Patent Application
20220218806
This application contains a “Sequence Listing” submitted as an electronic .txt file named “CS-138PCTUS_SEQ.txt.” The subject matter of the “Sequence Listing” is incorporated herein by reference.
The invention relates to peptide heteroconjugates useful in treating Rheumatoid Arthritis (RA). The peptide heteroconjugates include a portion of the Human aggrecan protein conjugated to an Immune Cell Binding Ligand (ICBL) by a direct bond or divalent linker. The peptide heteroconjugates including combinations thereof, can be used as a vaccination for RA when combined with an adjuvant and administered to a subject.
Autoimmune diseases are driven by the cytokines produced by T cells, B cells, macrophages, dendritic cells and other cells. The cytokines can define different T cell responses. Rheumatoid Arthritis (RA) is an autoimmune disorder characterized by systemic inflammation and progressive joint deterioration. Autoimmune diseases, such as RA, occur when antibodies produced by B cells mount an immune response against self antigens. According to the CDC, an estimated 1.5 million people in the US had RA in 2007, with an estimated 50 million worldwide having some form of arthritis. In 2012, three of the ten highest selling drugs in the US were RA biologics for treatment of symptoms or ablative treatment of activated cells or proinflammatory cytokines.
Vaccines protect against disease by stimulating the production of antibodies against a causative agent of the disease. Peptide vaccines can include useful peptides that can be incorporated into an immunogen and can have the ability to generate an appropriate immune response. There is a need for peptide vaccines that can be used to treat or vaccinate against RA. Ligand Epitope Antigen Presentation System (LEAPS) vaccines could be modulatory rather than ablative therapy. LEAPS is a novel immunization technology that enables the design and synthesis of relatively small peptide immunogens and determination of the resultant immune response. Further, determination of the T cell response driving the disease could allow personalized medicine for RA, with the choice of LEAPS vaccine determined by the T cell response and antigens involved.
Generating an effective immune response imposes certain requirements because not all immune responses are effective. Indeed, immune responses can be harmful, and some antigens can evoke or elicit antibodies when an immune response of a cellular nature is needed. Autoimmune arthritis imposes additional requirements since it is often a self-epitope. As such, a peptide epitope may sometimes need to be incorporated or linked to another peptide forming another larger component. The larger combination of peptides can then impose further restrictions because the components must be compatible and not interfere with one another. The known peptides include P49 which contains a key site for citrullination at arginine 305 (R305) that appears to be important in autoimmune arthritis in animal models and in man (Markovics, A., Ocskó, T., Katz, R. S., Buzás, E. I., Glant, T. T., & Mikecz, K. (2016), Immune Recognition of Citrullinated Proteoglycan Aggrecan Epitopes in Mice with Proteoglycan-Induced Arthritis and in Patients with Rheumatoid Arthritis. PloS One, 11(7), e0160284). However, epitope P49 has several undesirable features for commercial applications based on lower reactivity as seen in adjacent peptides when examined in pools for various responses (See also WO/2008/043157 (“Thomas”)). Similar studies of the same region albeit using a different starting position nomenclature of 280-292 for the same sequence and shifted away from the amino terminus of P49 to the carboxyl end just before the W301 in or W282 eliminating the two M and C support the same conclusion (Aggarwal, A., Srivastava, R., & Agrawal, S. (2013). T cell responses to citrullinated self-peptides in patients with Rheumatoid Arthritis. Rheumatology International, 33(9), 2359-63). Moreover, the known literature notes that an adjacent R285R286 did not contain D (Markovics, A., Ocskó, T., Katz, R. S., Buzás, E. I., Glant, T. T., & Mikecz, K. (2016) Immune Recognition of Citrullinated Proteoglycan Aggrecan Epitopes in Mice with Proteoglycan-Induced Arthritis and in Patients with Rheumatoid Arthritis. PloS One, 11(7), e0160284).
