WO2025024775A1 - Interféron-bêta-1 modifié ayant une immunogénicité réduite - Google Patents

Interféron-bêta-1 modifié ayant une immunogénicité réduite Download PDF

Info

Publication number
WO2025024775A1
WO2025024775A1 PCT/US2024/039763 US2024039763W WO2025024775A1 WO 2025024775 A1 WO2025024775 A1 WO 2025024775A1 US 2024039763 W US2024039763 W US 2024039763W WO 2025024775 A1 WO2025024775 A1 WO 2025024775A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
amino acid
modified
interferon
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2024/039763
Other languages
English (en)
Inventor
Eduardo Federico MUFARREGE
Sonia Ricotti
Alberto Sergio Garay
Marina Etcheverrigaray
Anna Searls DE GROOT
William D. Martin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Universidad Nacional del Litoral
Epivax Inc
Original Assignee
Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Universidad Nacional del Litoral
Epivax Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET, Universidad Nacional del Litoral, Epivax Inc filed Critical Consejo Nacional de Investigaciones Cientificas y Tecnicas CONICET
Priority to PCT/US2024/039763 priority Critical patent/WO2025024775A1/fr
Publication of WO2025024775A1 publication Critical patent/WO2025024775A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/215—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure generally relates to the development of therapeutic molecules of pharmaceutical interest for application to humans. More particularly, the present disclosure relates to modified interferon Beta-1 (IFNP-1) polypeptides, including IFNP-la, as well as to compounds and compositions. These modified IFNP-1 polypeptides display proven antiviral biological activity and reduced immunogenicity compared with wild-type IFNP-1. These modified IFNP-1 polypeptides, as well as related compounds and compositions, may be used for human therapy and treatments, including the treatment of multiple sclerosis.
  • IFNP-1 interferon Beta-1
  • MS Multiple sclerosis
  • RRMS relapsing-remitting form of MS
  • MS generally begins when autoreactive T lymphocytes trigger an inflammatory cascade in the central nervous system (CNS), which includes demyelination and axonal loss, culminating in neurodegeneration.
  • CNS central nervous system
  • IFNP-1 Interferon beta-1
  • IFNP-1 is a cytokine from the interferon family, secreted by various immune and non-immune cells, including macrophages, lymphocytes, fibroblasts, and endothelial cells. Interferons have immunomodulatory effects as well as antiviral and antitumor properties.
  • IFNP-la is a type 1 interferon naturally produced by fibroblasts and secreted in its mature form of 166 amino acids (SEQ ID No. 1) with N-glycosylation at the asparagine residue at position 80. Its synthesis begins as a 187-amino-acid precursor (SEQ ID No. 9) encoded by a single gene on the short arm of chromosome 9 (SEQ ID No. 12), which, after synthesis and secretion, removes a 21 -amino-acid signal peptide (SEQ ID No. 17).
  • recombinant proteins for therapeutic use are a routine part of medical practice, used to treat a wide variety of diseases.
  • the administration of recombinant human beta interferon (rhIFN beta) was the first disease-modifying therapy approved by the U.S. Food and Drug Administration (FDA) for the treatment of MS, due to its ability to reduce relapses and delay the onset of disability.
  • FDA U.S. Food and Drug Administration
  • IFN beta is not a cure for MS (no known cure exists to date), but it is the first line of treatment because it reduces the damage of neurological episodes by 30% and delays the definitive clinical signs of MS.
  • IFN beta biotherapeutics approved for the treatment of relapsing forms of MS: subcutaneous administration (SC) 1- IFN beta-lb Betaseron® (Bayer Healthcare Pharmaceuticals Inc), 2- Extavia® (Novartis Pharmaceuticals Corp), and 3- IFN beta-la Rebif® (EMD Serono Inc); and intramuscular administration (IM) 4- IFN beta- la Avonex® (Biogen Inc) and 5- PEG-interferon SC beta-la Plegridy® (Biogen Inc). Rebif and Avonex are produced in CHO (eukaryotic) cells, while Betaseron and Extavia are produced in bacterial cells.
  • Betaseron's recombinant IFN beta-lb is a mutated form where cysteine 17 is modified to a serine residue and is a non-glycosylated form of the protein.
  • rhIFN beta has played an important role in understanding the immunomodulatory mechanisms underlying the disease. Despite the approval of other therapies, the use of rhIFN beta persists due to its long-term beneficial effects, such as reduced disability progression and mortality rates and the relative safety for the fetus during pregnancy. Additionally, rhIFN beta has been used as a comparator in two large-scale randomized clinical trials to test innovative MS therapies. Despite its clinical efficacy, numerous reports have demonstrated the occurrence of immunogenicity events when rhIFN beta is administered to patients. In fact, all products containing rhIFN beta have triggered the formation of neutralizing antibodies (NAbs) that negatively impact the clinical efficacy of these drugs.
  • NAbs neutralizing antibodies
  • MS patients who do not respond to IFN beta therapy may be divided into three subgroups: genetic, pharmacological, and pathogenetic nonresponders.
  • Pharmacological non-responders show a lack of clinical efficacy due to the presence of serological factors (e.g., anti-IFN beta antibodies) that inhibit the biological activity of IFN beta.
  • serological factors e.g., anti-IFN beta antibodies
  • serological factors interfere with the interaction between IFN beta and its IFNAR receptor, which in turn blocks downstream IFN signaling, the transcription of interferon- stimulated genes (ISGs), and the expression of ISG products.
  • ISGs interferon- stimulated genes
  • neutralizing antibodies may occur due to various factors, which may be grouped into two main categories: extrinsic factors such as the route of administration, dosage, formulation, presence of aggregates and contaminants, or the presence and type of glycosylations; and intrinsic factors, among which the presence of immunogenic epitopes in the protein stands out. It is well known that the development of neutralizing antibodies is closely related to T helper cells.
  • the occurrence of an immunogenicity event from the administration of a recombinant IFNP-la occurs even when the therapeutic has an amino acid sequence identical to the autologous natural IFNP-la protein.
  • B lymphocyte activation may be mediated or not by T lymphocyte collaboration, resulting in T-dependent or T-independent responses, respectively.
  • T-independent responses develop as a result of the activation of a specific group of B lymphocytes, stimulated by certain structural characteristics of some molecules, such as polymeric repeats.
  • Antibodies developed as a result of this activation are primarily low-affinity IgM.
  • T-dependent lymphocyte activation is mainly associated with the protein’s primary sequence.
  • peptides when the molecule is endocytosed, processed, and the resulting peptides are presented on the surface of antigen- presenting cells (dendritic cells, macrophages, or B lymphocytes) in the context of Class II Major Histocompatibility Complex (MHC) molecules, some sequences may be recognized by “helper” (Th) or collaborating T lymphocytes (through their surface receptor, called T Cell Receptor or TCR). These specific lymphocytes, once activated, will trigger an immune response leading to B lymphocyte activation and consequent NAb production.
  • helper helper
  • T lymphocytes through their surface receptor, called T Cell Receptor or TCR.
  • the developed antibodies are IgG, have higher affinity, and are generated more prolonged and sustained over time than those generated without T lymphocyte participation.
  • T-dependent B lymphocyte activation begins with the interaction of a group of B lymphocytes with specific protein epitopes through their surface antigen receptors (IgM/IgD), constituting the first B lymphocyte activation signal.
  • This signal promotes protein internalization, which will then be processed into small peptide epitopes, ultimately exposed within the “groove” of Class II MHC molecules on the B lymphocyte surface.
  • B cells also co-express the CD40 molecule on their surface.
  • Th lymphocytes interact through their TCR and the CD40 ligand (CD 154) with epitope-MHC class II complexes and CD40 (on the B lymphocyte surface), they trigger the second activation signal.
  • T lymphocytes produce, among other cytokines, interleukin-4 (IL-4) (in a type 2 Th lymphocyte response) or interferon-gamma (IFN gamma) (type 1 Th lymphocytes), leading to the maturation of the immune response.
  • IL-4 interleukin-4
  • IFN gamma interferon-gamma
  • T lymphocytes without the participation of T lymphocytes, B lymphocytes undergo programmed cell death (apoptosis). For this reason, attenuating a T lymphocyte-mediated immune response has become the focus of attention in the process known as “de-immunization” of recombinant proteins for therapeutic purposes.
  • Immunogenicity risk assessment is an important component of biotherapeutic drug development, and part of the overall benefit risk assessment. A robust immunogenicity risk assessment process ensures that the most appropriate candidate molecules advance to the clinical stage, and that clinical immunogenicity is adequately monitored.
  • any protein that has been designed to change the amino acid sequence has linker regions that introduce new linear epitopes that are not present in either parent, or has unnatural or modified amino acids that may be at elevated risk of developing an immune response.
  • Most multi-species therapies contain these elements, and therefore these novel sequences must assess immunogenic risk using available tools such as in silico prediction, in vitro T cell assays, MHC binding assays, or ex vivo models.
  • the present disclosure provides modified IFNP-la polypeptides and related compounds displaying proven biological activity and having reduced immunogenicity compared to wild-type IFNP-la.
  • the modified IFNP-la polypeptides find use as a therapeutic in human subjects for a variety of reasons, such as better safety among patient populations, reduced immunogenicity and consistent biological activity.
  • the present disclosure has a potential use in the therapy of multiple sclerosis and other neurodegenerative diseases.
  • the present disclosure provides a modified interferon-pia polypeptide having interferon-pia activity, the polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and comprising one or more amino acid substitutions, the one or more amino acid substitutions at one or more of position 25, 28, 60, 65, 74, 111, 117, or 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the modified interferon-pia polypeptide having interferon-pia activity comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and comprising at least four amino acid substitutions, the at least four amino acid substitutions at positions 25, 28, 60, 65, 74, 111, 117, or 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the present disclosure provides a modified interferon-pia polypeptide having interferon-pia activity, the polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and comprising one or more amino acid substitutions, the one or more amino acid substitutions at one or more of position 25, 28, 60, 65, 74, 111, 117, or 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the present disclosure provides a modified interferon-pia polypeptide having interferon-pia activity, the polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and comprising at least four amino acid substitutions, the amino acid substitutions at one or more of positions 25, 28, 60, 65, 74, 111, 117, or 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the present disclosure provides a modified interferon-P la polypeptide having interferon-P la activity, the polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and comprising at least eight amino acid substitutions, the eight amino acid substitutions at one or more of positions 25, 28, 60, 65, 74, 111, 117, and 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the present disclosure provides a modified interferon-pia polypeptide having interferon-pia activity, the polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and comprising at least eight amino acid substitutions, the one or more amino acid substitutions at one or more of position 25, 28, 60, 65, 74, 111, 117, and 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the modified interferon-P la polypeptide has amino acid substitution(s) of N25P, L28P, Y60H, N65H, S74L, F111L, M117T, N158E, or a combination thereof.
  • the amino acid substitutions are N25P, Y60H, Ml 17T, and N158E.
  • the polypeptide also has an amino acid substitution of L28P, N65H, S74L, or Fl 1 IL.
  • the polypeptide also has amino acid substitutions of L28P, N65H, S74L, and Fl 1 IL.
  • the modified interferon-P la polypeptide has an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some aspects, the modified interferon-P la polypeptide has an amino acid sequence of SEQ ID NO: 2. In some aspects, the modified interferon-P la polypeptide has an amino acid sequence of SEQ ID NO: 3. In some aspects, the modified interferon-P la polypeptide has a reduced immunogenicity as compared to a wild type interferon- pia polypeptide of SEQ ID NO: 1. In some aspects, the modified interferon-P la polypeptide has a relative antiviral activity of about 10% to about 200% compared to a wild type interferon-P la polypeptide of SEQ ID NO: 1.
  • the modified interferon-P la polypeptide has a relative antiviral activity of about 100% to about 140% as compared to a wild type interferon-P la polypeptide of SEQ ID NO: 1.
  • the present disclosure provides a pharmaceutical composition comprising the modified interferon P-1 a polypeptide and a pharmaceutically acceptable excipient.
  • the present disclosure provides a method for treating multiple sclerosis in a subject in need thereof, by administering the pharmaceutical composition to the subject.
  • the present disclosure provides a nucleotide sequence encoding the modified interferon P-1 a polypeptide with interferon P-1 a activity.
  • the nucleotide sequence is SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the present disclosure provides a plasmid incorporating the nucleotide sequence encoding the modified interferon P-1 a polypeptide.
  • the present disclosure provides an expression vector incorporating the nucleotide sequence encoding the modified interferon P-1 a polypeptide.
  • the present disclosure provides a cell line for the expression of the modified interferon P-1 a polypeptide with interferon P-1 a activity, having a nucleotide sequence encoding the modified interferon P-la polypeptide with interferon P-la activity.
  • the cell line is selected from CHO-K1, HEK293, NSO, BHK, Sp2/0, CAP or CAP/T. In some aspects, the cell line is CHO-K1.
  • the present disclosure provides a method of preparing the modified interferon P-la polypeptide with interferon beta-la activity.
  • the method involves cloning a nucleotide sequence into a lentilviral vector.
  • the nucleotide sequences is selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
  • the method also involves transducing a Chinese hamster ovary cell line (CHO-K1) with the vector and expressing the modified interferon P-la polypeptide.
  • the modified interferon P-la polypeptide has an amino acid sequence selected from SEQ ID NO: 2 or SEQ ID NO: 3.
  • the method also involves collecting the supernatant and purifying the modified interferon P-la polypeptide from the supernatant using dye pseudo-affinity chromatography and high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • the method also involves concentrating the modified interferon P-la polypeptide with interferon beta- la activity by diafiltration.
  • FIG. 1 depicts the EpiMatrix MHC binding cluster immunogenicity scale.
  • IFNP wild type SEQ ID NO: 1
  • VARI deimmunized variants IFN-pia
  • VAR2 IFN-pia
  • the EpiMatrix cluster immunogenicity score represents the deviation in putative epitope content from baseline expectation based on a random peptide standard.
  • MHC binding clusters scoring above +10 are considered to be potentially immunogenic, while MHC binding clusters scoring lower than +10 are considered to have less potential to be immunogenic.
  • Some positive control peptides and proteins are also arranged by EpiMatrix score of immunogenicity, from highest (+80) to lowest (-50).
  • the Z score is an estimator of immunogenicity from the evaluation of the content of epitopes capable of triggering a T cell-dependent immune response.
  • FIG. 2 depicts a predicted lower pseudoenergy molecular structures of IFNP wild type (SEQ ID NO: 1) and the deimmunized variants IFN-pia(VARl) (SEQ ID NO:2) and IFN- pia(VAR2) (SEQ ID NO:3).
