are hereby incorporated by reference. These clones will be maintained by biweekly restimulation with irradiated NOD lymphocytes, the GAD peptide, and recombinant IL-2. For further analysis, BPI that have been substituted at predicted TCR-contact positions will be used to determine which of these BPI variants are most effective in the inhibition of proliferation and cytokine release from individual clones as analyzed above. Predicted positions that will be scanned with all amino acids except cysteine are amino acids 208, 213, and 216. These residues point toward the TCR in the recently solved crystal structure of the GAD65 peptide bound to I-Ag7 (Corper et al., 288 Science 505-511 (2000) and see
Results:
Importantly, this example shows the ability of a given BPI to modulate a functional immune response. It can be seen that mice treated with the BPI produce abundant IL-4, whereas the control mice did not produce this cytokine (illustrated in
As shown in
This example tested the capacity of the BPI to inhibit lymphocytic infiltration of pancreatic islets in NOD mice. Lymphocytic infiltration is a hallmark of insulitis and the development of type-1 diabetes.
Materials and Methods:
These studies sought to confirm that the BPI containing the immunodominant GAD65 TCR epitope (EGAD-BPI) was biologically active in vivo and inhibited the development of pancreatic inflammation. Groups of three normal glycemic NOD males (8 weeks old) were immunized with the GAD65 (208-217) peptide in CFA as described in Example 4. The control group received PBS, and separate experimental groups received either the EGAD-BPI, the GAD peptide alone (i.e., the T-cell receptor epitope), or the CD11a peptide alone(i.e., the second signal receptor moiety) by two intravenous injections as described in Example 4. On day 10, the pancreata were removed to 10% PBS-buffered formalin, embedded in paraffin, and five-micron serial sections were examined histologically for mononuclear cell infiltration as previously described by Yoon, et al., Control of Autoimmune Diabetes in NOD Mice by GAD Expression or Suppression in β Cells, 284 Science 1183-1187 (1999), the entirety of which is hereby incorporated by reference.
Results:
The results for this experiment are given in
This example tested BPI blocking of diabetes development in the well described intact immune system of immunologically reconstituted NOD.Scid mice to study diabetes progression.
Materials and Methods:
BPI proven active for modulation of TH1/TH2 responses against known immunodominant peptides of GAD65 for blocking T-cell initiated diabetes progression in a NOD mouse model was tested. The NOD.Scid adoptive transfer model, wherein CD25-depleted NOD spleen cells have been observed to induce diabetes as early as 2-4 weeks post adoptive transfer was used for this purpose. NOD.Scid adoptive transfers were performed by a modification of a protocol described by Solomon et al. in B7/CD28 Costimulation is Essential for the Homeostasis of the CD4+ CD25 Immunoregulatory T-cells That Control Autoimmune Diabetes, 12 Immunity 431-440 (2000), the content of which is incorporated herein by reference. NOD spleen cells from 8 week (non-diabetic) females were used to enrich a CD25-/CTLA4-depleted population by treatment with purified monoclonal antibody (mAb 7D4, PharMingen) followed by low-tox rabbit complement (Cedarlane) (80% depletion of CTLA4+ cells by flow analysis with PE-labeled UC10-4F10-11 mAb; not shown). These inducer cells (15×106 per mouse) were injected (i.v.) into 6 week NOD.Scid females (Jackson Labs) together with 3×106 CD4+ T-cells from mice either treated with the EGAD-BPI or treated identically except with PBS in place of the EGAD-BPI. In vitro clonal expansion was with either recombinant IL-2 (R&D systems), or ConA, as described for the ELISPOT experiments. All manipulations of animals and cells were performed in laminar flow hoods, and animals were maintained continuously behind laminar flow barriers on autoclaved food and water in microisolator cages. Some experiments will deplete specific subsets of the CD4+ cells using mAb to CD154, CD25, CD62L, CD152, etc. and magnetic particles prior to adoptive transfer. CD4+ cells from mice treated with the individual moieties of the BPI can be used as negative controls along with CD4+ cells from mice treated with saline alone. Co-transfer of CD4+ T-cells enriched from matched NOD mice treated with a given BPI demonstrates that BPI treatment leads to regulatory T-cells capable of delaying the onset of diabetes. Moreover, other diseases listed in Table 1 can be tested in this same manner (i.e., by adoptive transfer of regulatory T-cells).
