Patent Grant
12180264
Generally, the invention relates to the field of biological pharmaceuticals as well as their use in conditions associated with inflammatory disorders (e.g. rheumatoid arthritis, Crohn's disease, etc.), diabetes, cardiovascular disease and gout. More specifically, the invention relates to a heterodimeric IL-1R1/IL-1RAcP-derived composition that is capable of inhibiting IL-1(3 cytokine.
The interleukin-1 (IL-1) family of cytokines comprises 11 proteins (IL-1F1 to IL-1F11) encoded by 11 distinct genes in humans and mice. IL-1-type cytokines are major mediators of innate immune reactions, and blockade of the founding members IL-1 or IL-1β by the interleukin-1 receptor antagonist (IL-1RA) has demonstrated a central role of IL-1 in a number of human autoinflammatory diseases. IL-1 or IL-1β rapidly increase messenger RNA expression of hundreds of genes in multiple different cell types. The potent proinflammatory activities of IL-1 and IL-1β are restricted at three major levels: (i) synthesis and release, (ii) membrane receptors, and (iii) intracellular signal transduction. This pathway summarizes extracellular and intracellular signaling of IL-1 or IL-1β, including positive- and negative-feedback mechanisms that amplify or terminate the IL-1 response. In response to ligand binding of the receptor, a complex sequence of combinatorial phosphorylation and ubiquitination events results in activation of nuclear factor kappa-B signaling and the JNK and p38 mitogen-activated protein kinase pathways, which, cooperatively, induce the expression of canonical IL-1 target genes (such as IL-6, IL-8, MCP-1, COX-2, IB, IL-1, IL-1β, MKP-1) by transcriptional and posttranscriptional mechanisms. Of note, most intracellular components that participate in the cellular response to IL-1 also mediate responses to other cytokines (IL-18 and IL-33), Toll-like-receptors (TLRs), and many forms of cytotoxic stresses (see Weber A, et al., Sci Signal., 2010 Jan. 19; 3(105), the entire teachings of which are incorporated by reference herein).
IL-1 and IL-1β independently bind the type I IL-1 receptor (IL-1R1), which is ubiquitously expressed. A third specific ligand, the IL-1 receptor antagonist (IL-1RA), binds the IL-1RI with similar specificity and affinity but does not activate the receptor and trigger downstream signaling. The IL-1 receptor accessory protein (IL-1RAcP) serves as a co-receptor that is required for signal transduction of IL-1/IL-1RI complexes, and this co-receptor is also necessary for activation of IL-1R1 by other IL-1 family members, in particular IL-18 and IL-33. The type II IL-1 receptor (IL-1R2) binds IL-1 and IL-1β but lacks a signaling-competent cytosolic part and thus serves as a decoy receptor. The IL-1RA, the plasma membrane-anchored IL-1R2, and the naturally occurring “shed” domains of each of the extracellular IL-1 receptor chains (termed sIL-1RI, sIL-1RII, and sIL-1RAcP, where “s” stands for soluble) provide inducible negative regulators of IL-1 signaling in the extracellular space whose abundance, which is regulated by a combination of increased transcription and controlled release, can limit or terminate IL-1 effects.
The initial step in IL-1 signal transduction is a ligand-induced conformational change in the first extracellular domain of the IL-1RI that facilitates recruitment of IL-1RacP. Through conserved cytosolic regions called Toll- and IL-1R-like (TIR) domains, the trimeric complex rapidly assembles two intracellular signaling proteins, myeloid differentiation primary response gene 88 (MYD88) and interleukin-1 receptor-activated protein kinase (IRAK) 4. Mice lacking MYD88 or IRAK4 show severe defects in IL-1 signaling. Similarly, humans with mutations in the IRAK4 gene have defects in IL-1RI and Toll-like receptor (TLR) signaling. IL-1, IL-1RI, IL-RAcP, MYD88, and IRAK4 form a stable IL-1-induced first signaling module. This is paralleled by the (auto)phosphorylation of IRAK4, which subsequently phosphorylates IRAK1 and IRAK2, and then this is followed by the recruitment and oligomerization of tumor necrosis factor-associated factor (TRAF) 6. IRAK1 and 2 function as both adaptors and protein kinases to transmit downstream signals. Complexes of IRAK1, IRAK2, and TRAF6 dissociate from the initial receptor complex, and cells lacking these proteins have impaired activation of the transcription factors nuclear factor kappa-B (NF-kappa-B) and activator protein 1 (AP-1).
Overproduction of IL-1 is the cause of many inflammatory disorders. For example, IL-1 has been linked to the pathology of diabetes, cardiovascular disease, gout, certain types of arthritis (e.g. rheumatoid arthritis (RA)), as well as a number of less prevalent autoimmune diseases, such as familial Mediterranean fever (FMF), Behcet disease, etc. (Ozen S, Bilginer Y. “A clinical guide to autoinflammatory diseases: familial Mediterranean fever and next-of-kin”, Nat. Rev. Rheumatol. 2014 March; 10(3):135-47).