There is a need to provide a composition and methods for delivering a therapeutic goal in autoimmune inflammatory diseases such as Rheumatoid Arthritis to suppress responses against (auto)antigens present in one or more target organ. The need includes providing an antigen-specific therapy. The need further includes compositions and methods for having improved reactivity. The need can further include combination therapies and delivery of one more compositions to improve therapy. For increased efficacy of treatment, there is a need for multi-epitope vaccines incorporating distinct epitopes that are located in distant regions of the PG molecule involved in arthritis induction.
The first aspect of the invention is related to peptide heteroconjugates having a formula P1-x-P2 or P2-x-P1; wherein P1 is selected from the group consisting of SEQ ID NO.'s 3 and 7; wherein P2 is selected from the group consisting of SEQ ID NO.'s 1 and 2; and wherein x is a direct bond or divalent linker for covalently bonding P1 and P2.
In any embodiment, P1 can be SEQ ID NO. 3.
In any embodiment, P2 can be SEQ ID NO. 2.
In any embodiment, x can be a divalent linker comprising a sequence GGG.
The second aspect of the invention is related to a composition comprising at least one peptide heteroconjugate having a formula P1-x-P2 or P2-x-P1; wherein P1 is selected from the group consisting of SEQ ID NO.'s 3 and 7; wherein P2 is selected from the group consisting of SEQ ID NO.'s 1 and 2; wherein x is a direct bond or divalent linker for covalently bonding P1 and P2; and further comprising an adjuvant.
In any embodiment, the composition can further comprise a second peptide heteroconjugate.
In any embodiment, the second peptide heteroconjugate can have a formula P3-x-P4 or P4-x-P3; wherein P1 is selected from the group consisting of SEQ ID NO.'s 3 and 7; P3 is selected from the group consisting of SEQ ID NO.'s 3 and 7; wherein P4 is selected from the group consisting of SEQ ID NO.'s 8 and 9; and wherein x is a direct bond or divalent linker for covalently bonding P3 and P4.
In any embodiment, the second peptide heteroconjugate can be from PG, collagen, vimentin, and may include citrullination of arginine and/or glycosylation of other residues such as serine or threonine.
In any embodiment, P1 can be SEQ ID NO. 3.
In any embodiment, P2 can be SEQ ID NO. 2.
In any embodiment, x can be a divalent linker comprising a sequence GGG.
In any embodiment, the adjuvant can be selected from the group consisting of Seppic ISA51vg, Freund's incomplete adjuvant, Lipid A, MPL, AS01, AS03, AS04, Novasomes and Liposomes, MF59, QS21, IS01, IS03, IS04, or combinations thereof.
The third aspect of the invention is related to a method of treating a subject. In any embodiment, the method can comprise the steps of administering the composition of the second aspect of the invention to the subject.
In any embodiment, the composition can further comprise a second peptide heteroconjugate.
In any embodiment, the second peptide heteroconjugate can have a formula P3-x-P4 or P4-x-P3; wherein P1 is selected from the group consisting of SEQ ID NO.'s 3 and 7; P3 is selected from the group consisting of SEQ ID NO.'s 3 and 7; wherein P4 is selected from the group consisting of SEQ ID NO.'s 8 and 9; and wherein x is a direct bond or divalent linker for covalently bonding P3 and P4.
In any embodiment, the second peptide heteroconjugate can be from PG, collagen, vimentin, and may include citrullination of arginine and/or glycosylation of other residues such as serine or threonine.
In any embodiment, P1 can be SEQ ID NO. 3.
In any embodiment, P2 can be SEQ ID NO. 2.
In any embodiment, x can be a divalent linker comprising a sequence GGG.
In any embodiment, the adjuvant can be selected from the group consisting of Seppic ISA51vg, Freund's incomplete adjuvant, Lipid A, MPL, AS01, AS03, AS04, Novasomes and Liposomes, MF59, QS21, IS01, IS03, IS04, or combinations thereof.
In any embodiment, the subject can be human.
In any embodiment, the composition can be administered to the subject multiple times.
In any embodiment, the method can comprise administering between 1 mg and 50 mg of the peptide heteroconjugate each of the multiple times.
In any embodiment, a time period between each administration of the composition can be between 1 week and 1 year, between 1 week and 3 weeks, between 2 weeks and 3 weeks, between 3 weeks and 6 months, between 1 month and 6 months, between 3 months and 6 months, between 1 month and 1 year, or between 6 months and 1 year.