  • Peptide backbones are represented by ribbons and residues selected for mutation by Van der Waals spheres. All residues considered for mutation are labeled with a one-letter code to facilitate comparison.
  • FIGS. 3A-3B depicts a purity evaluation of different modified IFN-pia polypeptides. Specifically, FIG. 3 A depicts the protein purity of the variants as assessed by SDS-PAGE under reducing conditions, followed by Coomassie blue staining. The purity level of the purified IFNP- la and its de-immunized variants as provided herein exceeded 96.5%. FIG. 3B depicts the identity of the observed electrophoretic band, as confirmed by western blotting.
  • FIGS. 4A-4B depicts circular dichroism UV spectra of different modified IFN-pia polypeptides, expressed in molar ellipticity per residue. The spectra were obtained using a Jasco J-1500 spectropolarimeter (Jasco Inc., Easton, USA) at room temperature.
  • FIG. 4A displays results acquired in the range of 250-320 nm (near ultraviolet), while FIG. 4B displays results acquired in the range of 190 nm and 240 nm (far ultraviolet), using a scan speed of 20 nm/min and a response time of 1 second.
  • FIG. 5 illustrates the functional characterization of the purified IFNP-la variants through the quantification of their antiviral biological activity.
  • the biological activity (AB) values of each polypeptide were determined by comparison with the standard using the parallel lines test.
  • FIG. 6 depicts the specific antiviral biological activity of rhIFNP and de-immunized variants in a bar graph. The antiviral activity of the IFNP-la variants was assessed by their ability to inhibit the cytopathic effect of VSV on WISH cells, normalized to the concentration of each protein.
  • IFNP-la (VAR2) (SEQ ID NO: 3) showed similar antiviral activity to the wild type IFNP- la (SEQ ID NO: 1), while IFNP- la (VARI) (SEQ ID NO: 2) demonstrated a 40% increase in specific biological activity compared to the original molecule. Assays were conducted in quintuplets, and data is presented as the mean ⁇ SD of eight independent experiments (* p ⁇ 0.05).
  • FIGS. 7A-7F depict the quantification of total antibody titers across various plasma sample dilutions from animals 1-4 (FIG. 7A, FIG. 7C, FIG. 7E) and animals 5-8 (FIG. 7B, FIG. 7D, FIG. 7F) inoculated with IFNP- la WT (SEQ ID NO: 1), IFNP- la VARI (SEQ ID NO: 2), and IFNP- la VAR2 (SEQ ID NO: 3), using an indirect ELISA assay.
  • IFNP- la WT SEQ ID NO: 1
  • IFNP- la VARI SEQ ID NO: 2
  • IFNP- la VAR2 SEQ ID NO: 3
  • FIGS. 8A-8C illustrate box-and-whisper plots, illustrating the deimmunized variants exhibit markedly reduced in vivo immunogenicity.
  • FIG. 8A demonstrates splenocyte culture from IFNP- la (WT)-treated mice exhibited robust IFN-y release, which was expressed as stimulation index (SI).
  • FIG. 8B demonstrates that mice treated with IFNP-la(WT) developed higher titers of anti-IFNP-la (WT) BAbs.
  • FIG. 8C demonstrates that mice treated with IFNP-la (WT) developed higher titers of anti-IFNP-la (WT) NAbs.
  • FIG. 9A-9D illustrate the results from flow cytometric analysis using CD69 specific tags to activate memory (FIG. 9B, FIG. 9D) and naive (FIG.9A, FIG. 9C) T cells in PBMC samples from patients with multiple sclerosis. Values expressed as percentage of activated T lymphocytes with respect to the negative control (excipients).
  • FIGS. 10A-10D illustrate the results from flow cytometric analysis using CD69 (FIG. 10A, FIG. 10C) and CD 154 (FIG. 10B, FIG. 10D) specific tags to restimulate memory T lymphocytes in PBMC samples from patients with multiple sclerosis. Values expressed as percentage of activated T lymphocytes with respect to the negative control (excipients).
  • FIGS. 11A-11E illustrate the results from flow cytometric analysis using CD69 specific tags to activate memory and naive T cells in PBMC samples from multiple sclerosis patients from a cohort of 5 volunteer donors (Donor 7 (FIG. 11 A), Donor 8 (FIG. 11B), Donor 9 (FIG. 11C), Donor 10 (FIG. 11D), Donor 11 (FIG. HE)). Values expressed as percentage of activated T lymphocytes with respect to the negative control (excipients). Results obtained from flow cytometric analysis using CD69 specific tags.
  • FIGS. 12A-12F illustrate the results from flow cytometric analysis using CD69 (FIG. 12A, FIG. 12C, FIG. 12E) and CD 154 (FIG. 12B, FIG. 12D, FIG. 12F) specific tags to restimulate memory T lymphocytes in PBMC from a patient with multiple sclerosis. Values expressed as percentage of activated T lymphocytes with respect to the negative control (excipients).
  • modified IFNP-1 polypeptides including modified IFNP-la and modified IFNP-lb polypeptides
  • modified IFNP-1 polypeptides that present biological activity equivalent to recombinant IFNP-1 polypeptides and a reduction in its immunogenicity
  • nucleic acids that encode such modified IFNP-1 polypeptides expression cassettes, plasmids, vectors, cells comprising such nucleic acids, and pharmaceutical compositions and formulations comprising the modified IFNP-1 polypeptides.
  • these various compositions and formulations may be used in treating one or more diseases or conditions such as but not limited to neurodegenerative diseases, including but not limited to multiple sclerosis (MS).
  • MS multiple sclerosis
  • biological sample refers to any sample of tissue, cells, or secretions from an organism.
  • medical condition includes, but is not limited to, any condition or disease manifested as one or more physical and/or psychological symptoms for which treatment and/or prevention is desirable, and includes previously and newly identified diseases and other disorders.
  • immune response refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of cancerous cells, metastatic tumor cells, malignant melanoma, invading pathogens, cells or tissues infected with pathogens, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • the term “effective amount”, “therapeutically effective amount”, or the like of a composition, including modified interferon-P 1 compounds or compositions of the present disclosure is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount that results in the prevention of, or a decrease in, the symptoms associated with a disease that is being treated.
  • the amount of a compound or composition of the present disclosure administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compounds and compositions of the present disclosure may also be administered in combination with each other or with one or more additional therapeutic compounds.
  • T cell epitope means an MHC ligand or protein determinant, 7 to 30 amino acids in length, and capable of specific binding to human leukocyte antigen (HL A) molecules and interacting with specific T cell receptors (TCRs).
  • HL A human leukocyte antigen
  • TCRs T cell receptors
  • T cell epitopes are linear and do not express specific three-dimensional characteristics. T cell epitopes are not affected by the presence of denaturing solvents.
  • T cell epitopes The ability to interact with T cell epitopes may be predicted by in silico methods (De Groot AS et al., (1997), AIDS Res Hum Retroviruses, 13(7):539-41; Schafer JR et al., (1998), Vaccine, 16(19): 1880-4; De Groot AS et al., (2001), Vaccine, 19(31):4385-95; De Groot AR et al., (2003), Vaccine, 21(27-30):4486-504, all of which are herein incorporated by reference in their entirety.
  • T-cell epitope cluster refers to polypeptide that contains between about 4 to about 40 MHC binding motifs. In particular embodiments, the T-cell epitope cluster contains between about 5 to about 35 MHC binding motifs, between about 8 and about 30 MHC binding motifs; and between about 10 and 20 MHC binding motifs.
  • immune stimulating T-cell epitope polypeptide refers to a molecule capable of inducing an immune response, e.g., a humoral, T cell-based, or innate immune response.
  • B cell epitope means a protein determinant capable of specific binding to an antibody.
  • B cell epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • subject refers to any living organism in which an immune response is elicited.
  • subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • farm animals such as cattle, sheep, pigs, goats and horses
  • domestic mammals such as dogs and cats
  • laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • MHC complex refers to a protein complex capable of binding with a specific repertoire of polypeptides known as HLA ligands and transporting said ligands to the cell surface.
  • MHC Ligand means a polypeptide capable of binding to one or more specific MHC alleles.
  • HLA ligand is interchangeable with the term “MHC Ligand”.
  • APCs Antigen Presenting Cells
  • T Cell Receptor or “TCR” refers to a protein complex expressed by T cells that is capable of engaging a specific repertoire of MHC/Ligand complexes as presented on the surface of APCs.
  • MHC Binding Motif refers to a pattern of amino acids in a protein sequence that predicts binding to a particular MHC allele.
  • EpiBarTM refers to a 9-mer peptide that is predicted to be reactive to at least four different HLA alleles.
  • Immuno Synapse means the protein complex formed by the simultaneous engagement of a given T cell epitope to both a cell surface MHC complex and TCR.
  • polypeptide refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide.
  • a polypeptide is said to be “isolated” or “purified” when it is substantially free of cellular material when it is isolated from recombinant and non-recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized.
  • a polypeptide (e.g., a modified IFNP- la polypeptide) of the present disclosure, however, may be joined to, linked to, or inserted into another polypeptide (e.g., a heterologous polypeptide) with which it is not normally associated in a cell and still be “isolated” or “purified.”
  • a polypeptide When a polypeptide is recombinantly produced, it may also be substantially free of culture medium, for example, culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation.
  • polynucleotide and “nucleic acid sequence” are used interchangeably to refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.
  • polynucleotide is not intended to limit the present invention to polynucleotides comprising DNA.
  • polynucleotides may include ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues.
  • the polynucleotides as provided herein also encompass all forms of sequences including, but not limited to, singlestranded forms, double-stranded forms, and the like.
  • the terms “encoding” or “encoded” when used in the context of a specified polynucleotide mean that the polynucleotide comprises the requisite information to direct translation of the polynucleotide sequence into a specified polypeptide.
  • the information by which a polypeptide is encoded is specified by the use of codons.
  • a polynucleotide encoding a polypeptide may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA).
  • naturally interferon refers to a cytokine (e.g., polypeptide, nucleic acid, etc.) as it is found in nature (i.e., wild type), without having been subjected to any kind of artificial modification or mutation.
  • cytokine e.g., polypeptide, nucleic acid, etc.
  • amino acid substitution refers to the change of one amino acid in the primary sequence of a natural (i.e., wild type) protein, such as hIFNP-la, for another amino acid.
  • modified interferonP-la refers to molecules of a modified interferon beta- la molecule, containing changes to the amino acid or nucleic acid sequence as compared to the appropriate natural interferon, and in aspects includes at least one glycosylation site. In aspects, the molecules have reduced immunogenicity as compared to natural human interferon.
  • z-score indicates how many standard deviations an element is from the mean.
  • modified IFNP-1 polypeptides including modified IFNP-la and modified IFNP-lb polypeptides
  • a modified IFNP-1 polypeptide of the present disclosure is a modified IFNP-la polypeptide having IFNP-la activity and a reduced immunogenicity or a reduced propensity to elicit an immune response as compared to a wild type IFNP-la polypeptide of SEQ ID NO: 1.
  • the present disclosure provides a modified IFNP-1 polypeptide or nucleic acid encoding the modified IFNP-1 polypeptide having IFNP-1 activity (e.g., anti-viral activity) and reduced immunogenicity.
  • the modifications carried out in the natural amino acid sequence of human IFNP-la for obtaining the modified IFNP-la of the disclosure are a result of a modification of a gene encoding natural human interferon, such as wild-type IFNP-la. Further, the modifications are introduced in such a way that they reduce the immunogenicity of the amino acid sequence as compared to natural human interferon, while substantially maintaining or improving its biological activity (such as its antiviral biological activity).
  • the modified IFNP-la polypeptides and related modified IFNP-1 compounds and compositions of the present disclosure have reduced immunogenicity as compared to natural interferonP-1.
  • Mutations that reduce immunogenicity of a modified IFNP-la as compared to natural IFNP-la were identified by Interactive Screening and Protein Reengineering Interface (ISPRI) program analysis, provided by the company EpiVax, Inc. (Providence, Rhode Island), which is used to screen protein sequences for the presence of putative T cell epitopes. Input sequences are parsed into overlapping 9-unit (9-mer) frames where each frame overlaps the last by 8 amino acids.
  • ISPRI Interactive Screening and Protein Reengineering Interface
  • Each of the resulting frames is then scored for predicted binding affinity with respect to a panel of eight common Class II HLA alleles (DRB 1*0101, DRB 1*0301, DRB 1*0401, DRBl*0701, DRBl*0801, DRBl*1101, DRBl*1301 and DRBl*1501).
  • Raw scores are normalized against the scores of a large sample of randomly generated peptides.
  • the resulting “Z” score is reported.
  • any 9-mer frame peptide with an allele-specific EpiMatrixTM Z- score in excess of 1.64, theoretically the top 5% of any given sample is considered to have a high probability of binding to MHC Class II.
  • any 9-mer frame peptide with an allele-specific EpiMatrixTM Z-score in excess of 2.32, theoretically the top 1% of any given sample, is considered to have a high probability of binding MHC Class II and includes most of the published T cell epitopes.
  • Peptides containing clusters of putative T cell epitopes are more likely to test positive in validating in vitro and in vivo assays.
  • the results of the initial EpiMatrixTM analysis are further screened for the presence of putative T cell epitope “clusters” using a second proprietary algorithm known as ClustimerTM algorithm.
  • the ClustimerTM algorithm identifies sub-regions contained within any given amino acid sequence that contains a statistically unusually high number of putative T cell epitopes.
  • Typical T-cell epitope “clusters” are from about 9 amino acids in length and, considering their affinity to multiple alleles and across multiple 9-mer frames, may contain anywhere from about 4 to about 40 putative T cell epitopes.
  • Each epitope cluster identified an aggregate EpiMatrixTM score is calculated by summing the scores of the putative T cell epitopes and subtracting a correcting factor based on the length of the candidate epitope cluster and the expected score of a randomly generated cluster of the same length. EpiMatrixTM cluster scores in excess of +10 are considered significant.
  • the modified IFNP-1 molecules described herein contain one or more modifications (e.g., changes, substitutions, or mutations) within the T cell epitope clusters to reduce their immunogenicity.
  • modified IFNP-1 mutations for the instantly-disclosed modified IFNP-1 molecules are selected that not only reduce the immunogenicity of the molecule, but also do not significantly reduce its biological activity, such as its antiviral activity, and/or that do not affect its binding to receptors involved in the interferon’ s biological activity.
  • modifications for modified IFNP-1 molecules of the present disclosure are selected that do not disrupt the structure or function of the natural interferon and include substitution of one or more amino acids occupying select positions in the natural human IFNP-1 for any natural or non-naturally occurring amino acid, but in some aspects optionally for one or more of proline, histidine, leucine, threonine, or glutamic acid.
  • the modified IFNP-1 molecules of the present disclosure may include one or more modifications (e.g., changes, substitutions, or mutations) within the EpiBars® of the natural IFNP-1.
  • said modifications of the modified IFNP-1 molecules reduce the immunogenicity of the modified IFNP-1 molecules as compared to the natural IFNP-1.