Rilonacept is an IL-1 antagonist which includes an IL-1-specific fusion protein which comprises an IL-1 binding portion of the extracellular domain of human IL1-RAcP, an IL-1 binding portion of the extracellular domain of human IL-1RI, and a multimerizing component. This IL-1-specific fusion protein is described in U.S. Pat. No. 6,472,179, U.S. patent publication No. 2003/0143697, published 31 Jul. 2003, U.S. Pat. No. 7,361,350, and U.S. patent publication No. 2005/0197293, published 8 Sep. 2005 (all of which are incorporated by reference herein in their entirety). Rilonacept under the trade name ARCALYST was approved by U.S. Food and Drug Administration (FDA) for the treatment of Cryopyrin-Associated Periodic Syndromes (CAPS), including Familial Cold Auto-inflammatory Syndrome (FCAS) and Muckle-Wells Syndrome (MWS) in adults and children 12 and older. Further clinical trials of rilonacept are currently under way, i.e. for gout.
This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
In certain aspects, the present invention provides for a heterodimeric protein composition capable of binding human IL-1β (GenBank: AAH08678.1). The protein composition comprises a first polypeptide which includes a first amino acid sequence which contains amino acids 18 through 333 of human IL1-R1 (GenBank: AAM88423.1), and a second amino acid sequence which contains a first mutant of a Fc portion of human immunoglobulin gamma-1 Fc (GenBank: J00228.1). The protein composition also comprises a second polypeptide which includes another first amino acid sequence containing amino acids 21 through 358 of human IL1-RAcP (GenBank: BAA25421.1), and another second amino acid sequence which contains a second mutant of the Fc portion of human immunoglobulin gamma-1 Fc. In the protein composition, the first and second mutants are selected as to favor heterodimeric assembly between the first and second mutants over any homodimeric assembly. The protein composition may be capable of exhibiting human IL-1β/IL-1F2 binding activity with a Kd values of no more than about 10−11M. The first polypeptide of the protein composition may contain amino acid sequence of SEQ ID NO. 1, while the second polypeptide may contain amino acid sequence of SEQ ID NO. 2.
In certain aspects, the present invention provides for a heterodimeric protein composition, containing a first polypeptide including amino acid sequence of SEQ ID NO. 8 and a second polypeptide including amino acid sequence of SEQ ID NO. 9.
In certain aspects, the present invention provides for a therapeutic composition which contains a heterodimeric protein composition, including a first polypeptide containing amino acid sequence of SEQ ID NO. 8 and a second polypeptide containing amino acid sequence of SEQ ID NO. 9. The therapeutic composition may also contain about 6% (m/v) sucrose, about 3% (m/v) polyethylene glycol having an average molecular weight of 3350 Da, about 50 mM sodium chloride, and about 20 mM L-Histidine pH from about 4.5 to about 7.0. The pH value may be about 6.5.
In certain aspects, the present invention provides for a therapeutic composition which contains a heterodimeric protein composition, including a first polypeptide containing amino acid sequence of SEQ ID NO. 8 and a second polypeptide containing amino acid sequence of SEQ ID NO. 9. The therapeutic composition may also contain about 1.2% (m/v) sucrose, about 0.09% (m/v) polysorbate 80, about 3% (m/v) D-mannitol, about 38 mM glycine, and about 15 mM TRIS-HCl, pH may be from about 6.5 to about 8.5. The pH value may be about 7.5.
In certain aspects, the present invention provides for a therapeutic composition. The therapeutic composition comprises a heterodimeric protein composition capable of binding human IL-1β. The protein composition comprises a first polypeptide which includes a first amino acid sequence which contains amino acids 18 through 333 of human IL1-R1, and a second amino acid sequence which contains a first mutant of the Fc portion of human immunoglobulin gamma-1 Fc. The protein composition also comprises a second polypeptide which includes another first amino acid sequence containing amino acids 21 through 358 of human IL1-RAcP, and another second amino acid sequence which contains a second mutant of the Fc portion of human immunoglobulin gamma-1 Fc. In the protein composition, the first and second mutants are selected as to favor heterodimeric assembly between the first and second mutants over any homodimeric assembly.
The protein composition may be capable of exhibiting human IL-1β/IL-1F2 binding activity with a Kd values of no more than about 10−11M. The therapeutic composition may exhibit a half-life of the heterodimeric protein composition in systemic circulation in mice after a subcutaneous administration at a dose of 5 mg/kg of at least about 97 hours, as assayed by human Fc ELISA.
The therapeutic composition may exhibit a half-life of the heterodimeric protein composition in systemic circulation in Cynomolgus monkeys after a subcutaneous administration at a dose of 10 mg/kg of at least about 3 days, as assayed by human Fc ELISA. The therapeutic composition may comprise a heterodimeric protein comprised of a first polypeptide containing amino acid sequence of SEQ ID NO. 1 and a second polypeptide containing amino acid sequence of SEQ ID NO. 2. The therapeutic composition may also contain about 6% (m/v) sucrose, about 3% (m/v) polyethylene glycol with an average molecular weight of about 3350 Da, about 50 mM sodium chloride, and about 20 mM L-Histidine pH 6.5.