Results:
Significantly, we have now seen a clear difference in the development of hyperglycemia and diabetes between the two experimental groups. As can be seen in
Here, the predicted examples of BPI for other autoimmune diseases are briefly detailed. Specifically, BPI containing immunodominant TCR epitopes for collagen-induced arthritis (CIA) and myelin basic protein-induced experimental allergic encephalomyelitis (EAE) will be discussed. Also, the CD40L peptide mimic is predicted to favor TH2 immunity, as blocking the CD40 signal would be expected to decrease IL12 production (Ruedl, et al., The Antigen Dose Determines T Helper Subset Development by Regulation of CD40 Ligand, 30 Eur. J. Inmunol. 2056-2064 (2000)). Therefore, in these autoimmune models and in the NOD model, we will attempt to favor TH2 immunity by linking the appropriate TCR epitopes to the CD40L peptide mimic as well as the CD11 mimic used in diabetes inhibition. Mutations in this loop peptide have been shown to affect binding and function of CD40L.
Materials and Methods:
There are several autoimmune models which employ different cross-reactive immunodominant epitopes, different mouse strains, and tissues of analysis. These types of models are highly similar to our short-term model of diabetes development that we designed to test the EGAD-BPI. For CIA: We will induce the disease by a single peptide in CFA injection protocol as described by Murray et al., 24 Eur. J. Immunol. 2337-2344 (1994). As in the NOD system, these mice will receive either the BPI, PBS, or the single peptide moieties. The minimal immunodominant collagen-II epitope is listed above, and modifications to the BPI will be based upon the x-ray structure of this complex (Dessen et al., 7 Immunity 473-481 (1997). For EAE, the disease is induced by the method of Murray et al., 24 Eur. J. Immunol. 2337-2344 (1994). This method also uses a single peptide (the myelin basic protein (MBP) peptide, 85-101) injection in CFA, by methods analogous to those described in our NOD system.
Results:
We would anticipate that the BPI constructed with collagen and MBP would be effective in modulating immunity to these antigens to a TH2-dominated pattern. Since these diseases are thought to involve predominant TH1 immunopathology (see above references), such a switch would ostensibly delay or cure the disease in these mice (Murray et al., 24 Eur. J. Immunol. 2337-2344 (1994); Murray; 19 Immunology Today 157-163; and Schountz et al., 157 The Journal of Immunology 3893-3901 (1996). As in the NOD model, such results would target the development of analogous compounds for use in the human diseases of rheumatoid arthritis and multiple sclerosis, where these types of immune responses are clinically observed. Most importantly, BPI offer the capability of blocking autoimmune T-cell responses while maintaining host immunity to infectious agents and developing cancers.
This example describes predicted BPI for infectious diseases and certain cancers. Specifically, a general protocol for the testing of a given BPI containing immunodominant TCR epitopes of a specific human pathogen will be described using the example of HIV-1 p24 epitope (Harcourt, et. al., HIV-1 Variation Diminishes CD4 T Lymphocyte Recognition, 188 J. Exp. Med., 1785-1793 (1998)) (SEQ ID No. 8).
Materials and Methods:
By contrast to the previous experimental examples, these BPI will be primarily tested by their effects on long-term T-cell clones derived from human patients. Briefly, peripheral blood mononuclear cells (PBMCs) are prepared from patient and control whole blood, and CD8+ cells removed by the negative selection protocol with magnetic particles. These cells are grown in tissue culture medium at 4×106 cells in 1 ml for 6 days in the presence of 20 μM of the p24 peptide, and blast cells isolated by density gradient centrifugation. Secondary cultures of these cells contained recombinant human IL-2 (20 U/ml) and are continued for a maximum of 10-14 days. Lines were expanded by repeating the process of re-culturing with irradiated APC from histocompatible donors together with the p24 peptide and expansion of the T-cell blasts in IL-2. Clones were prepared by limiting dilution cloning in fresh plates containing irradiated APC, peptide and IL-2 (50 U/ml) (Murray et al., 24 Eur. J. Immunol. 2337-2344(1994); Murray; 19 Immunology Today 157-163; and Schountz et al., 157 The Journal of Immunology 3893-3901 (1996)). BPI will be tested for their ability to inhibit the proliferation and cytokine release of these established lines of human T-cells by our detailed methods described in Schountz et al., Unique T Cell Antagonist Properties of the Exact Self-Correlate of a Peptide Antigen Revealed by Self-Substitution of Non-Self-Positions in the Peptide Sequence, 168 Cellular Immunology 193-200 (1996). Here, we show that peptides changed by amino acid substitutions at key TCR-contact residues alter the proliferation and cytokine release of cloned T-cells. If a given BPI significantly inhibits or alters cytokine release to p24 peptide-specific clones, this will target the BPI for more stringent testing in vivo. Mouse models can be used to test candidate BPI for infectious diseases. These studies will be performed as described for the GAD65 BPI with the substitution of immunodominant peptides of the infectious agents for the GAD65 TCR epitope peptide. Positive results in these in vivo studies would substantiate animal studies with live pathogens. The Scid-human mouse model in which human tissues are adoptively transferred to the Scid mouse could be used to examine protective immunity to HIV (Jenkins, et al., Blood (1998) 8:2672, hepatitis-C virus (HCV) (Bronowicki, J., et al. Hepatology (1998)28:211), human pappiloma virus (HPV) (Tewari, et al. Gynecol. Oncol. (2000)77:137, and respiratory syncitial virus (RSV) (Nadal, et al. Clin. Exp. immunol. 85:358) infections.