  • said modifications of the modified IFNP-1 molecules additionally do not substantially disrupt the structure or function of the natural IFNP-1 activity.
  • modified IFNP-1 mutations are selected that do not significantly reduce its biological activity, such as its antiviral activity, and/or that do not affect its binding to receptors involved in the interferon’s biological activity.
  • modifications for modified IFNP-1 molecules of the present disclosure are selected that do not disrupt the structure or function of the natural interferon and include substitution of one or more amino acids occupying select positions in the natural human IFNP-1 optionally for proline, histidine, leucine, threonine, or glutamic acid.
  • OptiMatrix tool part of the EpiVax ISPRI toolkit for deimmunization. OptiMatrix begins with looking at “critical” residues, which contribute most to MHC binding affinity across multiple 9-mer frames and multiple HLA alleles. The program then iteratively substitutes all 19 alternative amino acids in any given position of a protein sequence (with operator-defined input that may limit the list to naturally conserved variants) and then reanalyzes the predicted immunogenicity of the sequence following that change. To avoid a negative effect on protein structure and consequently in biological activity, a comprehensive search in literature for critical residues was also conducted, which identified amino acids that were not candidates for modification.
  • said modifications of the modified IFNP-1 molecules reduce the immunogenicity of the modified IFNP-1 molecules as compared to the natural IFNP- 1.
  • said modifications of the modified IFNP-1 molecules additionally do not disrupt the structure or function of the natural IFNP-1 activity.
  • modified IFNP-1 mutations are selected that do not significantly reduce its biological activity, such as its antiviral activity, and/or that do not affect its binding to receptors involved in the interferon's biological activity.
  • modifications for modified IFNP-1 molecules of the present disclosure are selected that do not disrupt the structure or function of the natural interferon and include substitution of one or more amino acids occupying select positions in the natural human IFNP-1 optionally for proline, histidine, leucine, threonine, or glutamic acid.
  • a modified IFNP-1 polypeptide comprises the substitution of one or more amino acids in wild-type IFNP-la (SEQ ID NO: 1).
  • the substitutions are of one or more amino acids occupying a position selected from the group consisting of the following positions in the natural IFNP-la: 25, 28, 60, 65, 74, 111, 117, and/or 158.
  • the substitution includes the change of the amino acid from that position to an amino acid that may be any naturally occurring amino acid, any non-naturally occurring amino acid, or in some aspects and amino acid that is proline, histidine, leucine, threonine, or glutamic acid.
  • the substitution reduces the immunogenicity of the modified IFNP-1 polypeptide as compared to the natural human IFNP- 1.
  • a modified IFNP-1 molecule is a modified IFNP-la polypeptide.
  • a modified IFNP-1 polypeptide is a modified IFNP-lb polypeptide.
  • a modified IFNP-1 polypeptide includes an amino acid sequence with at least 60%, 70%, 80%, 90%, or 95% sequence identity to wild type IFNP-la (SEQ ID NO: 1).
  • a polypeptide may include one or more amino acid substitutions in any of the positions 25, 28, 60, 65, 74, 111, 117, and/or 158.
  • a modified IFNP-la polypeptide includes an amino acid sequence with at least 60%, 70%, 80%, 90%, or 95% identity to wild type IFNP-la (SEQ ID NO: 1) and therein includes one or more amino acid substitutions in any of the positions 25, 28, 60, 65, 74, 111, 117, and/or 158, wherein said substitution optionally is or includes the change of the amino acid of said position to proline, histidine, leucine, threonine, or glutamic acid.
  • a modified IFNP-la polypeptide incl dues an amino acid sequence with at least 60%, 70%, 80%, 90%, or 95% identity to wild type IFNP-la (SEQ ID NO: 1) and therein includes at least four amino acid substitutions in any of the positions 25, 28, 60, 65, 74, 111, 117, and/or 158.
  • a modified IFNP-la polypeptide includes an amino acid sequence with at least 60%, 70%, 80%, 90%, or 95% homology to wild type IFNP-la (SEQ ID NO: 1) and therein includes at least four amino acid substitutions in any of the positions 25, 28, 60, 65, 74, 111, 117, and/or 158, optionally wherein said substitution includes the change of the amino acid of said position to proline, histidine, leucine, threonine, or glutamic acid.
  • a modified IFNP-la polypeptide having IFNP-la activity includes an amino acid sequence with at least 60%, 70%, 80%, 90%, or 95% identity to wild type interferon-a2b (SEQ ID NO: 1) and therein includes one or more amino acid substitutions at positions 25, 60, 117, and/or 158, optionally wherein said substitutions include the change of the amino acid of the position to proline, histidine, leucine, threonine, or glutamic acid.
  • a modified IFNP-la polypeptide having IFNP-la activity includes an amino acid sequence with at least 60%, 70%, 80%, 90%, or 95% identity to wild type IFNP-la (SEQ ID NO: 1) and therein includes the mutations N25P, Y60H, Ml 17T and N158E.
  • the modified IFNP-la polypeptides have reduced immunogenicity or a reduced propensity to elicit an immune response as compared to a wild type IFNP-la polypeptide of SEQ ID NO: 1.
  • a modified IFNP-la polypeptide having IFNP-la activity includes an amino acid sequence with at least 60%, 70%, 80%, 90%, or 95% identity to wild type IFNP-la (SEQ ID NO: 1) and therein includes amino acid substitutions at positions 25, 28, 60, 65, 74, 111, 117, and/or 158, optionally wherein said substitutions include the change of the amino acid of the position to proline, histidine, leucine, threonine, or glutamic acid.
  • a modified IFNP-la polypeptide having IFNP-la activity includes an amino acid sequence with at least 60%, 70%, 80%, 90%, or 95% identity to wild type IFNP-la (SEQ ID NO: 1) and therein includes the mutations N25P, L28P, Y60H, N65H, S74L, F111L, M117T, and/or N158E.
  • the modified IFNP-la polypeptides have reduced immunogenicity or a reduced propensity to elicit an immune response as compared to a wild type IFNP-la polypeptide of SEQ ID NO: 1.
  • a modified IFNP-la polypeptide having IFNP-la activity is SEQ ID NO: 2 or SEQ ID NO: 3.
  • a modified IFNP-la polypeptide having IFNP-la activity includes an amino acid sequence of SEQ ID NO: 2 (IFNP-la(VARl)) form.
  • a modified IFNP-la polypeptide having IFNP-la activity includes an amino acid sequence of SEQ ID NO: 3 (IFNP- la(VAR2)).
  • the modified IFNP-la polypeptides have reduced immunogenicity or a reduced propensity to elicit an immune response as compared to a wild type IFNP-la polypeptide of SEQ ID NO: 1.
  • the instantly-disclosed modified IFNP-la polypeptides having IFNP-la activity such as the above-described modified IFNP-la polypeptides, have a relative antiviral activity of about 5% to about 200% as compared to a wild type IFNP-la polypeptide of SEQ ID NO: 1.
  • the instantly-disclosed modified IFNP-la polypeptides having IFNP-la activity such as the above-described modified IFNP-la polypeptides, have a relative antiviral activity of about 10% to about 175% as compared to a wild type IFNP-la polypeptide of SEQ ID NO: 1.
  • the instantly-disclosed modified IFNP-la polypeptides having IFNP-la activity such as the above-described modified IFNP-la polypeptides, have a relative antiviral activity of about 20% to about 150% as compared to a wild type interferon-a2b polypeptide of SEQ ID NO: 1.
  • the instantly-disclosed modified IFNP-la polypeptides having IFNP-la activity such as the above-described modified IFNP-la polypeptides, have a relative antiviral activity equal to or greater then a wild type interferon-a2b polypeptide of SEQ ID NO: 1.
  • the instantly-disclosed modified IFNP-la polypeptides having IFNP-la activity have a relative antiviral activity of about 100% to about 150% as compared to a wild type interferon-a2b polypeptide of SEQ ID NO: 1.
  • the instantly-disclosed modified IFNP-la polypeptides having IFNP-la activity such as the abovedescribed modified IFNP-la polypeptides, have a relative antiviral activity of about 100% to about 140% as compared to a wild type interferon-a2b polypeptide of SEQ ID NO: 1.
  • the instantly-disclosed modified IFNP-la polypeptides having IFNP-la activity such as the abovedescribed modified IFNP-la polypeptides, have a relative antiviral activity as compared to a wild type interferon-a2b polypeptide of SEQ ID NO: 1 of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or about 150%.
  • the present disclosure provides a polynucleotide or nucleic acid (e.g., DNA, including cDNA or RNA, including mRNA) encoding a modified IFNP-la polypeptide having IFNP-la activity, such as the above-described modified IFNP-la polypeptides.
  • a nucleic acid encoding for one or more one or more modified IFNP-la polypeptides includes one or more nucleic acid sequences of: SEQ ID NO: 5 or SEQ ID NO: 6.
  • a nucleic acid encoding a modified IFNP-la polypeptide includes a nucleic acid sequence of SEQ ID NO: 5, encoding (IFNP-la(VARl)). In aspects, a nucleic acid encoding a modified IFNP-la polypeptide includes a nucleic acid sequence of SEQ ID NO: 6, (IFNP-la(VAR2)).
  • the present disclosure provides a vector or plasmid comprising a nucleic acid of the present disclosure encoding one or more modified IFNP-la polypeptides of the present disclosure.
  • the vector or plasmid includes a nucleic acid (e.g., DNA or RNA) encoding a modified IFNP-la polypeptide, having a sequence comprising, consisting of, or consisting essentially of one or more of SEQ. ID NO: 7 (IFNP-la(VARl)) or SEQ ID NO: 8 (IFNP- la(VAR2)).
  • the present disclosure is directed to a cell comprising a vector or plasmid of the present disclosure. It will be appreciated that the nucleic acid sequence of the vector or plasmid may have slight variations based on codon use optimization to effect expression of the IFNP-la polypeptide in the cell.
  • a modified IFNP-la polypeptide as described herein is joined to or linked to (e.g., fused in-frame, chemically-linked, or otherwise bound) a heterologous polypeptide.
  • heterologous polypeptide is intended to mean that the one or more modified IFNP-la polypeptides of the instant disclosure are not included naturally in the heterologous polypeptide.
  • one or more of the instantly-modified IFNP-la polypeptides may be added to the C- terminus (with or without the use of linkers, as is known in the art), and/or added to the N-terminus (with or without the use of linkers, as is known in the art) of the heterologous polypeptide.
  • the present disclosure also provides chimeric or fusion polypeptides (which in aspects may be isolated, synthetic, or recombinant) wherein one or more of the instantly disclosed modified IFNP-la polypeptides is a part thereof.
  • the one or more modified IFNP-la polypeptides of the present disclosure may be joined or linked to (e.g., fused in-frame, chemically- linked, or otherwise bound) a small molecule, drug, or drag fragment, for example, but not limited to, a drug or drug fragment that is binds with high affinity to defined receptors.
  • two polypeptides are substantially homologous or identical when the amino acid sequences have a certain percentage or more identity, e.g., at least about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, optionally at least about 70-75%, optionally at least about 80-85%, optionally equal to or greater than about 90%, and optionally equal to or greater than 95% or more homologous or identical. Percent homology may be determined as is known in the art.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one polypeptide or nucleic acid molecule for optimal alignment with the other polypeptide or nucleic acid molecule).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid “identity” is equivalent to amino acid “homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Sequence homology for polypeptides is typically measured using sequence analysis software.
  • the present disclosure also encompasses polypeptides (e.g., modified IFNP-la polypeptides and modified IFNP-la compositions as disclosed herein) having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by a polypeptide encoded by a nucleic acid molecule as provided herein. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Vai, Leu, Met, and lie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Trp, and Tyr.
  • Guidance concerning which amino acid changes are likely to be phenotypically silent are found (Bowie, JU, et al., (1990), Science, 247(4948): 130610, which is herein incorporated by reference in its entirety).
  • amino acid sequences having the function of an interferon may be identified by performing a protein-protein BLAST (blastp) search of the non-redundant protein sequences (nr) database using the amino acid sequences of these proteins as query.
  • the search may be conducted on the National Center for Biotechnology Information (NCBI) website (http.//blast.ncbi. nlm.nih.gov) using default parameters.
  • NCBI National Center for Biotechnology Information
  • Fragments and variants of the disclosed modified IFNP-la polypeptides and polynucleotides are also encompassed by the present disclosure. “Fragment” is intended to mean a portion of the polypeptide or polynucleotide. Fragments of a polypeptide or a nucleotide sequence as disclosed herein may encode polypeptide fragments that retain the biological activity of the polypeptides of the instant disclosure, and hence have retain IFNP-la activity (e.g., antiviral biological activity) with reduced immunogenicity as compared to wild-type IFNP-la. In aspects, the present disclosure also encompasses fragments of the variants of the polypeptides and polynucleotides described herein.
  • a variant polypeptide may differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these.
  • Variant polypeptides may be fully functional (e.g., retain IFNP-la activity, such as antiviral biological activity) or may lack function in one or more activities.
  • Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions.
  • Functional variants may also contain substitution of similar amino acids that result in no change or an insignificant change in function (e.g., retain antiviral biological activity with reduced immunogenicity). Alternatively, such substitutions may positively or negatively affect function to some degree.
  • Non-functional variants typically contain one or more nonconservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
  • a modified IFNP-la polypeptide of the instant disclosure may differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these, provided said variants retain biological activity (e.g., IFNP-la activity, such an antiviral activity) and have reduced immunogenicity (as compared to wild-type IFNP-la).
  • fully functional variants of modified IFNP-la do not contain mutations at one or more critical residues or regions.
  • said one or more critical residues of modified IFNP-la that should not be mutated include: residues involved in or essential for biological activity, residues of functional hotspots that are heavily conserved between various wild type interferon alleles (such as between species), residues implicated in binding to the interferon’s natural receptor, residues involved in structural interactions that are important to the structural integrity of the natural interferon, residues engaged in disulfide bonds of the natural interferon (e.g., intramolecular disulfide bonds that occur in the natural interferon upon proper folding in its natural environment in vivo), and/or residues that are the site of glycosylation in the natural, wild type interferon (including N-glycosylation sites and O-glycosylation sites).
  • the instantly-disclosed modified IFNP-la polypeptides do not contain mutations at one or more critical residues or regions, wherein said one or more critical residues or regions are selected from the group comprising: residues of functional hotspots, residues that are heavily conserved between various wild type interferon alleles (such as between species), residues engaged in disulfide bonds of the natural interferon (e.g., intramolecular disulfide bonds that occur in the natural interferon upon proper folding in its natural environment in vivo), and/or residues that are the site of glycosylation in the natural, wild type interferon (including N-glycosylation sites and 0- glycosylation sites).
  • residues of functional hotspots residues that are heavily conserved between various wild type interferon alleles (such as between species), residues engaged in disulfide bonds of the natural interferon (e.g., intramolecular disulfide bonds that occur in the natural interferon upon proper folding in its natural environment in viv