In certain aspects, the present invention provides for a therapeutic composition which contains a heterodimeric protein composition, including a first polypeptide containing amino acid sequence of SEQ ID NO. 8 and a second polypeptide containing amino acid sequence of SEQ ID NO. 9. The therapeutic composition may also contain about 6% (m/v) sucrose, about 3% (m/v) polyethylene glycol having an average molecular weight of 3350 Da, about 50 mM sodium chloride, and about 20 mM L-Histidine pH from about 4.5 to about 7.0. The pH value may be about 6.5. Alternatively, the therapeutic composition may also contain about 1.2% (m/v) sucrose, about 0.09% (m/v) polysorbate 80, about 3% (m/v) D-mannitol, about 38 mM glycine, and about 15 mM TRIS-HCl, pH may be from about 6.5 to about 8.5. The pH value may be about 7.5.
In certain aspects, the present teachings provide for a substance or a composition containing a heterodimeric protein assembly including a polypeptide of SEQ ID NO. 8 and another polypeptide of SEQ ID NO. 9 for use in the treatment of certain disorders or diseases associated with IL-1β modulation, including, but not limited to, arthritis, gout, rheumatoid arthritis, cryopyrin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergy, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-elevation myocardial infarction (STEMI), acute respiratory distress syndrome/cytokine release storm (ARSD/CRS), Schnitzler syndrome, postoperative incisional pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet disease, breast cancer, non-small cell lung cancer, or stroke.
In certain aspects, the present teachings provide for a method of treating or preventing a disease or condition associated with modulation of activity of human IL-1β. The method includes administering to a patient in need for treating or preventing a disease associated with modulation of activity of human IL-1β a therapeutically effective amount of a pharmaceutical composition including a heterodimeric protein containing a first polypeptide including amino acid sequence of SEQ ID NO. 8 and a second polypeptide comprising amino acid sequence of SEQ ID NO. 9. Diseases associated with IL-1β modulation, include, but are not limited to, arthritis, gout, rheumatoid arthritis, cryopyrin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergy, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-elevation myocardial infarction (STEMI), acute respiratory distress syndrome/cytokine release storm (ARSD/CRS), Schnitzler syndrome, postoperative incisional pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet disease, breast cancer, non-small cell lung cancer, or stroke.
The following drawings and descriptions are provided to aid in the understanding of the invention:
The teachings disclosed herein are based, in part, upon engineering of a heterodimeric protein assembly that is capable of binding to human IL-1β and attenuating its function. The heterodimeric protein assembly of the present teachings comprises extracellular portions of IL1-R1 (GenBank: AAM88423.1) and of IL-1RAcP (GenBank: BAA25421.1), or functional fragments thereof. Each, the IL1-R1 portion and the IL-1RAcP portion, is fused to a distinct mutant of Fc portion of the human Ig Gamma-1 (GenBank: J00228.1). The two distinct Fc mutants in the heterodimeric protein assembly are engineered as to favor the heteromeric dimer formation between the two Fc mutants over any homomeric assembly. To enable recombinant production of the heterodimeric protein assembly of the present teachings, a DNA expression vector has been constructed for overproducing the heterodimeric protein assembly in a heterologous protein expression system, and mammalian cells have been prepared stably expressing the heterodimeric protein assembly to a high expression level. A protein purification procedure has been devised allowing obtaining a physiologically relevant substantially pure preparation of the heterodimeric protein assembly of the present teachings. Thus, purified protein molecule demonstrates a high degree of specific activity in an in vitro Enzyme-Linked Immunosorbent Assay (ELISA) using human IL-1β (GenBank: AAH08678.1). Unexpectedly, the protein molecule exhibits an acceptable pharmacokinetics profile upon subcutaneous animal administration, while not resulting in any body weight loss or adverse clinical events. Design, preparation and preliminary characterization of composition of matter of the present teachings are disclosed, in part, in an International Patent Application Publication No. WO/2014/035361, published on Mar. 6, 2014, and International Patent Application Serial No. PCT/US/2013/026349, filed on Feb. 15, 2013, both of which are incorporated herein by reference in their entirety.
The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used. “About” and “approximately” shall generally mean an acceptable to a skilled person degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
The methods of the invention may include steps of comparing sequences to each other, including wild-type sequence to one or more mutants (sequence variants). Such comparisons typically comprise alignments of polymer sequences, e.g., using sequence alignment programs and/or algorithms that are well known in the art (for example, BLAST, FASTA and MEGALIGN, to name a few). The skilled artisan can readily appreciate that, in such alignments, where a mutation contains a residue insertion or deletion, the sequence alignment will introduce a “gap” (typically represented by a dash, or “A”) in the polymer sequence not containing the inserted or deleted residue.
The methods of the invention may include statistical calculations, e.g. determination of IC50 or EC50 values, etc. The skilled artisan can readily appreciate that such can be performed using a variety of commercially available software, e.g. PRISM (GraphPad Software Inc, La Jolla, CA, USA) or similar.
“Homologous,” in all its grammatical forms and spelling variations, refers to the relationship between two proteins that possess a “common evolutionary origin,” including proteins from super families in the same species of organism, as well as homologous proteins from different species of organism. Such proteins (and their encoding nucleic acids) have sequence homology, as reflected by their sequence similarity, whether in terms of percent identity or by the presence of specific residues or motifs and conserved positions. However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
The term “sequence similarity,” in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.