In any embodiment, the subject can be a mouse.
In any embodiment, the method can comprise administering between 0.05 μg and 0.1 μg, between 0.1 μg and 10 μg, between 10 μg and 100 μg, or between 100 μg and 300 μg of the peptide heteroconjugate.
In any embodiment, the subject can be a rat.
In any embodiment, the method can comprise administering between 0.05 μg and 3 mg of the peptide heteroconjugate.
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 trademarked term “L.E.A.P.S.” (also referred to as “L.E.A.P.S.” or LEAPS) refers to “Ligand Epitope Antigen Presentation System” and stands for a peptide based antigen delivery technology for directing immune responses toward a desired outcome.
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 “citrulline” indicates an α-amino acid. The name is derived from citrullus. Citrulline has the idealized chemical formula H2NC(O)NH(CH2)3CH(NH2)CO2H, and is a key intermediate in the urea cycle, the pathway by which mammals excrete ammonia. Citrulline is made from ornithine and carbamoyl phosphate in one of the central reactions in the urea cycle. It is also produced from arginine as a by-product of the reaction catalyzed by NOS family (NOS; EC 1.14.13.39). Arginine is first oxidized into N-hydroxyl-arginine, which is then further oxidized to citrulline concomitant with release of nitric oxide. Citrulline is formed in the protein, after protein synthesis, by enzymatic or chemical conversion of arginine to citrulline. The in-situ process is catalyzed by one or more variants (1-5) of the enzyme referred to PAD for peptidyl-dinimido-amino peptide.
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.
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 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.
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.
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 terms “peptide” and “peptide construct” broadly refer to any molecule or part of a molecule including two or more amino acid residues linked by a peptide bond. The term “peptide construct” can also include 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.
The term “subject” or “patient” refers to an animal, including mice and humans, to which a therapeutic agent is administered.
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 a composition containing one or more antigens that stimulate an immune response when administered to vertebrates in vivo.
Table 1 shows peptides of one of the major proteins (aggrecan or Proteoglycan) thought to be involved as epitopes and antigens in RA and animal models from several publications including the Thomas R. patent applications. PCT/AU2007/001555 and PCT/AU2013/000303.
L ATTGHVYLAW QAGMDMCSAG
WLAD
SV YP ISKA
PNCGG NLLGVRTVYV HANQTGYPDP SSRYDAICYT
The novel peptides of this invention include peptide heteroconjugates having the following formulae (I) or (II):
P1-x-P2 (I)
P2-x-P1 (II)
where P1 is an immune cell binding ligand (ICBL), P2 is either PG275 or PG275Cit, and x is a direct bond or divalent linker for covalently bonding P1 and P2. Attachment of an ICBL to a T cell epitope containing peptide determines the resultant response.
A key consideration in developing an immunization is to preserve the key position of the R305 known to be involved in binding to both antibodies and T cells based on the effects of substitutions with citrulline which enhances the efficacy of the peptide and to position the R305 in an appropriate location and maintain the local proximity of net charge and hydrophobicity around this “305” site.
One concern regarding peptide vaccines is the use of only a limited number of epitopes because an agent responsible for a disease process can oftentimes involve multiple epitopes. Another concern is a possibility of escape such as due to genetic drift if only a single epitope is used. However, several factors related to the criteria used in the epitope selection process can mitigate the concerns. Moreover, combinations of peptides vaccines and/or peptide vaccines containing multiple epitopes can confer high efficacy. In particular, combinations of peptides vaccines and/or peptide vaccines containing multiple epitopes may be of a benefit if one epitope is missing or altered. The effective use of numerous therapeutic monoclonal antibodies supports the use of combinations of peptides vaccines and/or peptide vaccines containing multiple epitopes insofar as monoclonal antibodies by definition have a single epitope specificity. The likelihood of both epitopes being altered or missing is generally lower, hence supporting a concept of combinations of peptides vaccines and/or peptide vaccines containing multiple epitopes. Further, two epitopes distant from one another can minimize the likelihood of a genetic missing element. That not all epitopes induce the same type of Th2/Treg protective response and use of two epitopes can also minimize the probability that both may be missing.