  • polypeptides of the instant disclosure may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the instantly-disclosed polypeptides may be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192: Walker and Gaastra, eds.
  • polypeptides may include, for example, modified forms of naturally occurring amino acids such as D-stereoisomers, non-naturally occurring amino acids, amino acid analogs, and mimetics.
  • modified IFNP- la polypeptides of the present disclosure will vary widely, depending upon the nature of the various elements comprising the molecule.
  • the modified IFNP- la polypeptides may be purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods.
  • the synthetic procedures may be selected so as to be simple, provide for high yields, and allow for a highly purified stable product.
  • polypeptides of the instant disclosure may be produced either from a nucleic acid disclosed herein, or by the use of standard molecular biology techniques, such as recombinant techniques, mutagenesis, or other known means in the art.
  • An isolated polypeptide may be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis techniques.
  • a polypeptide of the instant disclosure is produced by recombinant DNA or RNA techniques.
  • a polypeptide of the instant disclosure may be produced by expression of a recombinant nucleic acid of the instant disclosure in an appropriate host cell. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression cassette or expression vector, the expression cassette or expression vector introduced into a host cell and the polypeptide expressed in the host cell. The polypeptide may then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
  • polypeptide may be produced by a combination of ex vivo procedures, such as protease digestion and purification.
  • polypeptides of the instant disclosure may be produced using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et al., 2012, Protein Engineering Methods and Applications, intechopen.com, which are herein incorporated by reference in their entirety).
  • the present disclosure is also directed to a method of synthesizing modified IFNP-la polypeptides.
  • the instantly-disclosed modified the modified IFNP-la polypeptides with the improved properties may be created through genetic modification in one of a variety of ways that are described herein.
  • the term “the modified IFNP- la” as used herein may refer to the group of instantly disclosed modified the modified IFNP-la polypeptides having an intentionally altered amino acid sequence, i.e., a “non-wild type” amino acid sequence, or to a microbial organism (depending upon placement of either term as an adjective) having a genome that has been intentionally altered as to (at least) the specific, modified IFNP-la molecules described herein, or both.
  • Such alterations may be accomplished via recombinant technology, wherein one or more genes are transferred from a second, different microbial organism into a target microbial organism.
  • Recombinant technology may be accomplished using fully synthetic DNA that is transferred to the target microbial organism using conventional methods.
  • Such alterations may also be accomplished via engineered technology, wherein the nucleic acids within the target microbial organism are altered, generally via site- directed mutagenesis, resulting in the conversion of at least one nucleic acid to a different nucleic acid and therefore modification of one or more enzymes. Combinations of any of the above methods and those described throughout the application may also be employed.
  • the instantly disclosed modified IFNP-la molecule may be produced either in vivo, i.e., by a genetically modified microorganism, or in vitro.
  • the present disclosure provides a method for generating said amino acid substitutions to reduce immunogenicity.
  • Said method optionally includes the generation of point mutations in the nucleotide sequence of the gene encoding the human natural interferon (e.g., natural IFNP-la), by means of a site-directed mutagenesis technique in said gene.
  • the method includes the following steps: 1) cloning a gene encoding natural human interferon (e.g IFNP-la) in a suitable plasmid; 2) generating mutations required for producing the modified IFNP-la of the present disclosure using a site-directed mutagenesis technique; and 3) cloning the modified gene from step 2, into a suitable expression vector.
  • the expression vector is capable of carrying the gene of the present disclosure and further containing the necessary elements for expressing the gene of interest in eukaryotic cells.
  • the site-directed mutagenesis technique of the present disclosure involves the use of oligonucleotides specifically designed to that end.
  • This technique includes two stages. In the first stage, two PCR reactions are carried out separately using oligonucleotides that hybridize to the terminal ends of the fragment cloned into a suitable vector, and oligonucleotides carrying a point mutation corresponding to an amino acid substitution that reduces immunogenicity (as described here) that hybridize to the internal region of the gene where the mutation is to be introduced.
  • a reaction mixture is obtained in tube a using a reverse external oligonucleotide and the direct oligonucleotide mut a.
  • Another reaction mixture is obtained in tube b with a direct external oligonucleotide and the reverse oligonucleotide mut b.
  • PCR products from both reactions are purified by agarose gel electrophoresis and used as a template for the second stage.
  • This second stage includes a second PCR reaction using direct and reverse external oligonucleotides. The first three cycles are carried out without the addition of primers to allow hybridization and elongation of the complete product and finally these are added for the amplification.
  • said modified IFNP-la is constructed sequentially as follows: first, a modified IFNP-la with amino acid substitution site is generated, using a site-directed mutagenesis technique, and then said modified IFNP-la is used as a starting template for generating a new amino acid substitution site.
  • the present disclosure also provides for nucleic acids (e.g., DNA, RNA, vectors, viruses, or hybrids thereof, all of which may be isolated, synthetic, or recombinant) that encode in whole or in part one or more modified IFNP-la polypeptides of the present disclosure and/or chimeric or fusion polypeptide compositions of the present disclosure.
  • nucleic acids e.g., DNA, RNA, vectors, viruses, or hybrids thereof, all of which may be isolated, synthetic, or recombinant
  • the nucleic acid further includes, or is contained within, an expression cassette, a plasmid, and expression vector, or recombinant virus, wherein optionally the nucleic acid, or the expression cassette, plasmid, expression vector, or recombinant virus is contained within a cell, optionally a human cell or a non-human cell, and optionally the cell is transformed with the nucleic acid, or the expression cassette, plasmid, expression vector, or recombinant virus.
  • cells are transduced, transfected, or otherwise engineered to contain within one or more of e.g., polypeptides (modified IFNP-la polypeptides) of the present disclosure; isolated, synthetic, or recombinant nucleic acids, expression cassettes, plasmids, expression vectors, or recombinant viruses as disclosed herein; and/or isolated, synthetic, or recombinant chimeric or fusion polypeptide compositions as disclosed herein.
  • the cell may be a mammalian cell, bacterial cell, insect cell, or yeast cell.
  • the nucleic acid molecules of the present disclosure may be inserted into vectors and used, for example, as expression vectors or gene therapy vectors.
  • Gene therapy vectors may be delivered to a subject by, e.g., intravenous injection, local administration (U.S. Pat. No. 5,328,470) or by stereotactic injection (Chen S H et al., (1994), Proc Natl Acad Sci USA, 91(8):3054-7, which are herein incorporated by reference in their entirety).
  • the pharmaceutical preparation of the gene therapy vector may include the gene therapy vector in an acceptable diluent, or may include a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation may include one or more cells that produce the gene delivery system.
  • compositions may be included in a container, pack, or dispenser together with instructions for administration.
  • the present disclosure is directed to a cell comprising a vector of the present disclosure.
  • the cell may be a mammalian cell, bacterial cell, insect cell, or yeast cell.
  • a “variant” includes a deletion and/or addition of one or more nucleotides at one or more internal sites within the polynucleotide sequences of the instant disclosure and/or a substitution of one or more nucleotides at one or more sites in the polynucleotide sequences of the instant disclosure.
  • variants of the polynucleotides of this disclosure will be constructed such that the open reading frame is maintained.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides as provided herein.
  • Naturally occurring allelic variants such as these may be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below.
  • Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a polynucleotide having the desired activity as provided herein (i.e., encoding a polypeptide that possesses the desired biological activity, that is, antipathogenic activity, antifungal activity, antialgal activity, and/or enzymatic activity against chitin and/or polyglucuronic acid as described herein).
  • variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
  • Variants of a particular polynucleotide of the present disclosure may also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides may be calculated using sequence alignment programs and parameters described elsewhere herein.
  • the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • RNA, DNA, expression cassettes, vectors, viruses or hybrids thereof that encode in whole or in part one or more polypeptides of the present disclosure may be isolated from a variety of sources, genetically engineered, amplified, synthetically produced, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids may be individually isolated or cloned and tested for a desired activity. Any recombinant expression system may be used, including e.g. in vitro, bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.
  • polynucleotides provided herein are synthesized in vitro by well-known chemical synthesis techniques (as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Pat. No.
  • the present disclosure is directed to a characterized cell line comprising the nucleic acid that encodes for a modified IFNP-la as provided herein.
  • said cell line is suitable for the production of a modified IFNP-la as provided herein.
  • a cell line suitable for the production of a modified interferon-a2 as provided herein is selected from the set of CHO-K1, HEK293, NS0, BHK, Sp2/0, CAP, and CAP/T.
  • the present disclosure is also directed to a method for obtaining a eukaryotic cell line for producing a modified IFNP-la as provided herein by transformation or transfection of a cell line containing said gene encoding a modified IFNP-la as provided herein, inserted in a suitable expression vector.
  • the eukaryotic cell line is a CH0.K1 cell line.
  • the present disclosure is directed to a method for producing a modified IFNP-la as provided herein, said method including the steps of a) culturing said transformed or transfected eukaryotic cell line with an expression vector containing the gene encoding a modified IFNP-la polypeptide as disclosed herein, and b) isolating the expressed and secreted modified IFNP-la polypeptide from the culture medium.
  • modified IFNP-la compounds or compositions of the present disclosure may be purified to homogeneity or partially purified. It is understood, however, that preparations in which the modified IFNP-la compositions are not purified to homogeneity are useful. The critical feature is that the preparation allows for the desired function of the modified IFNP-la, even in the presence of considerable amounts of other components.
  • the language “substantially free of cellular material” includes preparations of the modified interferon-a2 having less than about 30% (by dry weight) other proteins (e.g., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, less than about 5% other proteins, less than about 4% other proteins, less than about 3% other proteins, less than about 2% other proteins, less than about 1% other proteins, or any value or range there between.
  • other proteins e.g., contaminating protein
  • a modified IFNP-la compound or composition of the present disclosure is recombinantly produced, wherein said modified IFNP-la composition may also be substantially free of culture medium, for example, culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the modified IFNP-la polypeptide, nucleic acid, or chimeric or fusion polypeptide preparation.
  • culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the modified IFNP-la polypeptide, nucleic acid, or chimeric or fusion polypeptide preparation.
  • substantially free of chemical precursors or other chemicals includes preparations of the polypeptide, nucleic acid, or chimeric or fusion polypeptide in which it is separated from chemical precursors or other chemicals that are involved in the synthesis of the modified IFNP-la.
  • substantially free of chemical precursors or other chemicals may include, for example, preparations of modified IFNP-la polypeptide, nucleic acid, or chimeric or fusion polypeptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, less than about 5% chemical precursors or other chemicals, less than about 4% chemical precursors or other chemicals, less than about 3% chemical precursors or other chemicals, less than about 2% chemical precursors or other chemicals, or less than about 1% chemical precursors or other chemicals.
  • a modified IFNP-la compound or composition of the present disclosure may be produced by standard recombinant DNA or RNA techniques as are known in the art.
  • DNA or RNA fragments coding for the different polypeptide sequences may be ligated together in-frame in accordance with conventional techniques.
  • the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR polymerase chain reaction
  • anchor primers which give rise to complementary overhangs between two consecutive nucleic acid fragments which may subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence
  • polypeptides e.g., modified IFNP- la polypeptide
  • one or more polypeptides may be inserted into a heterologous polypeptide or inserted into a non-naturally occurring position of a polypeptide through recombinant techniques, synthetic polymerization techniques, mutagenesis, or other standard techniques known in the art.
  • protein engineering by mutagenesis may be performed using site-directed mutagenesis techniques, or other mutagenesis techniques known in the art (see e.g., James A. Brannigan and Anthony J. Wilkinson., 2002, Protein engineering 20 years on. Nature Reviews Molecular Cell Biology 3, 964-970; Turanli-Yildiz B. et al., 2012, Protein Engineering Methods and Applications, intechopen.com, which are herein incorporated by reference in their entirety).
  • fusion moiety e.g., a GST protein
  • a nucleic acid molecule encoding a modified IFNP-la of the invention may be cloned into such an expression vector such that the fusion moiety is linked in- frame to the at least one modified IFNP-la.
  • Such linking of the fusion moiety may be done, for example, to improve protein purification yields.
  • one or more modified IFNP-la polypeptides, chimeric polypeptides, polynucleotides, microorganism that expresses one or more polypeptides or polynucleotides, expression cassettes, plasmids, expression vectors, and/or recombinant viruses of the present disclosure may be comprised in a pharmaceutical composition or formulation.
  • pharmaceutical compositions or formulations generally include a modified interferonP- la compound or composition of the present disclosure and a pharmaceutically-acceptable carrier and/or excipient.
  • said pharmaceutical compositions are suitable for administration.
  • compositions for administering the instantly disclosed modified interferonP-la compositions (see, e.g., Remington’s Pharmaceutical Sciences, (18th ed., 1990), Mack Publishing Co., Easton, PA Publ)).
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • compositions, carriers, excipients, and reagents are used interchangeably and represent that the materials are capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