The terms “protein” and “polypeptide” are used interchangeably. The polypeptides described herein may be comprised of more than one contiguous amino acid chain, thus forming dimers or other oligomeric formations. In general, the polypeptides of the present teachings for use in mammals are expressed in mammalian cells that allow for proper post-translational modifications, such as CHO or HEK293 cell lines, although other mammalian expression cell lines are expected to be useful as well. It is therefore anticipated that the polypeptides of the present teachings may be post-translationally modified without substantially effecting its biological function.
In certain aspects, functional variants of the heterodimeric protein assemblies of the present teachings include fusion proteins having at least a biologically active portion of the human IL1-R1 or IL-1RAcP or a functional fragment thereof, and one or more fusion domains. Well known examples of such fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chain constant region (e.g., an Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, the IL1-R1 or IL-1RAcP polypeptide portions may be fused with a domain that stabilizes the IL1-R1 or IL-1RAcP polypeptides in vivo (a “stabilizer” domain), optionally via a suitable peptide linker. The term “stabilizing” means anything that increases the half life of a polypeptide in systemic circulation, regardless of whether this is because of decreased destruction, decreased clearance, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on certain proteins. Likewise, fusions to human serum albumin can confer desirable properties. Other types of fusion domains that may be selected include multimerizing (e.g., dimerizing, tetramerizing) domains and functional domains that confer an additional biological function, e.g. promoting accumulation at the targeted site of action in vivo.
In certain aspects, the heterodimeric protein assemblies of the present teachings comprise an extracellular portion of IL1-R1, or a functional fragment thereof, fused with a IgG-Fc domain, and an extracellular portion IL-1RAcP, or a functional fragment thereof, fused with another IgG-Fc domain. The IgG-Fc domain and the another IgG-Fc domain are chosen as to favor a heterodimeric protein assembly over any homodimeric protein assembly. The extracellular portion of IL1-R1 may be fused with the IgG-Fc domain via a flexible linker, while IL-1RAcP, or a functional fragment thereof, may be fused with the another IgG-Fc domain via the flexible linker of the same amino acid sequence or via another flexible linker.
In an example embodiment, illustratively shown in
In certain aspects, the present teachings provides for a recombinant DNA molecule having an open reading frame coding for a polypeptide comprising the leading 333 amino acids of the human IL1-R1 fused with IgG-Fc domain (Fc-II) via a flexible linker, and for another recombinant DNA molecule having an open reading frame coding for another polypeptide comprising the leading 358 amino acids of the human IL-1RAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker.
In an example embodiment, the polypeptide comprising the leading 333 amino acids of the human IL1-R1 fused with IgG-Fc domain (Fc-II) via a flexible linker comprises the amino acid sequence of SEQ. ID NO. 3. The corresponding to it DNA molecule may comprise the nucleotide sequence of SEQ ID NO. 4. The another polypeptide comprises the leading 358 amino acids of the human IL-1RAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker may comprise the amino acid sequence of SEQ. ID NO. 5. The corresponding to it DNA molecule may comprise the nucleotide sequence of SEQ ID NO. 6.
In certain aspects, the present invention provides for a recombinant mammalian expression plasmid for high expression of a polypeptide comprising the leading 333 amino acids of the human IL1-R1 fused with IgG-Fc domain (Fc-II) via a flexible linker, and for another recombinant DNA molecule having an open reading frame coding for another polypeptide comprising the leading 358 amino acids of the human IL-1RAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker. This plasmid comprises two cytomegalovirus (CMV) promoters to drive transcription of the two genes coding for said polypeptide and said another polypeptide, each followed by a transcription termination sequence and a polyadenylation sequence. The plasmid also contains an origin of replication and a gene conferring ampicillin resistance, for supporting plasmid propagation and selection in bacteria. The plasmid further contains a gene for Glutamine synthetase, a selectable marker widely used for establishing stable CHOK1 and NSO cell lines.
In an example embodiment, the mammalian expression plasmid of the present teachings comprises the nucleotide sequence of SEQ ID NO. 7.
In certain aspects, the present teachings provide for a mammalian expression system for production of a heterodimeric protein assembly comprising a polypeptide comprising amino acid residues 18 through 333 of the human IL1-R1 fused with IgG-Fc domain (Fc-II) via a flexible linker, and another polypeptide comprising amino acid residues 21 through 358 of the human IL-1RAcP fused with another IgG-Fc domain (Fc-V) via a flexible linker.
In an example embodiment, the mammalian expression system of the present teachings comprises Chinese hamster ovary cells (CHO-K1) harboring a plasmid comprising nucleotide sequence of SEQ ID NO. 7.
In certain aspects, the mammalian expression system of the present teachings yields a heterodimeric protein assembly comprising a polypeptide of SEQ ID NO. 8 and another polypeptide of SEQ ID NO. 9.
In certain aspects, the present teachings provide for a substance or a composition, comprising a heterodimeric protein assembly comprising a polypeptide of SEQ ID NO. 8 and another polypeptide of SEQ ID NO. 9, for use in the treatment of certain disorders or diseases associated with IL-1β modulation, including, but not limited to, arthritis, gout, rheumatoid arthritis, cryopyrin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergy, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-elevation myocardial infarction (STEMI), acute respiratory distress syndrome/cytokine release storm (ARSD/CRS), Schnitzler syndrome, postoperative incisional pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet disease, breast cancer, non-small cell lung cancer, or stroke.