Based on the concerns, two core analogues of P291 are identified:
The two core analogues, SEQ ID NO. 1 and SEQ ID NO. 2, can be conjugated with an immune cell binding ligand (ICBL) to promote the immunogenicity of an epitope. Examples of ICBLs include:
Peptide J is derived from human beta-2-microglobulin. J-LEAPS vaccines activate mouse and human precursors to differentiate into dendritic cells (DCs) that produce IL12p70 (Rosenthal, K S, and Zimmerman, D H. J-LEAPS vaccines elicit antigen specific Th1 responses by promoting maturation of type 1 dendritic cells (DC1). 2017 AIMS Allergy and Immunology 1(2): 89-100). J-LEAPS conjugate vaccines or J-LEAPS induced DCs have been shown to be protective in HSV-1, Influenza A, Murine Her2Neu cancer, EAM and CIA challenge mode (Zimmerman, et al., 2010, Int Immunopharmacol. 2010 10(4):412-21; Rosenthal et al., 2017, J Immunol Res. 2017; 2017:361; Rosenthal K S, Taylor P, Zimmerman D H. J-LEAPS peptide and LEAPS dendritic cell vaccines. Microb Biotechnol. 2012 March; 5(2):203-13; Taylor P R, Koski G K, Paustian C C, Bailey E, Cohen P A, Moore F B, Zimmerman D H, Rosenthal K S. J-LEAPS vaccines initiate murine Th1 responses by activating dendritic cells. Vaccine. 2010 Aug. 2; 28(34):5533-42; Taylor P R, Paustian C C, Koski G K, Zimmerman D H, Rosenthal K S. Maturation of dendritic cell precursors into IL12-producing DCs by J-LEAPS immunogens. Cell Immunol. 2010; 262(1):1-5; Zimmerman D H, Taylor P, Bendele A, Carambula R, Duzant Y, Lowe V, O'Neill S P, Talor E, Rosenthal K S. CEL-2000: A therapeutic vaccine for rheumatoid arthritis arrests disease development and alters serum cytokine/chemokine patterns in the bovine collagen type II induced arthritis in the DBA mouse model. Int Immunopharmacol. 2010 April; 10(4):412-21; and Cihakova D, Barin J G, Baldeviano G C, Kimura M, Talor M V, Zimmerman D H, Talor E, Rose N R. L.E.A.P.S. heteroconjugate is able to prevent and treat experimental autoimmune myocarditis by altering trafficking of autoaggressive cells to the heart. Int Immunopharmacol. 2008 May; 8(5):624-33).
Peptide Der-G is derived from the beta chain of human MHC II. Der-G-LEAPS vaccines induce immune responses with Th2 associated IgG isotypes and cytokine profiles. The DerG-LEAPS vaccines are not protective or therapeutic in the HSV, influenza, EAM and CIA models. DerG-LEAPS vaccine is therapeutic for the PGIA and GIA RA models. Proteoglycan-induced arthritis and recombinant human proteoglycan aggrecan G1 domain-induced arthritis in BALB/c mice resemble two subtypes of Rheumatoid Arthritis. Arthritis and Rheumatism, 63(5), 1312-21). The conjugation of the core analogues of P291 with the ICBLs give the peptide heteroconjugates having the formulae P1-x-P2 or P2-x-P1. One non-limiting example, SEQ ID NO. 3 is conjugated with SEQ ID NO. 2 to give the peptide heteroconjugate:
Alternatively, SEQ ID NO. 3 can be conjugated with SEQ ID NO. 1 to give the peptide heteroconjugate:
As described, peptide J can be used as the ICBL an conjugated to SEQ ID NO. 1 or SEQ ID NO. 2. Table 2 shows the peptide sequences used, along with two peptides derived from PG70, which may be combined with the PG275 peptides for vaccination, as described:
To treat or immunize subjects against RA, the described peptide heteroconjugates can be administered to the subject in an adjuvant. Any adjuvant capable of enhancing antigen-specific immune responses in combination with the described peptide heteroconjugates can be used. Non-limiting examples of adjuvants can include Seppic ISA51vg, Freund's incomplete adjuvant, Lipid A, MPL, AS01, AS03, AS04, Novasomes and Liposomes, MF59, QS21, IS01, IS03, IS04, or combinations thereof.