Results:
It is hypothesized that incorporation of the CD28/CTLA4 peptide or the Fas-ligand peptide as the second signal moiety will shift immunity toward the TH1 subset and induce or enhance CTL responses. (Padrid, P., et al. Am. J. Respir. Cell. Mol. Biol. (1998)18:453). CTLA4 is a negative regulator of T-cell activation and signaling through CD28 has been linked to TH2 differentiation (Rulifson, I., et al. J. Immunol. (1997)158:658). Given the evidence that the LFA-1 signal is inhibitory to TH2 differentiation and that CD28 signaling favors TH2 differentiation, we will proceed with these studies using this selective differentiation paradigm to further modify BPI design. Similarly, the evidence suggests that CD95 (also called Fas) interaction with the CD95-ligand (FasL) favors TH1 differentiation (Chattergoon, et al. Nature Biotech. (2000)18:974). BPI with the peptide mimic of the FasL, which is from the Y218 loop of FasL, predicted to interact with Fas at the T-cell:APC interface, will be used to favor TH1 activation and CTL responses against chronic TH2-dominated diseases.
Here we describe several possible BPI that contain the TCR epitopes of described allergen sequences synthetically linked to the predicted CD28/CTLA4 peptide mimic and/or the predicted FasL peptide mimic.
Materials and Methods:
These studies will again use a combination of analyses using human cloned T-cell lines and animal models of allergy to these well-known allergic agents. Allergies affect a very large population of humans, with estimates of many allergies and asthma reaching 10% of otherwise healthy individuals. We will use our previously described 7-10 day protocol to investigate BPI modulation of the immune response to the allergen peptides described in the table. As previously described, this protocol involves the injection of the allergen peptide in CFA on day 1, followed by i.v. injection of the BPI on day 1 and on day 6, 7, 8 or 9. On day 7, 8, 9 or 10, the draining lymph nodes are cultured and compared for TH1 versus TH2 cytokines by ELISPOT (see Example 4 for detailed methods). Once candidate BPI for a given allergen have been identified by this relatively short-term experiment, we will test these BPI for the alteration of CD4 T-cell response using cloned lines from atopic patients. Briefly, CD4 T-cell clones will be established from patient peripheral blood lymphocytes as described under Example 8. We will test the capacity of a given BPI to inhibit or alter cytokine release by these cells using the ELISPOT as described previously. Finally, we will move the examination of a given BPI toward clinical testing using identically described procedures with these specific allergens in human MUC (HLA) transgenic mice (Svetlana, P. et al. J. Immunol. (1998)161:2032). These mice offer the advantage of having the human MHC molecules and thus, BPI binding will be very similar in this model as in the human allergic condition. If a given BPI blocks TH2 activation in these mice against the native allergen, then adoptive transfer studies with human Scid mice (similar to those used in Example 8) can be used as a precursor to human clinical trials.
Results:
Blocking the CTLA4/CD28 pathway in preference for LFA-1 signaling will favor TH1 differentiation against these allergens in the presence of the BPI. Also, BPI with the FasL moiety should favor type-1 immunity. It has been a long-held practice to “desensitize” atopic patients with allergens given by injection and desensitization is thought to operate via a shift toward TH1 responses to the specific allergens (Holt, et al., Nature (1999) 402:6760 suppl:B12-17). Therefore, as in the case of the chronic TH2-linked infectious diseases, our working hypothesis is to use the TH1-promoting capacity of LFA-1 signaling together with the possibility to inhibit TH2 signaling via the CTLA4/CD28 molecules to effect this result. We will also examine the effects of allergen epitopes linked to the Fas peptide mimic, as dual expression of Fas and antigen within APC has been shown to favor TH1 immunity and CTL induction (Chattergoon, M., et. al. Nat. Biotech. (2000)18:974). It maybe possible to induce cell death of APC presenting allergen epitopes, thereby limiting the activation of TH2-type regulatory cells in allergy and asthma, including RSV-precipitated childhood and geriatric bronchiolitis.
| Number | Name | Date | Kind |
|---|---|---|---|
| 5540926 | Aruffo et al. | Jul 1996 | A |
| Number | Date | Country |
|---|---|---|
| WO 98044129 | Oct 1998 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20050107585 A1 | May 2005 | US |