In certain aspects, the present teachings provide for a method of treating or preventing a disease or condition associated with modulation of activity of human IL-1β. The method includes administering to a patient in need for treating or preventing a disease associated with modulation of activity of human IL-1β a therapeutically effective amount of a pharmaceutical composition including a heterodimeric protein including a first polypeptide including amino acid sequence of SEQ ID NO. 8 and a second polypeptide comprising amino acid sequence of SEQ ID NO. 9. Diseases associated with IL-1β modulation, include, but are not limited to, arthritis, gout, rheumatoid arthritis, cryopyrin-associated periodic syndromes (CAPS), scleroderma, diabetes, atherosclerosis, dry eye syndrome, ocular allergy, uveitis, recurrent pericarditis, familial Mediterranean fever (FMF), ST-elevation myocardial infarction (STEMI), acute respiratory distress syndrome/cytokine release storm (ARSD/CRS), Schnitzler syndrome, postoperative incisional pain, chronic kidney disease (CKD), PFAPA (Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis) syndrome, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), pyoderma gangrenosum, Kawasaki disease, acne vulgaris, atopic dermatitis, Behcet disease, breast cancer, non-small cell lung cancer, or stroke.
The following Examples illustrate the forgoing aspects and other aspects of the present teachings. These non-limiting Examples are put forth so as to provide those of ordinary skill in the art with illustrative embodiments as to how the compounds, compositions, articles, devices, and/or methods claimed herein are made and evaluated. The Examples are intended to be purely exemplary of the inventions disclosed herein and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for.
hIL1-R1-hIgG1-Fc polypeptide of SEQ ID NO. 1 and hIL-1RAcP-hIgG1-Fc polypeptide of SEQ ID NO. 2 were co-expressed in CHO-K1 using molecular biology, cell culture and protein biochemistry techniques known in the art and described in PCT Publication WO/2014/035361, and PCT Application Serial No. PCT/US/2013/026349. Essentially, CHO-K1 cells expressing the polypeptides were harvested and lysed utilizing well established protocols. After cell lysate clarification, the supernatant containing expressed polypeptides was first applied to a Protein A affinity column. The pH adjusted Protein A column eluate was further purified by anion-exchange chromatography (AIEX) utilizing Q Sepharose resin. The AIEX flowthrough was analyzed by size-exclusion HPLC (SEC-HPLC), SDS-PAGE and other analytical techniques, as appropriate.
For subsequent studies, a therapeutic composition comprising hIL1-R1-hIgG1-Fc and hIL-1RAcP-hIgG1-Fc polypeptides was formulated to contain 40 mg/ml of the polypeptides, 6% (m/v) sucrose, 3% (m/v) polyethylene (PEG) 3350, 50 mM sodium chloride, and 20 mM L-Histidine pH from about 4.5 to about 7.0, preferably about 6.5.
The sequences of the polypeptides contained in the final product were analyzed as outlined in the following example. Unexpectedly, the polypeptides in the final product predominantly contained hIL1-R1-hIgG1-Fc polypeptide of SEQ ID NO. 8 and hIL-1RAcP-hIgG1-Fc polypeptide of SEQ ID NO. 9. This polypeptide containing solution was lyophilized and subsequently, reconstituted and for formulated to contain 80 mg/ml of the polypeptides, 1.2% (m/v) sucrose, 0.09% (m/v) polysorbate 80, 3% (m/v) D-mannitol, 38 mM glycine, and about 15 mM TRIS-HCl pH from about 6.5 to about 8.5, preferably about 7.5.
Three samples of the product prepared essentially as described in the forgoing example were analyzed as described below. First, the molecular masses of the two intact polypeptides contained in each sample were determined by Liquid Chromatography-Mass Spectrometry (LC-MS). Then peptide mapping was performed by Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS). Lastly, terminus peptide sequencing was performed.
For intact peptide mass determination by LC/MS, protein samples were reduced and deglycosylated following well established protocols know in the art.
Peptide mapping was performed essentially as follows:
Samples were digested with LysC, Trypsin and Chymotrypsin. Each sample was analyzed by LC-MS/MS.
1) 40 μg of sample was denatured, reduced and digested with trypsin according to an established protocol (Cat #VS280, Promega Corporation, Madison, WI).
2) 40 μg of sample was denatured, reduced and digested with LysC according to an established protocol (Cat #VA1170, Promega Corporation, Madison, WI).
3) 40 μg of each sample was denatured, reduced and digested with Chymotrypsin according to an established protocol (Cat #VA106A, Promega Corporation, Madison, Wis).
4) High pressure liquid chromatography utilizing an Agilent 1900 UPLC system (Agilent Technologies, Santa Clara, CA) was performed as follows:
5) Tandem Mass Spectrometry Analysis—Spectra were acquired using a QTOF 6550 mass spectrometer (Agilent Technologies, Santa Clara, CA). The mass spectrometer was operated in positive ion mode. Mass spectra were acquired over m/z 350-2000 at 20,000 resolution (m/z 1521) and data-dependent acquisition selected the top 10 most abundant precursor ions for tandem mass spectrometry by CID fragmentation using an isolation width of 4.0 Da, formula of (slope)*(m/z)/100+offset was used for collision energy. Dynamic exclusion was used to minimize redundancy of MS/MS collection and maximize peptide identifications.