The amount of the peptide heteroconjugate administered to the subject can vary with size, age, and species of the subject. In human subjects, between 1 mg and 50 mg of the peptide heteroconjugate can be administered per dose. Generally, it is preferable with human subjects to spread out the doses. For example, the peptide heteroconjugate can be administered to the subject every week, every 2 weeks, every three weeks, or longer. In any embodiment, the time period between each administration can be is between 1 week and 1 year, between 1 week and 3 weeks, between 2 weeks and 3 weeks, between 3 weeks and 6 months, between 1 month and 6 months, between 3 months and 6 months, between 1 month and 1 year, between 6 months and 1 year, or longer than 1 year between administrations.
For administration to mice, between 0.05 μg and 300 μg of the peptide heteroconjugate can be administered, including between 0.05 μg and 0.1 μg, between 0.1 μg and 10 μg, between 10 μg and 100 μg, or between 100 μg and 300 μg of the peptide heteroconjugate. In rats, between 0.05 μg and 3 mg of the peptide heteroconjugate can be administered.
The vaccines may be administered with an adjuvant on a regular regimen by any route, including intradermal, intramuscular, subcutaneous or as a cutaneous transdermal or nasal delivery.
In certain embodiments, a combination vaccine may be administered. The combination vaccine can include the peptide heteroconjugates using PG275 and PG275Cit in combination with a peptide heteroconjugate using PG70. Previously, A DerG LEAPS conjugate of PG70 (Cel-4000) was shown to be therapeutic via up modulation of Th2 and Treg, i.e. up modulation of IL4, IL10 and CD4+CD25+ FoxP3 cells (Mikecz et al. 2017 Vaccine 35:4048-4056). A multi-epitope combination vaccine can have certain advantages over a single epitope peptide vaccine. A multi-epitope vaccine may cover the different epitopes involved in the disease process of a pathogen or autoimmune antigen. However, based on the single epitope action of many monoclonal antibodies, single epitope activity may be effective. A multi-epitope vaccine may still be effective in an individual with responses to a different epitope or an altered epitope, especially if the other epitope is presented together or in the same context. With a single epitope vaccine, if the epitope selected is missing in the antigen in the affected individual or animal the vaccine may be ineffective. These concerns may be substantially overcome if a two epitope vaccine is used especially if the two epitopes are not closely linked or in proximity to one another and even more so if a three epitope vaccine with a third conjugate is used. Another advantage of a two epitope approach is in a possible event that one pathway is missing in the T cell recognition of antigen or the repertoire of immune response having a second redundant pathway or recognition. PG70 and PG275 incorporate distinct epitopes that are located in distant regions of the PG molecule involved in arthritis induction.
There are certain concerns with multi-epitope vaccines as well. The response to a different epitope may compete or interfere with that against the first conjugate. Further, one epitope could initiate an inappropriate response. However, neither of these concerns is found for the PG70 and PG275 vaccines. As described, the two epitopes appeared to act independently to induce different but therapeutic, and probably based on involvement of DerG binding to CD4, similar Th2/Treg, responses. DerG-PG70 induced antibodies against the epitope and DerG components. DerG-PG275Cit induced antibodies against neither the DerG element nor the PG275Cit element. The cytokine response elicited in vitro and the ICS response upon antigenic stimulation were different for each epitope and yet both appear to act via the Th2/Treg pathway based on the DerG affinity for CD4+ T cells.