  • pharmaceutically-acceptable excipient means, for example, an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • exemplary carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the modified interferonP-la compounds or compositions of the present disclosure and as previously described above, use thereof in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.
  • a modified interferonP-la compound or composition of the present disclosure is formulated to be compatible with its intended route of administration.
  • the modified interferonP- la compounds or compositions of the present disclosure may be administered by parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; vaginally; intramuscular route or as inhalants.
  • modified interferonP-la compounds or compositions of the present disclosure may be injected directly into a particular tissue.
  • intramuscular injection or intravenous infusion may be used for administration of modified interferonP-la compounds or compositions of the present disclosure.
  • modified interferonP-la compounds or compositions of the present disclosure are administered as a sustained release composition or device, such as but not limited to a MedipadTM device.
  • modified interferonP-la compounds or compositions of the present disclosure may optionally be administered in combination with other agents that are at least partly effective in treating various medical conditions as described herein.
  • modified interferonP-la pharmaceutical compositions or formulations of the present disclosure may also be administered in conjunction with other agents that stimulate antiviral activity of the immune system, improve pharmacokinetic parameters of the composition, enhance and/or compliment the natural biological activity of interferonP-la, and/or reduce immunogenicity of the composition.
  • solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include, but are not limited to, a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents
  • antibacterial compounds such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • the pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • excipients may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, water, ethanol, DMSO, glycol, propylene, dried skim milk, and the like.
  • the composition may also contain pH buffering reagents, and wetting or emulsifying agents.
  • the parenteral preparation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions or formulations suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition is sterile and should be fluid to the extent that easy syringeability exists. It is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • modified interferonP-la formulations may include aggregates, fragments, breakdown products and post-translational modifications, to the extent these impurities have reduced immunogenicity and high relative antiviral activity that is similar to pure modified interferonP-la.
  • the carrier may be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity may be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic compounds e.g., sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, may be included in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition a compound that delays absorption, e.g., aluminum monostearate and gelatin.
  • sterile injectable solutions may be prepared by incorporating the modified interferonP-la compounds or compositions of the present disclosure in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the binding agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • modified interferonP-la compounds or compositions of the present disclosure may be administered in the form of a depot injection or implant preparation that may be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • solutions or suspensions of pharmaceutical compositions or formulations are maintained at a pH at which the modified interferonP-la polypeptide is in its natural structural conformation.
  • said pH is maintained below pH 10.
  • said pH is maintained below pH 7.
  • said pH is maintained between pH 3-10.
  • said pH is maintained between pH 4-9.
  • said pH is maintained between pH 5-8.
  • said pH is maintained between pH 6-7.5.
  • a buffer is provided to maintain the pH at a desired level.
  • said buffer is a phosphate buffer.
  • said buffer is an acetate buffer.
  • solutions or suspensions of pharmaceutical compositions or formulations include surface adsorption inhibitors.
  • said surface adsorption inhibitors are provided that inhibit the adsorption of components of the pharmaceutical compositions or formulations by surfaces that enclose the compositions or formulations (such as ampoules, syringes, or vials made of glass or plastic).
  • said surface adsorption inhibitors are provided that inhibit the adsorption of one or more modified interferonP-la polypeptides by glass surfaces that enclose the compositions or formulations.
  • the pharmaceutical compositions or formulations are enclosed in ampoules, syringes, or vials made of borosilicate glass, and the pharmaceutical compositions or formulations include a surface adsorption inhibitor (e.g., a surface adsorption inhibitor that inhibits the adsorption of one or more modified interferonP-la polypeptides by the borosilicate glass surface).
  • a surface adsorption inhibitor e.g., a surface adsorption inhibitor that inhibits the adsorption of one or more modified interferonP-la polypeptides by the borosilicate glass surface.
  • said surface adsorption inhibitor is Polysorbate 80.
  • said surface adsorption inhibitor is albumin.
  • solutions or suspensions of pharmaceutical compositions or formulations include degradation inhibitors.
  • degradation inhibitors are provided that inhibit the degradation of a modified interferonP-la polypeptide.
  • degradation inhibitors are provided that inhibit the oxidative degradation of a modified interferonP-la polypeptide.
  • degradation inhibitors are provided that inhibit the oxidative degradation of a modified interferonP-la polypeptide, wherein said degradation inhibitor is benzyl alcohol.
  • compositions or formulations include a sterile powder for the extemporaneous preparation of sterile injectable solutions or dispersion, wherein said sterile powder comprises: a dry powder formulation of one or more modified interferonP- la polypeptides, a bulking agent, and a surface adsorption inhibitor.
  • said bulking agent is glycine.
  • said surface adsorption inhibitor is albumin.
  • said sterile powder further comprises one or more antimicrobial preservatives.
  • said one or more antimicrobial preservatives are selected from the group comprised of: m-cresol, benzyl alcohol, and phenol.
  • said sterile powder further comprises sodium phosphate dibasic and sodium phosphate monobasic.
  • said sterile powder is provided as a tablet-like solid that is whole, in pieces, and/or in a loose powder.
  • said dry powder formulation of one or more modified interferonP- la polypeptides is a lyophilized powder.
  • said one or more modified interferonP- la polypeptides are provided that have a desired specific activity.
  • said sterile powder is stored at a cold temperature prior to administration to a subject.
  • said sterile powder is stored at a temperature in the range of 2°C-8°C prior to administration to a subject.
  • said sterile powder prior to administration to a subject, is reconstituted with a diluent to provide a sterile solution.
  • said reconstitution is accomplished by dissolving the sterile powder in the diluent (e.g., by stirring, swirling, inverting, shaking, vortexing, or other means known and understood in the art) to produce the sterile solution.
  • said diluent comprises one or more components selected from the group comprised of: sterile water, sodium chloride, sodium phosphate dibasic, sodium phosphate monobasic, EDTA, polysorbate 80, and m-cresol.
  • said resuspension is performed in a single-use vial, ampoule, or syringe.
  • said sterile solution provides one or more modified interferonP- la polypeptides at a desired concentration.
  • said desired concentration of modified interferonP- la polypeptide is 1-100 million lU/mL.
  • said desired concentration of a modified interferonP- la polypeptide is 10-50 million HJ/mL.
  • said desired concentration of a modified interferonP- la polypeptide is 1-10 million lU/mL.
  • said desired concentration of a modified interferonP- la polypeptide is decreased for a maintenance dose during maintenance treatment of a condition in a subject.
  • said sterile solution is stored at a cold temperature prior to administration to a subject.
  • said sterile solution is stored at a temperature in the range of 2°C-8°C prior to administration to a subject.
  • compositions or formulations include solutions or suspensions comprising one or more modified interferonP- la polypeptides and one or more components, wherein said components are selected from the group comprised of: sterile water, sodium chloride, sodium phosphate dibasic, sodium phosphate monobasic, EDTA, one or more surface adsorption inhibitors (e.g., polysorbate 80), one or more antimicrobial preservatives (e.g., m- cresol), one or more bulking agents, and one or more degradation inhibitors.
  • sterile water sodium chloride, sodium phosphate dibasic, sodium phosphate monobasic, EDTA, one or more surface adsorption inhibitors (e.g., polysorbate 80), one or more antimicrobial preservatives (e.g., m- cresol), one or more bulking agents, and one or more degradation inhibitors.
  • said solution or suspension comprises: one or more modified interferonP- la polypeptides, sterile water, sodium chloride, sodium phosphate dibasic, sodium phosphate monobasic, EDTA, polysorbate 80, and m-cresol.
  • said one or more modified interferonP- la polypeptides are provided that have a desired specific activity.
  • said solution or suspension provides said one or more modified interferonP- la polypeptides at a desired concentration.
  • said desired concentration of a modified interferonP- la polypeptide is 1-100 million lU/mL.
  • said desired concentration of a modified interferonP- la polypeptide is 10-50 million lU/mL.
  • said desired concentration of a modified interferonP- la polypeptide is 1-10 million lU/mL. In aspects, said desired concentration of a modified interferonP- la polypeptide is decreased for a maintenance dose during maintenance treatment of a condition in a subject.
  • said solution or suspension is stored at a cold temperature prior to administration to a subject. In aspects, said solution or suspension is stored at a temperature in the range of 2°C-8°C prior to administration to a subject.
  • solutions or suspensions of pharmaceutical compositions or formulations comprise: one or more modified interferonP- la polypeptides, a salt, and a buffer.
  • said buffer is provided to maintain the pH at a desired level.
  • said buffer is phosphate buffer and said salt is sodium chloride.
  • compositions or formulations include a sterile powder for the extemporaneous preparation of sterile injectable solutions or dispersion, wherein said sterile powder comprises a dry powder formulation of one or more modified interferonP- la polypeptides.
  • said sterile powder further comprises one or more components selected from the group comprised of: dibasic sodium phosphate anhydrous, monobasic sodium phosphate dihydrate, sucrose, and polysorbate 80.
  • said sterile powder is provided as a tablet-like solid that is whole, in pieces, and/or in a loose powder.
  • said dry powder formulation of one or more modified interferonP- la polypeptides is a lyophilized powder.
  • said one or more modified interferonP- la polypeptides are provided that have a desired specific activity.
  • said sterile powders are stored at a cold temperature prior to administration to a subject.
  • said sterile powders stored at a temperature in the range of 2°C-8°C prior to administration to a subject.
  • said sterile powders are stored at room temperature prior to resuspension.
  • said sterile powders stored at a temperature in the range of 15°C-30°C prior to resuspension.
  • prior to administration to a subject said sterile powder is reconstituted with a diluent to provide a sterile solution.
  • said reconstitution is accomplished by dissolving the sterile powder in the diluent (e.g., by stirring, swirling, inverting, shaking, vortexing, or other means known and understood in the art) to produce the sterile solution.
  • said diluent comprises sterile water.
  • said resuspension is performed in a singleuse vial, ampoule, or syringe.
  • said resuspension is performed in a dual-chamber cartridge, wherein a first chamber contains said sterile powder and a second chamber contains said diluent, and wherein, prior to injection, the components of the two chambers are combined to produce a sterile solution.
  • said dual chamber cartridge is used to inject said sterile solution into a subject via an injection apparatus that is a part of the dual-chamber cartridge.
  • said sterile solution provides said one or more modified interferonP- la polypeptides at a desired concentration.
  • said desired concentration of a modified interferonP- la polypeptide is 50-500 mcg/mL.
  • said desired concentration of a modified interferonP- la polypeptide is 100-300 mcg/mL.
  • said desired concentration of a modified interferonP-la polypeptide is 100-2000 mcg/mL.
  • said desired concentration of a modified interferonP- la polypeptide is 400-1200 mcg/mL.
  • said sterile solution is stored at a cold temperature prior to administration to a subject.
  • said sterile solution is stored at a temperature in the range of 2°C-8°C prior to administration to a subject.
  • compositions or formulations of a modified interferonP- la compound or composition of the present disclosure are co-administered with one or more other pharmaceutical compositions of formulations.
  • said one or more other pharmaceutical compositions or formulations are selected from the group consisting of ribavirin (e.g., REBETOL®), Pegintron ®, and INTRON-A®.
  • oral compositions generally include an inert diluent or an edible carrier and may be enclosed in gelatin capsules or compressed into tablets.
  • the binding agent may be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding compounds, and/or adjuvant materials may be included as part of the composition.
  • the tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating compound such as alginic acid, Primogel or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening compound such as
  • modified interferonP-la compounds or compositions of the present disclosure may be delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • systemic administration of modified interferonP-la compounds or compositions of the present disclosure may also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration may be accomplished through the use of nasal sprays or suppositories.
  • the modified interferonP-la compounds or compositions of the present disclosure may be formulated into ointments, salves, gels, or creams and applied either topically or through transdermal patch technology as generally known in the art.
  • modified interferonP-la compounds or compositions of the present disclosure may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • modified interferonP-la compounds or compositions of the present disclosure are prepared with carriers that protect the modified interferonP-la compositions against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems.
  • a controlled-release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers may be used, such as, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials may also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions may also be used as pharmaceutically-acceptable carriers. These may be prepared according to methods known to those skilled in the art (U.S. Pat. No. 4,522,811, which is herein incorporated by reference in its entirety).
  • the modified interferonP-la compounds or compositions of the present disclosure may be implanted within or linked to a biopolymer solid support that allows for the slow release of the modified interferonP-la compositions to the desired site.
  • dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of binding agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the instant disclosure are dictated by and directly dependent on the unique characteristics of the binding agent and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such modified interferonP-la compounds or compositions of the present disclosure for the treatment of a subject.
  • modified IFNP-la compounds or compositions of the present disclosure find use in protecting/treating against neurodegenerative diseases, including, but not limited to, multiple sclerosis and chronic inflammatory demyelinating polyneuropathy; autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; inflammatory diseases, including, but not limited to, idiopathic pulmonary fibrosis, dermatomyositis, and polymyositis; cancer; and viral diseases, such as hepatitis C and hepatitis B.