6) Data Analysis—the raw data was extracted and searched by using Spectrum Mill v5.01 and Hunter (Agilent Technologies). The collected MS and MS/MS spectra were analyzed against protein database+decoy sequence databases. The enzyme parameter was limited with a maximum miscleavage of 2 for Trypsin, 2 for LysC and 5 for Chymotrypsin. Additional non-enzyme search was performed for the N-term peptides. All other search parameters were set to the default settings of Spectrum Mill (carbamidomethylation of cysteines, +/−20 ppm for precursor ions, +/−50 ppm for fragment ions, and a minimum matched percent scored peak intensity (SPI %) of 50%). A concatenated forward-reverse database was constructed to calculate the in situ false discovery rate (FDR). Cutoff scores were dynamically assigned to each data set to maintain the false discovery rate at less than 0.1% at the peptide level. Manual inspection was also applied for every uniquely identified peptides of each of the analyzed samples.
Two well resolved major peaks (each greater than 98% purity) were detected in intact mass analysis, first with corresponding to a MW=61,505.9±0.1 Da, and second—to MW=64,753.3±0.1 Da. The first peak corresponds to SEQ ID NO. 8 (theoretical MW is 61,495 Da), the second to SEQ ID NO. 9 (theoretical MW is 64,743 Da). The difference in MW (˜11 Da) is most likely due to deamination of an Asn residue after deglycosylation.
Peptide mapping and C-terminal sequence analysis further confirmed these sequences with high degree of confidence.
The binding affinity of prepared polypeptides of IL1R-FcV-RAcP-FcII heterodimer to IL-1β/IL-1F2 (NCBI Accesion #NP_000567) was measured using a specially designed Surface Plasmon Resonance (SPR) assay. The assay was carried out using capturing method where anti-human IgG were cross-linked to the surface of sensor chip for capturing IL1R-FcV-RAcP-FcII heterodimer via its IgG (Fc) fragments. Series of different concentrations of IL-1β/IL-1F2 were used for calculation of the dissociation constant (Kd).
Anti-Human IgG Conjugation:
Conjugation procedure for anti-human IgG (Fc) was carried out according manufacturer's protocol using conditions below.
1. CMS Sensor Chip was placed into the instrument and primed with Biacore running buffer, 1× HBS-EP, for 6 min at 10 μl/min, repeated twice. All steps were carried out at 25° C. Channels 1 and 2 was used for the experiment and channels 3 and 4 were reserved as a backup;
2. Anti-Human IgG from the kit, 0.5 mg/ml in 0.15 M NaCl, was diluted 20-fold in Immobilization Buffer (10 mM Na-acetate pH 5.0) to a final concentration of 25 μg/ml;
3. Reagents for immobilization procedure were prepared as follows: EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide)-0.4 M in Milli-Q water; NHS (N-hydroxysuccinimide)-0.1 M in Milli-Q water; 1 M Ethanolamine-HCl pH 8.5 in Milli-Q water;
4. Standard protocol for surface activation and immobilization was used;
5. Activation: EDC and NHS were mixed at 1:1 ratio and injected into the chip at 10 μl/min for 7 min;
6. Immobilization: Anti-Human IgG were injected into the chip at 10 μl/min for 5 min;
7. Deactivation: Unreacted active groups were blocked by injection of 1 M Ethanolamine-HCL at 10 μl/min for 7 min;
8. After antibody conjugation, the chip was washed with 1× HBS-EP 2 times at 10 μl/min for 6 min and then the “dry” working cycle without addition of any protein component was run twice. The working cycle consisted of Ligand (IL1R-FcV-RAcP-FcII heterodimer) Loading Step of 1 min, Wash Step of 3 min, Sample (IL-1) Loading Step of 1 min, Wash step of 16.7 min, Chip Regeneration Step, 1 min, 3 M MgCl2. All steps were run at 10 μl/min except Sample Loading Step that was run at 30 μl/min;
Affinity evaluation of IL1R-FcV-RAcP-FcII heterodimer/IL-1β/IL-1F2 interaction.
The goal of this experiment was to measure association constant for IL1R-FcV-RAcP-FcII heterodimer and IL-1β/IL-1F2. Anti-human IgG were covalently immobilized on CM5 Sensor Chip then IL1R-FcV-RAcP-FcII heterodimer was loaded and followed by various concentrations of human IL-1β/IL-1F2. Series of sensograms were generated and used for calculation of Kd value.
1. In preliminary series of experiments, several different concentrations (1, 10 and 100 μg/ml) of IL1R-FcV-RAcP-FcII heterodimer were prepared and tested for their association with immobilized anti-human IgG. It was found that at 1 μg/ml, IL1R-FcV-RAcP-FcII heterodimer produced sufficient signal within the range of ˜100 RU and this concentration was used for the entire assay;
2. Parameters for binding/dissociation cycles were optimized in series of pilot runs and are summarized in Table 1;
3. Human IL-1β/IL-1F2 were used at the concentrations specified in Table 2 where concentration of 3.676 nM was run two time independently as an internal control for the instrument reproducibility;
4. Series of sensograms corresponding to different concentrations IL-1β/IL-1F2 were generated. The data were normalized by subtraction of ‘buffer only’ sensorgam. The buffer-normalized sensograms are shown in
Experimental conditions were optimized to enable accurate use of curve fit algorithms. As evident from the sensograms (
As an alternative way for Kd calculation, Steady-State data analysis using 1:1 Langmuir binding model was used. According to this method, Kd is calculated from series of plots of steady-state analyte binding levels (Req) against concentration. The obtained data are summarized in Table 2.