To study the efficacy of the described peptide heteroconjugates, LEAPS therapy in the cartilage proteoglycan (PG) induced arthritis (PGIA) model of RA using these new peptides and conjugates were examined Studies for efficacy were determined in a challenge disease model of human RA. The animal model of RA is induced by immunization of aging female BALB/c mice with human cartilage proteoglycan. The animal model is well documented and resembles human disease with spondyloarthropathy, the presence of RF and ACPA, and propensity for occurrence in older females (von Delwig, A., Locke, J., Robinson, J. H., & Ng, W.-F. (2010)). Response of Th17 cells to a citrullinated arteriogenic aggrecan peptide in patients with Rheumatoid Arthritis. Arthritis and Rheumatism, 62(1), 143-9). Immunization with PG or the G1 domain (containing most arthritogenic/dominant epitopes of human cartilage) induces PGIA or GIA disease (Glant, T. T., Radacs, M., Nagyeri, G., Olasz, K., Laszlo, A., Boldizsar, F., Mikecz, K. (2011). Proteoglycan-induced arthritis and recombinant human proteoglycan aggrecan G1 domain-induced arthritis in BALB/c mice resemble two subtypes of Rheumatoid Arthritis. Arthritis and Rheumatism, 63(5), 1312-21 and Glant et al. 2004 Autoimmunity: Methods and Protocols 102:313). PGIA and GIA disease are characterized by predominant Th1 IFNg cytokine production. Retired breeder female BALB/c mice received 3 intraperitoneal injections of 40 μg rhG1 in DDA adjuvant 3 weeks apart. Once the mice in the study reached a desired mean disease (GIA) score of 3 for the intended groups, the mice were randomized and assigned to the treatment groups and were vaccinated on days 0 and 14 subcutaneously with one of four treatments. The first group received ISA51vg adjuvant emulsified with PBS (adjuvant control); the second group received CEL-4000 in adjuvant, the third group received CEL-4000 and DerG-PG275Cit in adjuvant, and the fourth group received DerG-PG275Cit in adjuvant. The vaccines were formulated by one investigator and given under code to a second investigator immunizing the animals.
After the onset of disease, mice were divided in several (as indicated) separate equally size groups, each with a similar mean arthritis index (AI). One group was vaccinated with adjuvant only, and the other groups with one of two LEAPS conjugates of a PG epitope 70 (J-PG70 or DerG-PG70) in adjuvant or both DerG-PG70 AND DerG-PG275Cit. The same vaccination was repeated two weeks later. AI were measured by visual scoring of each limb as done before (Mikecz et al. 2017 and Zimmerman et al. 2010) every other day for a total of about 5 weeks since the start of therapeutic immunization. Upon euthanization of the mice in the groups A, C, D and E, the proportions of Th1, Th2, Th17, and regulatory T cell (Tregs) in the spleen cells were determined by flow cytometry. as well as these cells were examined for cytokine secretion, in addition for all Groups (A-E) sera collected and examined for cytokines and antibodies to the vaccine and limbs process for histological examination.
Epitope PG275 was synthesized as a citrullinated to form PG275Cit and conjugated to DerG to form DerG-PG275Cit. Citrullination is a common posttranslational modification to RA antigens. PG275cit occurs in 27% of RA patients. CEL-4000 and other peptides were supplied with free amino terminus and amidated C-terminus as a lyophilized acetate salt at >90% purity (RP-HPLC and MS+/−2amu). Neutravidin Microplates were purchased from Thermo Fisher and Biotin labeled peptides of DerG-PG70 (CEL-4000), PG70, DerG, DerG-PG275Cit, PG275Cit and Ova as a specificity control from Biomatik, prepared from dry peptide, dissolved, stored frozen, diluted and loaded onto plates at mg/mL just before conducting an immuno-assay. Each conjugate vaccine was evaluated alone and in combination. Multiple parameters were evaluated, as described.
To determine a visual arthritis score (VAS), swelling and redness of the paws were scored visually (scores: 0-4/paw, 0-16/mouse). One of skill in the art will understand that the terms “visual arthritis score (VAS),” visual score (VA),” and “visual arthritis index (VAI)” can be used interchangeably. Mice were sorted into 5 groups, each of a similar mean VAS (day 0). The scoring was done 3 times a week by two separate investigators independently of each other. Data was analyzed by a third investigator that determined if the two scoring investigators agreed. If the two investigators disagreed by more than 0.5 in animal's score, the scores were sent back for another scoring before analysis could proceed.