  • neurodegenerative diseases including, but not limited to, multiple sclerosis and chronic inflammatory demyelinating polyneuropathy; autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; inflammatory diseases, including, but not limited to, idiopathic pulmonary fibrosis, dermatomyositis, and polymyositis; cancer; and viral diseases, such as
  • the present disclosure provides the use of a modified IFNP-la compounds or compositions of the present disclosure, such as disclosed herein, for manufacturing a medicament for the treatment of neurodegenerative diseases, including, but not limited to, multiple sclerosis and chronic inflammatory demyelinating polyneuropathy; autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; inflammatory diseases, including, but not limited to, idiopathic pulmonary fibrosis, dermatomyositis, and polymyositis; cancer; and viral diseases, such as hepatitis C and hepatitis B.
  • neurodegenerative diseases including, but not limited to, multiple sclerosis and chronic inflammatory demyelinating polyneuropathy; autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; inflammatory diseases, including, but not limited to, idiopathic pulmonary fibrosis, dermatomyositis,
  • the present disclosure is directed to methods of preventing or treating one or more medical conditions in a subject comprising administering one or more modified IFNP-la compounds or compositions of the present disclosure, and preventing or treating the medical condition in a subj ect by said step of administering said one or more modified IFNP- 1 a compounds or compositions of the present disclosure.
  • the medical condition can be, for example against neurodegenerative diseases, including, but not limited to, multiple sclerosis and chronic inflammatory demyelinating polyneuropathy; autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; inflammatory diseases, including, but not limited to, idiopathic pulmonary fibrosis, dermatomyositis, and polymyositis; cancer; and viral diseases, such as hepatitis C and hepatitis B.
  • the modified IFNP-la compounds or compositions of the present disclosure can be used with in conjunction with other proteins or compounds used for treating a subject with the medical condition in order to reduce adverse events or enhance the efficacy of the co-administered compound.
  • the present disclosure is directed to, for example, methods of treating multiple sclerosis, said method comprising administering one or more modified IFNP-la compounds or compositions of the present disclosure, and preventing or treating IFNP-la in a subject by said step of administering said one or more modified IFNP-la compounds or compositions of the present disclosure.
  • the modified IFNP-la compounds or compositions of the present disclosure can be used with in conjunction with other proteins or compounds used for treating a subject with multiple sclerosis in order to reduce adverse events or enhance the efficacy of the co-administered compound.
  • the modified IFNP-la compounds or compositions of the present disclosure demonstrate high relative antiviral activity with reduced immunogenicity in multiple sclerosis treatment.
  • the present disclosure is directed to, for example, methods of treating chronic inflammatory demyelinating polyneuropathy, said method comprising administering one or more modified IFNP-la compounds or compositions of the present disclosure, and preventing or treating chronic inflammatory demyelinating polyneuropathy in a subject by said step of administering said one or more modified IFNP-la compounds or compositions of the present disclosure.
  • the modified IFNP-la compounds or compositions of the present disclosure can be used with in conjunction with other proteins or compounds used for treating a subject with chronic inflammatory demyelinating polyneuropathy in order to reduce adverse events or enhance the efficacy of the co-administered compound.
  • the modified IFNP-la compounds or compositions of the present disclosure demonstrate high relative antiviral activity with reduced immunogenicity in chronic inflammatory demyelinating polyneuropathy treatment.
  • said modified IFNP-la compounds or compositions of the present disclosure are co-administered with one or more other pharmaceutical compositions or formulations.
  • said one or more other pharmaceutical compositions or formulations are selected from the group consisting of: AVONEX®, REBIF®, glatiramer acetate ofatumumab, alemtuzumab, natalizumab, ocrelizumab, cladribine, dimethyl fumarate, diroximel fumarate, monomethyl fumarate, fmgolimod, siponimod, ozanimod, ponesimod, and teriflunomide.
  • kits comprising at least one pharmaceutical formulation or composition for treatment and/or prevention of a disease as described herein (including a melanoma or viral infection and/or related diseases), which can be conveniently used, e.g., in clinical settings to treat subjects exhibiting symptoms or family history of a medical condition described herein.
  • the kit further comprises instructions for use of the at least one modified interferon-a2 composition of the instant disclosure to treat subjects exhibiting symptoms or family history of a medical condition described herein.
  • T cells specifically recognize epitopes presented by antigen presenting cells (APCs) in the context of MHC (Major Histocompatibility Complex) Class II molecules.
  • APCs antigen presenting cells
  • MHC Major Histocompatibility Complex
  • T-helper epitopes can be represented as linear sequences comprising 7 to 30 contiguous amino acids that fit into the MHC Class II binding groove.
  • the EpiMatrixTM system (EpiVax, Buffalo, Rhode Island) is a set of predictive algorithms encoded into computer programs useful for predicting class I and class II HLA ligands and T cell epitopes.
  • the EpiMatrixTM system uses 20 x 9 coefficient matrices in order to model the interaction between specific amino acids (20) and binding positions within the HLA molecule(9).
  • the EpiMatrixTM System first parses the input protein into a set of overlapping 9-mer frames where each frame overlaps the last by eight amino acids.
  • Each frame is then scored for predicted affinity to one or more common alleles of the human HLA molecule; typically DRB 1*0101, DRB 1*0301, DRBl*0401, DRBl*0701, DRBl*0801, DRBl*1101, DRBl*1301, and DRBl*1501 (Mack et al., (2013), Tiss Antig, 81(4): 194-203). Briefly, for any given 9-mer peptide specific amino acid codes (one for each of 20 naturally occurring amino acids) and relative binding positions (1-9) are used to select coefficients from the predictive matrix.
  • T cell epitopes are not randomly distributed throughout protein sequences but instead tend to “cluster.” T cell epitope “clusters” range from 9 to roughly 30 amino acids in length and, considering their affinity to multiple alleles and across multiple frames, contain anywhere from 4 to 40 binding motifs.
  • the result set produced by the EpiMatrixTM algorithm was screened for the presence of T cell epitope clusters and EpiBarsTM by using a proprietary algorithm known as ClustimerTM. Briefly, the EpiMatrixTM scores of each 9- mer peptide analyzed are aggregated and checked against a statistically derived threshold value. High scoring 9 mers are then extended one amino acid at a time.
  • the scores of the extended sequences are then re-aggregated and compared to a revised threshold value. The process is repeated until the proposed extension no longer improves the overall score of the cluster.
  • Regions of high immunogenic potential defined as having a score above 10 (including multiple ‘hits’ against many different HLA DR alleles), were identified as T cell epitope clusters. They contain significant numbers of putative T cell epitopes and EpiBarsTM indicating a high potential for MHC binding and T cell reactivity.
  • OptiMatrix tool part of the EpiVax ISPRI toolkit for deimmunization. OptiMatrix begins with looking at “critical” residues, which contribute most to MHC binding affinity across multiple 9- mer frames and multiple HLA alleles. The program then iteratively substitutes all 19 alternative amino acids in any given position of a protein sequence (with operator-defined input that may limit the list to naturally conserved variants) and then re-analyzes the predicted immunogenicity of the sequence, following that change. To avoid a negative effect on protein structure and consequently in biological activity a comprehensive search in literature for critical residues was also conducted, which identified amino acids that were not candidates for modification.
  • Peptide binding to HLA molecules is the first step required for a T cell response.
  • the strength of peptide binding to MHC molecules is particularly important in determining protein immunogenicity.
  • wild-type IFNP-la SEQ ID NO: 1
  • the complete amino acid sequence was screened using EpiMatrix. As shown in FIG. 1, wild-type human IFNP-la demonstrates a very high potential for immunogenicity, having a Z-score of 101.81.
  • IFNP-la SEQ ID NO: 1
  • OptiMatrix iteratively substituted all 19 alternative amino acids at the given positions in the amino acid sequence protein sequence and then reanalyzed the predicted immunogenicity of the sequence.
  • the following mutations were selected for the IFNP-la variants: N25P; L28P; Y60H; N65H; S74L; F111L; M117T; N158E.
  • IFNP(VARl) SEQ ID NO:2
  • IFNP-la(VAR2) SEQ ID NO: 3
  • Immunogenicity scores for each of the variants was calculated using EpiMatrix, as described above. As shown in FIG. 1, the EpiMatrix immunogenicity global score for each variant is markedly reduced in comparison with the original molecule. Table 1 summarizes the IFNP variants.
  • each variant was assessed by calculating AAG using dg_monomer4 software within the Rosetta5 Bioinformatics package.
  • IFNP-la(VAR2) SEQ ID NO:3
  • IFNP-la(VARl) SEQ ID NO:2
  • AAG 18.569
  • FIG. 2 Molecular structures of IFNP-la(WT) and the de-immunized variants are depicted in FIG. 2, highlighting residues considered for mutation represented as Van der Waals spheres on their peptide ribbon structures. Residues selected for mutation are labeled using one-letter codes to facilitate structural comparison.
  • Example 2 Production, purification, and characterization of IFNP-la de-immunized variants
  • Wild-type IFNP-la (SEQ ID NO:1), IFNP-la(VARl) (SEQ ID NO:2), and IFNP-la (VAR2) (SEQ ID NO:3) were expressed in CHO-K1 cells using nucleotide sequences containing the coding sequence and signal peptide.
  • nucleotide sequences were optimized for codon usage specific for the different de-immunized IFNP-la variants IFNP-la(VARl) (SEQ ID NO: 15) and IFNP-la (VAR2) (SEQ ID NO: 16).
  • the nucleotide sequences were synthesized artificially.
  • CHO-K1 Chinese hamster ovary (CHO-K1) cells were grown in the basal culture medium as previously described (Kratje R B, Wagner R, (1992), Biotechnol. Bioeng. 39: 233-242) supplemented with 5% (v/v) fetal calf serum (FCS) (PAA, Argentina) and supplemented with 5% (v/v) fetal calf serum (FCS) (PAA, Argentina).
  • FCS fetal calf serum
  • FCS fetal calf serum
  • MDBK Madine Darby bovine kidney
  • Bioassays were performed using MEM supplemented with 2% (v/v) FCS (assay medium).
  • Vector Construction Production and purification ofIFNfl-la variants.
  • a third-generation lentiviral strategy was carried out which included the packaging construct (pMDLg/pRRE), VSV-G expressing construct (pMD.G), and the Rev. expressing construct (pRSV-Rev) (Addgene, USA; Plasmid numbers #12251, #12259, #12253, respectively)
  • the IFNP- la de-immunized variants were synthetically generated by Gene UniversalTM (USA) and cloned into a self-inactivating (SIN) promoter containing the expression vector. The sequences of all of the newly designed de-immunized constructs were confirmed by DNA sequencing.
  • LVPs Lentiviral particles containing supernatants were collected 72 h after transfection. Simultaneously, EGFP-containing LVPs were also assembled. IFNP- la-containing LVPs titers were estimated based on the titer obtained from EGFP transduced cells by flow cytometry. The titer of LVPs was used to calculate the necessary supernatant dilutions to transduce cells at a similar multiplicity of infection (MOI).
  • MOI multiplicity of infection
  • Transductions were carried out by incubating 6.0 x io 4 cells per well seeded onto 6-well plates (Greiner) with LVPs at a final MOI of sixty. Twenty-four hours post-transduction, media were replaced with fresh media. To eliminate the remaining wild-type cells, 96 h post-transduction a selective pressure process was started by replacing supernatants with fresh growth mediums containing 10 pg/ml puromycin (Invivogen, USA). The selective medium was changed every 3- 4 days until controlled cell death occurred.
  • Transduced cells were grown for IFNP-la production until confluence was obtained in 500 cm 2 triple flasks using a growth medium. The medium was then changed to basal medium supplemented with 0.5% (v/v) FCS (production medium). Every 48 or 72 h, the conditioned medium was harvested and replaced with a fresh production medium. Harvests were clarified by centrifugation and then stored at -20 °C until protein purification. This procedure was repeated between 8 and 10 times. Every harvest was assayed for IFNP-la concentration by a noncommercial (in-house) ELISA sandwich assay.
  • IFNP-la deimmunized variants were synthesized and cloned into a third-generation lentiviral vector as described herein, followed by production in CHO cells. Cell culture supernatants were initially screened for antiviral activity, demonstrating robust production levels of each IFNP-la protein (data not shown).
  • Purification of the secreted IFNP-la variants from individual harvests involved a three- step purification process: Blue Sepharose chromatography, followed by RP-HPLC-C4 chromatography, and protein concentration with simultaneous buffer exchange via diafiltration.
  • IFNP-la variants were purified by Blue Sepharose chromatography followed by RP- HPLC-C4 chromatography. Culture supernatants were passed through a column of Blue Sepharose 6 Fast Flow (GE Healthcare, Buckinghamshire, UK), previously equilibrated with Buffer A (20 mM sodium phosphate, 0.15 M NaCl, pH 7.2). The flow through was collected and the column was washed with Buffer A (35 mL) and 35 mL of Buffer B (20 mM sodium phosphate, 2 M NaCl, pH 7.2). IFN was eluted with Buffer B containing 50% ethylene glycol.
  • Buffer A 20 mM sodium phosphate, 0.15 M NaCl, pH 7.2
  • IFNP-la protein variants were eluted at 1 mL/min using a linear gradient of Buffer A (0% acetonitrile (ACN), 0.1% TFA, pH 2.0) into Buffer B (90% ACN, 0.1% TFA, pH 2.0). Purified proteins were then concentrated and diafiltrated against NaAc 100 mM, and Mannitol 300 mM (pH 3.8).
  • the wild type IFNP-la (SEQ ID NO: 1) and the mutated forms IFNP-la (VAR 1)(SEQ ID NO:2) and IFNP-la(VAR2) (SEQ ID NO:3) were obtained in purified form. SDS-PAGE and western blotting
  • the protein purity was analyzed by SDS-PAGE under reducing conditions followed by Coomassie blue staining.
  • concentration of purified IFNP-la and its de-immunized variants was analyzed by spectrophotometry considering a molar absorptivity index of 1.45.
  • Electrophoretic migration comparison by SDS-PAGE revealed similar banding patterns for IFNP-la (WT) and IFNP-la (VARI), characterized by a dominant band at approximately 22 kDa, consistent with the original molecular size.
  • IFNP-la VAR2
  • IFNP-la VAR2
  • Identity verification of these bands was confirmed via western blot analysis, which demonstrated the presence of a single band with the molecular size expected for all rhIFNP variants (FIG. 3B).
  • the UV-far and UV-near circular dichroism (CD) spectra were analyzed to assess changes in secondary and tertiary structure composition, respectively.
  • CD spectra were recorded in the ranges of 250-320 nm (near-UV) and 190-240 nm (far- UV) using a Jasco J-1500 spectropolarimeter (Jasco Inc., Easton, USA) at room temperature, with a scan speed of 20 nm/min and a response time of Is.
  • IFNP, IFNP(VARl), and IFNP(VAR2) protein samples were prepared at a concentration of 0.33 mg/ml in a cuvette with a 0.1 cm path length. Each spectrum represents the average of three scans, corrected by subtracting the buffer signal.