Experimental data are summarized in Table 3 and are shown in
Polypeptides of IL1R-FcV-RAcP-FcII heterodimer (SEQ ID NO. 1 and SEQ ID NO. 2) were co-expressed and purified essentially as described in the forgoing examples. For administration into animals, the polypeptides were formulated in the following buffer: 1% w/v Sucrose, 100 mM Sodium Chloride, 20 mM L-Arginine Hydrochloride, 25 mM Sodium Bicarbonate, pH 6.3. The dosing stock concentration used was 0.5 mg/mL of the polypeptide.
Fourteen male DBA/1 mice were randomized by body weight into seven groups of two animals on Day 0 of the study. A single dose of IL1R-FcV-RAcP-FcII heterodimer (5 mg/kg in 10 ml/kg) was administered subcutaneously (dorsally) on Day 0 to mice in six of the groups. The mice in the remaining group remained untreated and were bled via cardiac puncture for plasma preparation on Day 0 of the study. Plasma was prepared from blood samples collected from mice in the treated groups via the orbital sinus or terminal cardiac puncture at specified times throughout the study. Body weights were recorded for all animals on the treatment day (Day 0) and then three times per week, including the termination day of each group. Body weight change was not measured in groups culled for sample collection at 0 hours and within 36 hours of dose administration. Mean body weight loss between Day 0 and termination of the groups culled between 96 hours and 21 days post-dose was minimal. No mice lost body weight exceeding ethical limits. Following the in-life phase of the study, plasma samples were analyzed by Enzyme Linked Immunosorbent Assay (ELISA) for Hu-Fc proteins. Quantification of Hu-Fc in mouse plasma samples by ELISA was used as a read-out for circulating levels of IL1R-FcV-RAcP-FcII heterodimer. The assay was performed on samples from all mice in the study.
The polypeptides (detected as Human-Fc protein) were detected in the plasma of animals at all time-points post-dose. One Phase Decay Model equation using Prism 5.0c (GraphPad Software Inc, La Jolla, CA, USA) was then used to determine pharmacokinetics of the polypeptides as detected by Hu-Fc ELISA. Peak circulating level of Hu-Fc (Cmax) was determined to be 1.284 μg/mL, and time to peak circulating levels (Tmax) was 24 hours post-dose. The half-life (T½) was 97 hours, 31 minutes and the rate constant (K) was 0.0071 hr-1. Hu-Fc was below the level of detection in the plasma of the untreated animals. The results of the study are summarized in Table 4.
#Bleed via terminal cardiac puncture
For the purpose of this study, initially three naïve male Cynomolgus monkeys were used. The animals were approximately 2-4 years old and weighed approximately 2 kg. The animals received a single dose of overexpressed and purified IL1R-FcV-RAcP-FcII heterodimer (SEQ ID NO. 1 and SEQ ID NO. 2), formulated essentially as described in the foregoing Example 1, by subcutaneous administration at a dose level of 10 mg/kg on Day 1 of the study. The results of the bioanalysis from the initial set of three animals are shown in
There were no clinical signs noted during the course of the study. Body weight profiles were considered satisfactory. Results from the initial PK data analysis showed the Cmax and Tmax following single subcutaneous administration at 10 mg/kg were to be approximately 24-48 h. PK results from the additional 3 animals showed, the IL1R-FcV-RAcP-FcII heterodimer was quantifiable in plasma until at least 10 days for 2 of the 3 animals, and up to day 14 for one animal. The pharmacokinetics parameters for the follow-up set of three monkeys were determined using a non-compartmental model in WinNonLin 6.3 software package and are summarized in Table 5.
1harmonic mean
2 median [min-max]
All animals were widely exposed to IL1R-FcV-RAcP-FcII heterodimer. The observed inter-individual variability was relatively high with a CV % of about 60%. The latter was explained by the lowest drug exposure found in animal F1290 (
IL1R-FcV-RAcP-FcII is a heterodimer comprised of soluble portions of human IL-1R and IL-1RAcP each linked to a unique IgG1 Fc portion. Sequence alignment of the 333 amino acid portion of the human IL-1R with relevant portions from several species demonstrates only a modest sequence identity (˜64%) with IL-1R portions from rodents (mouse, rat). However, the sequence identity is much higher between human IL-1R and those of other primates (e.g. 91% with marmoset monkey). Further presented below are protein binary sequence alignments of the 358 amino acid portion of the human IL-1RAcP, forming a part of IL1R-FcV-RAcP-FcII heterodimer molecule, with relevant portions from several species. Cross-species sequence identity of this portion of IL1R-FcV-RAcP-FcII heterodimer is somewhat higher. Higher sequence identity is also observed comparing the 358 amino acid portion of the human IL-1RAcP with its ortholog from Macaca mulatta (92%) vs comparing with the ortholog from Mus musculus (85%).