Animals were euthanized on day 35 after the first control or LEAPS vaccination, and sera was collected. For histopathology, hind limbs (from 5 mice/treatment groups plus 3 normal mice limbs) were dissected and fixed in 10% buffered formalin. The specimens were embedded in paraffin and sectioned. Adjacent tissue sections were stained separately with TB and HE. Microscopic analysis and scoring of joint damage were carried out independently under code by Bolder BioPath. Serum anti-LEAPS peptide antibody levels were determined by ELISA using Neutavidin plates and Biotin labeled peptides for loading onto plates. To determine cytokine production by spleen cell cultures, after a 4-day stimulation of spleen cells with 7.5 μg/ml rhG1 (or no treatment as control), cytokines secreted into the culture media were assayed by MagPix as 9-plex or 6-plex kits from R&D Systems. For intracellular cytokines and FoxP3 detection, spleen cells from 5 mice from each group were cultured with rhG1 for 4 days. The cells were treated with PMA, ionomycin, and GolgiStop for 4 hours, stained for cell surface markers and intracellular cytokines or Foxp3. Samples were run on a FACS Canto flow cytometer and the results analyzed by FACS Diva software. Statistical analysis of data was done using GraphPad Prism 7 software package. Nonparametric and multiple comparison test were conducted as described.
In addition to studying PG275 and PG275Cit, PG70 and PG70Cit were also studied. PG70 is defined as proteoglycan aggrecan (G1) residues 70-84, and has the following sequence:
The citrulline version of PG70, called PG70Cit was also studied and has the following sequence:
In any heteroconjugate of the invention, one non-limiting embodiment of the linker can be a divalent linker between the ICBL and the epitope. In another non-limiting embodiment, the divalent linker can be a triple glycine linker. In other embodiments, the linker of the invention can be a direct link together in any order (i.e., N-terminal of one to C-terminal of other or vice versa). The peptide of the invention can be covalently bonded by a spacer or linker molecule by any method or composition known to those of skill in the art. With regard to linkers between the two domains, suitable examples include a thioether bond between an amino terminus bromoacetylated peptide and a carboxyl terminus cysteine, often preceded by a diglycine sequence (Zimmerman et al., supra), carbodiimide linkages, a multiple glycine, e.g., from 3 to 6 glycines, such as triglycine, with or without one or two serines, separation between the two entities, e.g., GGGS (SEQ ID NO. 7), GGGSS (SEQ ID NO. 8), GGGGS (SEQ ID NO. 9), GGGGSS (SEQ ID NO. 10), GGGSGGGS (SEQ ID NO. 11), etc., and other conventional linkages, such as, for example, the direct linkages such as, EDS, SPDP, and MBS, as disclosed in the aforementioned U.S. Pat. No. 5,652,342.
Based on the results, both DerG-PG70 and DerG-PG275Cit are therapeutic in the GIA model of RA with high efficacy, and may act by different mechanisms. DerG-PG275Cit does not induce antibodies to either DerG or PG275Cit or the conjugate. DerG-PG70 induces antibodies to itself that recognize DerG or PG70. DerG-PG70 appears to act by up modulation of Th2 and Treg cells and corresponding cytokines. DerG-PG275 does not appear to up modulate Th2 or Treg or Th1 cells or cytokines, but likely binds to CD4+ T cells. Thus, while either DerG-PG275 or DerG-PG70 can provide protection alone, a combination vaccine containing both DerG LEAPS conjugates could offer advantages in case one epitope or another was missing or mutated beyond recognition in the disease inducing situation or if the host did not recognize one epitope due to T cell repertoire.
In certain embodiments, other or additional conjugates can be included. For example, epitopes from PG, collagen, or vimentin can be used. The conjugates can include regular or post translational modifications, such as citrullination of arginine or glycosylation of other residues such as serine or threonine.
It will be apparent to one skilled in the art that various combinations and/or modifications and variations can be made in the dialysis system depending upon the specific needs for operation. Moreover, features illustrated or described as being part of an aspect of the invention can be included in the aspect of the invention, either alone or in combination
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/032376 | 5/11/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62846609 | May 2019 | US |