  • CD data were expressed as molar ellipticity, [0], in degrees cm 2 /dmol, calculated based on a mean residue weight of 116.76 Da per amino acid residue. Spectra were further corrected by subtracting the excipient signal. CD spectra deconvolution was performed to dissect the contributions from different secondary structures within the molecule. Using reference spectra from proteins with known structures, various empirical algorithms were applied, based on the linear independence and additivity of different components to produce the net spectrum. The CD spectra deconvolution in this study was conducted using the SELCOM software, accessed via the DichroWeb online server. Predictions utilized reference database 7, comprising 48 proteins.
  • the far-UV CD spectra of all three variants display characteristics consistent with alpha-helix structures, evidenced by negative bands at 222 nm and 208 nm and a positive band at 193 nm.
  • the spectra of IFNP-la (VARI) and IFNP-la (VAR2) differ from that of IFNP-la (WT).
  • Example 4 Determination of the antiviral biological activity of the different variants of IFNp ⁇ la de-immunized.
  • IFNP-la vesicular stomatitis virus
  • WISH cells ATCC, USA.
  • the experimental procedure was as follows:
  • the cells were stained using a crystal violet solution (0.75% w/v in 40% v/v methanol) for 15 minutes. Excess stain was washed off with water, and the stained cells were subsequently treated with acetic acid (20% v/v). The intensity of the staining was measured at 540 nm. Each experiment was conducted in quintuplicate.
  • the antiviral biological activity of the IFNP- la variants obtained in Example 2 was quantified by evaluating their ability to inhibit viral propagation or replication. This was measured by determining the ability of the interferon variants to protect susceptible cells from the cytopathic effects of a lytic virus over a range of cytokine concentrations.
  • the biological antiviral activities of the parent IFNP-la and the mutated variants IFNP- la(VARl) and IFNP-la(VAR2) were specifically determined by their ability to inhibit the cytopathic effect caused by VSV on WISH cells.
  • 2.5 x io 5 WISH cells were grown overnight at 37 °C, and the culture supernatants were removed.
  • Serial 1 :2 dilutions of the WHO International Standard for rhIFNP (NIBSC 00/572) ranging from 20 U/ml to 0.16 U/ml, along with serial dilutions of the IFNP-la variants, were added to the assay medium.
  • the supernatants were removed, and the cells were infected with VSV at a ratio of 1.6 PFU/cell. Once the cytopathic effect was evident in the control cells (without IFN), the cells were stained with a crystal violet solution (0.75% w/v in 40% v/v methanol) for 15 minutes. After washing with water, the stained cells were treated with acetic acid (20% v/v), and the signal intensity was measured at 540 nm. Determinations were performed in quintuplicate.
  • the functional characterization of the purified IFNP- la variants involved quantifying their specific antiviral biological activity. Absorbance data at 540 nm were plotted against the IFNP-la activity values (standard) and sample dilutions on a logarithmic scale. The biological activity (AB) values for each polypeptide were calculated by comparison with the standard using the parallel lines test (FIG. 5). The specific biological activity (ABE) values of each protein were determined by calculating the ratio between the AB and the protein concentration in the samples.
  • the percentage biological activity value was determined by calculating the ratio between the mean ABEs of each de-immunized variant and the mean ABEs of IFNP-la WT (33.93 ⁇ 4.00 Ul/ng).
  • the relative antiviral activity results (%) for the IFNP- la variants are shown in Table 2.
  • the specific antiviral activity of IFNP-la VARI shows a statistically significant 40% higher than the natural variant, while IFNP- la VAR2 presents a similar activity to natural IFNP- la, as shown in FIG. 6.
  • Example 5 IFNp ⁇ la(VARl) and IFNp ⁇ la(VAR2) exhibit a markedly reduced in vivo immunogenicity
  • HLA-DRB 1*03:01 mice humanized for both HLA-A2 and HLA-DR3, and deleted for H-2 class I and II molecules 2m-/- H-2Db-/- IA0-/- IAa-/- IE0-/-), were provided by the Pasteur Institute and developed by Francois Lemonnier.
  • Transgenic mice aged 6-10 weeks were housed in pathogen-free conditions at the animal facilities of the Faculty of Veterinary Sciences, Universidad Nacional del Litoral (UNL). All animal handling procedures and protocols were approved by the UNL Research Ethics Committee, code 2021-21CE-C.
  • mice were immunized three times via intraperitoneal (i.p.) injection with PBS or 25 pg of IFN0-la (wild-type), IFN0-la(VARl), or IFN0-la(VAR2) emulsified in complete Freund's adjuvant (CFA) at a final volume of 200 pl.
  • CFA complete Freund's adjuvant
  • Total antibody titers (BAbs) against IFN0-la, VARI, and VAR2 were determined by indirect ELISA. 96-well plates were coated with 1 pg/ml IFN0 protein, blocked with 1% BSA in PBS, and incubated with serial dilutions of plasma samples. After incubation with peroxidase- conjugated anti-mouse Igs, titers were defined as the dilution exceeding the limit (OD for the negative control) plus three standard deviations.
  • Neutralizing antibody titers were determined by antiviral bioassays. These results are depicted in Table 3. Serial dilutions of mouse plasma were pre-incubated with a constant concentration of IFN0-la variants, then added to WISH cell cultures. The reduction in antiviral activity was measured by the degree of cell lysis. Titers were expressed as the serum dilution that reduced the initial IFN antiviral activity by 50% (IC50).
  • mice treated with IFNP-la(WT) developed higher titers of anti-IFNP- la BAbs.
  • a significant reduction in BAbs and NAbs titers was observed in animals treated with the de-immunized variants, consistent with the reduced T cell responses.
  • Statistical analysis using the Kruskal-Wallis test revealed significant differences between the medians of the total anti- IFNP-la antibody titers, with a p-value ⁇ 0.05, confirming these differences at a 95% confidence level.
  • Plasma from mice inoculated with IFNP-la(WT) showed high neutralizing capacity for IFNP-la antiviral activity in vitro.
  • plasma from mice treated with the de-immunized variants exhibited negligible NAb activity.
  • Spleen cells were seeded in 96-well plates and evaluated under four conditions: negative control (culture media), positive control (Phaseolus vulgaris lectins), excipient control, and IFNP- 1 a variants. After 72 hours, culture supernatants were harvested, and mouse IFN-y and IL-4 levels were assayed by sandwich ELISA.
  • the Stimulation Index (SI) was defined as the ratio of cytokine concentration from protein-challenged samples to excipient-treated samples.
  • splenocyte samples treated with IFNP-la(WT) demonstrated robust IFN-y stimulation index (SI).
  • SI IFN-y stimulation index
  • T cell immune responses were barely detectable in mice treated with the de-immunized IFNP-la variants.
  • mIL-4 was not detected in the splenocyte culture supernatants (data not shown), suggesting that IFNP-la primarily induces T cell polarization towards the Thl profile.
  • BAbs binding antibodies
  • Example 6 Ex vivo immunogenicity assays of the different variants of IFNP-la de-immunized in samples of human peripheral blood mononuclear cells (PMBC).
  • PMBC peripheral blood mononuclear cells
  • IFNP-la variants developed in Example 2. This study employed an ex vivo assay with samples of human peripheral blood mononuclear cells (PBMCs) from patients with multiple sclerosis.
  • PBMCs peripheral blood mononuclear cells
  • IFNP-la WT specifically activated naive and memory T cells in PBMC samples from both donors. The values are expressed as a percentage of activated T lymphocytes relative to the negative control (excipients).
  • Activated memory T lymphocytes were isolated and restimulated with IFNP-la WT, IFNP-la VAR 1, and IFNP-la VAR 2.
  • FIGS. 10-11 indicate that IFNP-la WT induced a higher level of lymphocyte activation compared to the de-immunized variants. This was observed for T lymphocyte activation markers CD69 and CD154.
  • IFNP-la(VAR2) exhibited lower lymphocyte activation than IFNP-la VAR 1, correlating with the in silico analysis results.
  • FIG. 11 summarizes lymphocyte activation based on the CD69 marker. Except for donor 10, PBMC samples from multiple sclerosis patients showed greater lymphocyte activation with IFNP-la WT compared to the de-immunized variants. These results demonstrate that mutations in the IFNP-la WT sequence reduce immunogenicity.
  • FIG. 12 demonstrates that restimulation with IFNP-la WT resulted in a higher percentage of activation compared to the de-immunized proteins.
  • the restimulation results for IFNP-la VAR 1 and IFNP-la VAR 2 showed a similar pattern to FIG. 10, demonstrating the effectiveness of the de-immunization strategy.
  • the present disclosure relates to a modified interferon-pia polypeptide having interferon-pia activity, the polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and comprising one or more amino acid substitutions, the one or more amino acid substitutions at one or more of position 25, 28, 60, 65, 74, 111, 117, or 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the present disclosure relates to a modified interferon-pia polypeptide having interferon-pia activity, the polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and comprising at least four amino acid substitutions, the one or more amino acid substitutions at one or more of position 25, 28, 60, 65, 74, 111, 117, or 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the present disclosure relates to a modified interferon-pia polypeptide having interferon-pia activity, the polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and comprising one or more amino acid substitutions, the one or more amino acid substitutions at one or more of position 25, 28, 60, 65, 74, 111, 117, or 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the present disclosure relates to a modified interferon-pia polypeptide having interferon-pia activity, the polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and comprising at least four amino acid substitutions, the one or more amino acid substitutions at one or more of position 25, 28, 60, 65, 74, 111, 117, or 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the present disclosure relates to a modified interferon-pia polypeptide having interferon-pia activity, the polypeptide comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and comprising at least eight amino acid substitutions, the one or more amino acid substitutions at one or more of position 25, 28, 60, 65, 74, 111, 117, and 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the present disclosure relates to a modified interferon-pia polypeptide having interferon-pia activity, the polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and comprising at least eight amino acid substitutions, the one or more amino acid substitutions at one or more of position 25, 28, 60, 65, 74, 111, 117, and 158, optionally wherein the substitution comprises changing the amino acid at the position to proline, histidine, leucine, threonine, or glutamic acid.
  • the present disclosure relates to a modified interferon-pia polypeptide, wherein the amino acid substitutions are N25P, L28P, Y60H, N65H, S74L, F111L, M117T, N158E, or a combination thereof.
  • the present disclosure relates to a modified interferon-pia polypeptide, wherein the amino acid substitutions comprise N25P, Y60H, Ml 17T, and N158E.
  • the present disclosure relates to a modified interferon-pia polypeptide, further comprising an amino acid substitution of L28P, N65H, S74L, or Fl 1 IL.
  • the present disclosure relates to a modified interferon-pia polypeptide, wherein the amino acid substitutions comprise L28P, N65H, S74L, and Fl 1 IL.
  • the present disclosure relates to a modified interferon-pia polypeptide, wherein the amino acid sequence is SEQ ID NO: 2 or SEQ ID NO: 3.
  • the present disclosure relates to a modified interferon-pia polypeptide, wherein the modified interferon-pia polypeptide has a reduced immunogenicity as compared to a wild type interferon- pia polypeptide of SEQ ID NO: 1.
  • the present disclosure relates to a modified interferon-pia polypeptide, wherein the modified interferon-pia polypeptide has a relative antiviral activity of about 10% to about 200% compared to a wild type interferon-pia polypeptide of SEQ ID NO: 1.
  • the present disclosure relates to a modified interferon-pia polypeptide, wherein the modified interferon-pia polypeptide has a relative antiviral activity of about 100% to about 140% as compared to a wild type interferon-pia polypeptide of SEQ ID NO: 1.
  • the present disclosure relates to a modified interferon-pia polypeptide, wherein the amino acid sequence is SEQ ID NO: 2.
  • the present disclosure relates to a modified interferon-pia polypeptide, wherein the amino acid sequence is SEQ ID NO: 3.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising modified interferon P- la polypeptide and a pharmaceutically acceptable excipient.
  • the present disclosure relates to a method for treating multiple sclerosis in a subj ect in need thereof, the method comprising administering the pharmaceutical composition to the subject.
  • the present disclosure relates to a nucleotide sequence encoding the modified interferon P-1 a polypeptide with interferon beta- la activity.
  • the present disclosure relates to a nucleotide sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.
  • the present disclosure relates to a plasmid comprising a nucleotide sequence.
  • the nucleotide sequence is optionally selected from the set comprised of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
  • the present disclosure relates to an expression vector comprising a nucleotide sequence encoding the modified interferon beta-la polypeptide.
  • the nucleotide sequence is optionally selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
  • the present disclosure relates to a cell line for the expression of the modified interferon P- la polypeptide with interferon beta- la activity, comprising a nucleotide sequence encoding the modified interferon beta- 1 a polypeptide with interferon beta- 1 a activity.
  • the nucleotide sequence is optionally selected from the set comprised of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
  • the present disclosure relates to a cell-line selected from a list comprised of CHO-K1, HEK293, NSO, BHK, Sp2/0, CAP and CAP/T.
  • the present disclosure relates to a CHO-K1 cell line.
  • the present disclosure relates to a method of preparing the modified interferon P-la polypeptide with interferon beta- la activity, the method comprising cloning a nucleotide sequence selected from the group comprised of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 into a lentilviral vector; transducing a Chinese hamster ovary cell line (CHO-K1) with the vector; expressing the modified interferon P-la polypeptide, the modified interferon P-la polypeptide having an amino acid sequence selected from SEQ ID NO: 2 or SEQ ID NO: 3; collecting supernatant; purifying the modified interferon P-la polypeptide from the supernatant using dye pseudo-affinity chromatography and high-performance liquid chromatography (HPLC); and concentrating the modified interferon P-la polypeptide with interferon beta- la activity selected from the group comprised of
  • Patents, publications, and applications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents, publications, and applications are incorporated herein by reference to the same extent as if each individual patent, publication, or application was specifically and individually incorporated herein by reference.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des compositions comprenant un polypeptide d'interféron bêta-1 modifié ayant une activité d'interféron bêta-1a et une immunogénicité réduite. Selon certains aspects, les polypeptides d'interféron bêta-1a modifiés ont une activité biologique antivirale similaire, identique ou supérieure à celle de l'interféron bêta-1a naturel. L'invention concerne également des séquences d'acides nucléiques codant pour le polypeptide d'interféron bêta-1 modifié avec une activité d'interféron bêta-1a. La présente divulgation concerne des compositions comprenant une lignée cellulaire d'expression de protéine recombinante comprenant la séquence d'acide nucléique codant pour le plypeptide d'interféron bêta-1a modifié avec une activité d'interféron bêta et une immunogénicité réduite; la lignée cellulaire comprenant un plasmide ou un vecteur contenant la séquence d'acide nucléique. L'invention concerne également des formulations pharmaceutiques comprenant de l'interféron-bêta modifié ayant une immunogénicité réduite pour le traitement de la sclérose en plaques.
PCT/US2024/039763 2023-07-26 2024-07-26 Interféron-bêta-1 modifié ayant une immunogénicité réduite Pending WO2025024775A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2024/039763 WO2025024775A1 (fr) 2023-07-26 2024-07-26 Interféron-bêta-1 modifié ayant une immunogénicité réduite