In order to comparatively evaluate the functional (inhibitory) properties of a novel drug candidate IL1R-FcV-RAcP-FcII heterodimer (SEQ ID NO. 1 and SEQ ID NO. 2) the following study was performed. Assays were carried out using human, Macaca Rhesus and murine IL-1β IL-1F2 orthologs. Human vs. Mouse IL-1β/IL-1F2 were compared in Mouse Embryo Fibroblasts. Human vs. M. Rhesus IL-1β IL-1F2 were compared in MRC5 human lung fibroblasts. As a functional comparator, previously characterized mouse monoclonal antibodies against human IL-1β IL-1F2 and goat polyclonal antibodies against mouse IL-1β IL-1F2 were used. Quantification of IL-1(3 IL-1F2-induced IL-6 production by MRC5 cells or MEFs was used for determination of inhibitory properties (IC50 values) for all three orthologs.
Materials and Reagents
Cells
MRC5 cells, Human Lung Fibroblasts, ATCC Cat #CCL-171, Lot #59474707.
Mouse Embryo Fibroblasts (MEFs) used for the experiments.
Medium
DMEM, Dulbecco's Modification of Eagle's Medium, high glucose (4.5 g/L), Invitrogen, Cat #11995-065, Lot #1237317, supplemented with L-glutamine and 1× penn/strep and 10% Benchmark Fetal Bovine Serum, Gemini Bioproducts, Cat #100-106, Lot #A78D00E.
Reagents
IL1R-FcV-RAcP-FcII heterodimer, Preparation of 1.5 mg/ml.
IL-1β IL-1F2, Human recombinant, E. coli-derived, Ala117-Ser269, Accession #NP_000567, R&D systems, Cat #201-LB, Lot #AD1412111
IL-1β IL-1F2, M. Rhesus recombinant, E. coli-derived, Ala117-Ser269, Accession #P48090, R&D systems, Cat #1318-RL, Lot #GUG0110111
IL-1β/IL-1F2, Mouse recombinant, E. coli-derived, Vla118-Ser269, Accession #NP_032387, R&D systems, Cat #401-ML-005, Lot #BN0713032
Mouse monoclonal antibodies against human IL-1β/IL-1F2, clone #8516, R&D systems, Cat #MAB201, Lot #AWE1011081
Goat polyclonal antibodies against mouse IL-1β/IL-1F2, clone #8516, R&D systems, Cat #AF-401-NA, Lot #NP2812121
IL-6 Quantakine Immunoassay, R&D systems, Cat #D6050, Lot #308916
Mouse IL-6 Quantakine Immunoassay, R&D systems, Cat #M6000B, Lot #309487
Procedure
Cell Maintenance
Centrifuge the supernatants at 300×g for 10 min, collect cleared supernatants and use them for ELISA either directly (MEFs) or with ⅕ dilution (MRC5) if appropriate according to pilot experiments.
ELISA
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-6 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL-6 present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for IL-6 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of IL-6 bound in the initial step. The color development is stopped and the intensity of the color is measured.
Experimental Data
The goal of three series of experiments was to identify suitable cell line for measuring IL6 secretion upon treatment with human and mouse orthologs of IL-1β/IL-1F2. Several preliminary pilot experiments were carried out to identify mouse cells that respond to mouse-IL-1β/IL-1F2 treatment by robust secretion of IL6. On the basis of these preliminary experiments, MEFs were chosen as a model cell line for IL1R-FcV-RAcP-FcII heterodimer titration experiments. IL1R-FcV-RAcP-FcII heterodimer titration curve of mouse IL6 secretion induced by mouse IL-1B/IL-1F2 in MEFs is shown in
The experimental data indicates that IL1R-FcV-RAcP-FcII heterodimer is an efficient inhibitor of human IL-1β/IL-1F2, but not mouse IL-1B/IL-1F2 signaling pathway: IL1R-FcV-RAcP-FcII heterodimer IC50 value for human IL-1B/IL-1F2 is 0.19 ng/ml and for mouse IL-1B/IL-1F2→200 ng/ml (0.95 pM and >1000 pM respectively, assuming molecular mass of IL1R-FcV-RAcP-FcII heterodimer as 200 kDa). IL1R-FcV-RAcP-FcII heterodimer titration curve of human IL6 secretion induced by human IL-1B/IL-1F2 in MRC5 cells is shown in
Thus, stimulation of IL-6 production upon treatment of mouse or human cells with IL-1B/IL-1F2 was used a functional test for inhibitory properties of a novel drug candidate IL1R-FcV-RAcP-FcII heterodimer against human, mouse and M. Rhesus orthologs of IL-1B/IL-1F2. Suitable cell lines were identified and experimental conditions including cell density, treatment duration linear range for IL6 detection and were optimized for all three orthologs. The obtained data are summarized in Table 6.
M. Rhesus
All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
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| Number | Date | Country | |
|---|---|---|---|
| 20210403534 A1 | Dec 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2020/034114 | May 2020 | WO |
| Child | 17474718 | US | |
| Parent | 15129412 | US | |
| Child | 17474718 | US |