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AR2358219 2023-07-26
PCT/US2024/039763 WO2025024775A1 (fr) 2023-07-26 2024-07-26 Interféron-bêta-1 modifié ayant une immunogénicité réduite

Publications (1)

Publication Number Publication Date
WO2025024775A1 true WO2025024775A1 (fr) 2025-01-30

Family

ID=94371410

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/039763 Pending WO2025024775A1 (fr) 2023-07-26 2024-07-26 Interféron-bêta-1 modifié ayant une immunogénicité réduite

Country Status (1)

Country Link
WO (1) WO2025024775A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080003202A1 (en) * 2006-03-28 2008-01-03 Thierry Guyon Modified interferon-beta (IFN-beta) polypeptides
US20090087411A1 (en) * 2006-02-03 2009-04-02 Fuad Fares Long-acting interferons and derivatives thereof and methods thereof
US20090311216A1 (en) * 2006-08-08 2009-12-17 Deborah Johnson-Jackson Recombinant interferon-beta with enhanced biological activity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090087411A1 (en) * 2006-02-03 2009-04-02 Fuad Fares Long-acting interferons and derivatives thereof and methods thereof
US20080003202A1 (en) * 2006-03-28 2008-01-03 Thierry Guyon Modified interferon-beta (IFN-beta) polypeptides
US20090311216A1 (en) * 2006-08-08 2009-12-17 Deborah Johnson-Jackson Recombinant interferon-beta with enhanced biological activity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RICOTTI SONIA; GARAY ALBERTO SERGIO; ETCHEVERRIGARAY MARINA; AMADEO GABRIEL IGNACIO; DE GROOT ANNE S.; MARTIN WILLIAM; MUFARREGE E: "Development of IFNβ-1a versions with reduced immunogenicity and full in vitro biological activity for the treatment of multiple sclerosis", CLINICAL IMMUNOLOGY, vol. 257, 4 November 2023 (2023-11-04), AMSTERDAM, NL , XP087438909, ISSN: 1521-6616, DOI: 10.1016/j.clim.2023.109831 *

Similar Documents

Publication Publication Date Title
JP4937132B2 (ja) 免疫原性の低下したｉｌ−７変種
US20250282843A1 (en) Compositions comprising an interleukin construct
CA2986388C (fr) Proteine d'interleukine-7 modifiee et ses utilisations
CN111615396A (zh) 白介素-2的部分激动剂
EP1292621B1 (fr) Compositions pour le traitement des maladies auto-immunes
EP2572734B1 (fr) Protéines de fusion à base de cytokine pour le traitement des troubles immunitaires
JP7786010B2 (ja) 免疫原性が低下した修飾インターフェロン－アルファ－２
PL213710B1 (pl) Kompozycja farmaceutyczna zawierajaca IL-7 i sposób jej wytwarzania
WO2025024775A1 (fr) Interféron-bêta-1 modifié ayant une immunogénicité réduite
US20240409596A1 (en) Novel immune regulator
KR20240128579A (ko) Cd38 또는 이의 단편을 포함하는 자가면역질환 치료용 조성물
US10525101B2 (en) Methods of treating inflammatory or autoimmune disorders with compounds that bind macrophage migration inhibitory factor
EP4667486A1 (fr) Composition comprenant cd38 ou un fragment de celui-ci pour le traitement de maladies auto-immunes
Ricotti et al. Development of IFNβ-1a versions with reduced immunogenicity and full in vitro biological activity for the treatment of multiple sclerosis
US20260092095A1 (en) Interleukin-2 for use in treating autism spectrum disorder
CN113423731B (zh) 抗hiv抗体及其制造方法
EP4230220A1 (fr) Utilisation d'un triple activateur ayant une activité sur tous les récepteurs du glucagon, du glp-1 et du gip pour le traitement des séquelles d'une maladie infectieuse respiratoire
JP2023538002A5 (fr)
JP2023538002A (ja) 制御性ｔ細胞エピトープ

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24846564

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE