Patent Grant
11945855
This disclosure relates to recombinant therapeutic proteins, particularly modified partial MHC molecules, and their use in the treatment of inflammatory disease.
Multiple sclerosis (MS) is the most prevalent chronic inflammatory disease of both white and grey matter of the central nervous system, affecting more than 2 million people worldwide and at least 400,000 in the United States (GBD 2015 Neurological Disorders Collaborator Group, Lancet Neurol. 16:877-897, 2017). The inflammatory process in MS is mediated by autoimmune driven demyelination, often accompanied by neurodegenerative injuries. Currently there is no cure for MS. Thus, MS patients have to rely on disease modifying therapies in order to ameliorate some of the disease signs (Wingerchuk et al., Mayo Clin. Proc. 89:225-240, 2014). About 15 disease modifying medications are available for the relapsing and remitting form of MS (Reich et al., N. Engl. J. Med. 378:169-180, 2018). However, there is a continuing need for improved therapies for MS.
Disclosed herein are modified major histocompatibility (MHC) class II DRα1 polypeptides. These modified DRα1 polypeptides exhibit altered affinity for CD74 and ability to compete with macrophage migration inhibitory factor (MIF) for binding to CD74, without apparent effects on the structure of the molecule.
In some embodiments, disclosed are recombinant polypeptides that include a MHC class II DRα1 domain including a glutamine residue at a position corresponding to amino acid position 14 of SEQ ID NO: 1 (referred to as L14Q mutation in some instances or as L50Q when in the context of DRα1-MOG-35-55 constructs). The glutamine residue replaces the native leucine residue at this position (
In other embodiments, disclosed are recombinant polypeptides that include a DRα1 domain including a glutamine residue at a position corresponding to amino acid position 14 of SEQ ID NO: 1, an antigenic peptide, and optionally a linker sequence. In one example, the linker sequence includes a first glycine-serine spacer, a thrombin cleavage site and a second glycine-serine spacer. The antigenic peptide may be a myelin oligodendrocyte glycoprotein (MOG) polypeptide, such as human or mouse MOG-35-55. In some embodiments, the sequence of the recombinant polypeptide includes or consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.
Also provided are nucleic acid molecules encoding the recombinant polypeptides disclosed herein. Exemplary nucleic acid molecules include SEQ ID NOs: 4-6. In some embodiments, the nucleic acid is included in an expression construct and/or a vector. Also disclosed are cells or cell lines that include the expression construct or vector.
In some embodiments the disclosed recombinant polypeptides or nucleic acids encoding the recombinant polypeptides are included in a pharmaceutical composition, for example, with a pharmaceutically acceptable carrier. In some examples, the composition includes at least 5 mg/kg of the recombinant polypeptide.
Further disclosed herein are methods of treating inflammatory disorders that include administering a disclosed recombinant polypeptide or pharmaceutical composition (such as a composition including the polypeptide of any one of SEQ ID NOs: 1-3) to a subject that is suffering from the inflammatory disorder. In some examples, the subject has multiple sclerosis (or its mouse model, EAE).
The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
Any nucleic acid and amino acid sequences listed herein or in the accompanying sequence listing are shown using standard single letter abbreviations for nucleotide bases and amino acids, as defined in 37 C.F.R. § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
The Sequence Listing is submitted as an ASCII text file in the form of the file named Corrected_Sequence_Listing.txt, which was created on May 12, 2021, and is 28,237 bytes, which is incorporated by reference herein.
MEVGWYRPPFSRVVHLYRNGK
GGGGS
LVPR
GSGGGGIKEEHVIIQAEFYQNPDQSGEF
MEVGWYRSPFSRVVHLYRNGK
GGGGS
LVPR
GSGGGGIKEEHVIIQAEFYQNPDQSGEF
In both the animal model of MS, experimental autoimmune encephalomyelitis (EAE), and in MS subjects, multiple lines of evidence support the role of autoreactive, myelin-specific, CD4+ T cells in the autoimmune disease process (Steinman, Cell 85:299-302, 1996; Zamvil et al., Annu. Rev. Immunol. 8:579-621, 1990; Chitnis, Int. Rev. Neurobiol. 79:43-72, 2007). Key cytokines thought to drive the early inflammatory stage of MS to a chronic progressive phase are macrophage migration inhibitory factor (MIF), the first described cytokine, and its ancestral functional homolog, D-dopachrome tautomerase (D-DT) (Chitnis, Int. Rev. Neurobiol. 79:43-72, 2007). MIF and D-DT levels are increased and have been implicated as markers of clinical worsening in MS and as a requirement for disease progression in EAE (Benedek et al., Proc. Natl. Acad. Sci. USA 114:E8421-E8429, 2017; Niino et al., J. Neurol. Sci. 179(S1-2):127-131, 2000; Powell et al., J. Immunol. 175:5611-5614, 2005; Meza-Romero et al., J. Immunol. 192:4164-4173, 2014).
A potent biological therapy called RTL1000 and a second-generation derivative, DRα1-human(h)MOG-35-55, that bind tightly to the MIF/D-DT receptor, CD74, and competitively inhibit MIF/D-DT binding and downstream signaling through phosphorylated extracellular signal-related kinases (p)ERK1/2 has previously been developed (Vandenbark et al., J. Immunol. 171:127-133, 2003; Benedek et al., J. Neuroinflammation 12:123, 2015). RTL1000 and DRα1-hMOG-3-55 can treat mice with EAE after onset of clinical signs, resulting in the inhibition of T cell and macrophage activation and migration into the central nervous system and reduced disease severity (Vandenbark et al., J. Immunol. 171:127-133, 2003; Benedek et al., Eur. J. Immunol. 43:1309-1321, 2013; Meza-Romero et al., J. Immunol. 192:4164-4173, 2014). RTL treatment was also found to enhance anti-inflammatory macrophage/microglia cell numbers, promote re-myelination, and reduce the severity of acute and chronic EAE (Meza-Romero et al., J. Immunol. 192:4164-4173, 2014).
In the present disclosure, the inventors have identified an amino acid substitution of glutamine for leucine at position 50 (L50Q) of the DRα1-hMOG-35-55 construct (termed DRhQ) that alters the affinity of the construct for CD74, with a 8-10-fold increase in binding capacity. This substitution did not affect the structure of the molecule as evaluated by circular dichroism and antibody probing and the increased binding affinity translated into a commensurate ability of DRhQ to competitively inhibit MIF binding to its cognate CD74 receptor. Treatment of WT C57BL/6 mice with DRhQ reduced pERK1/2 phosphorylation in vitro in splenocytes to a background level. Finally, the L50Q substitution significantly enhanced the ability of the construct to treat ongoing clinical signs of severe EAE.
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Hence “comprising A or B” means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. All GenBank Accession numbers mentioned herein are incorporated by reference in their entirety as present in GenBank on Oct. 5, 2018. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:
Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term “antigen” includes all related antigenic epitopes. “Epitope” or “antigenic determinant” or “antigenic peptide” refers to a site on an antigen to which B and/or T cells respond. In one embodiment, T cells respond to the epitope, when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 8 amino acids (such as about 8-50 or 8-23 amino acids) in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
An antigen can be a tissue-specific antigen, or a disease-specific antigen. These terms are not exclusive, as a tissue-specific antigen can also be a disease-specific antigen. A tissue-specific antigen is expressed in a limited number of tissues, such as a single tissue. A tissue-specific antigen may be expressed by more than one tissue, such as, but not limited to, an antigen that is expressed in the central or peripheral nervous system.
CD74: Also known as CD74 molecule, major histocompatibility complex, class II invariant chain or Ii. CD74 is a chaperone regulating antigen presentation. It is also a cell surface receptor for macrophage migration inhibitory factor (MIF). Nucleic acid and protein sequences for CD74 are publicly available. For example, GenBank Accession Nos. NM_001025158, NM_004355, and NM_001025159 disclose exemplary human CD74 nucleic acid sequences, and GenBank Accession Nos. NP_001020329, NP_004346, and NP_001020330 disclose exemplary human CD74 amino acid sequences. Similarly, GenBank Accession Nos. NM_001042605 and NM_010545 disclose exemplary mouse CD74 nucleic acid sequences, and GenBank Accession Nos. NP_001036070 and NP_034675 disclose exemplary mouse CD74 amino acid sequences. Each of these sequences is incorporated herein by reference as present in GenBank on Oct. 5, 2018.
Control: A “control” refers to a sample or standard used for comparison with an experimental sample. In some embodiments, the control is a sample obtained from a healthy subject or population of healthy subjects. In other embodiments, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of samples that represent baseline or normal values, such as the level of CD74 expression or activity in healthy subjects). In further examples, the control is from a subject prior to treatment (such as CD74 expression or activity level prior to treatment with an MHC class II β1α1 polypeptide or an MHC class II α1 domain polypeptide).
Domain: A discrete part of an amino acid sequence of a polypeptide or protein that can be equated with a particular function. For example, the α and β polypeptides that constitute a MHC class II molecule are each recognized as having two domains, α1, α2 and β1, β2, respectively. The various domains are typically joined by linking amino acid sequences. In one embodiment, the entire domain sequence is included in a recombinant molecule by extending the sequence to include all or part of the linker or the adjacent domain. For example, when selecting the α1 domain of an MHC class II molecule, the selected sequence may extend from amino acid residue number 1 of the α chain, through the entire α1 domain to amino acid 84 at the carboxy terminus of the α1 domain. The precise number of amino acids in the various MHC molecule domains varies depending on the species of mammal, as well as between classes of genes within a species. The selection of a sequence for use in a recombinant molecule requires maintenance of the domain function rather than a precise structural definition based on the number of amino acids. One of ordinary skill in the art will appreciate that domain function may be maintained even if somewhat less than the entire amino acid sequence of the selected domain is utilized. For example, a number of amino acids at either the amino or carboxy termini of the α1 domain may be omitted without affecting domain function. The functional activity of a particular selected domain may be assessed in the context of the MHC class II polypeptides provided by this disclosure (e.g., the α1 polypeptides), for example, T cell proliferation and/or CD74 binding assays.
Effective amount: A dose or quantity of a specified compound sufficient to inhibit advancement, or to cause regression of a disease or condition, or which is capable of relieving symptoms caused by the disease or condition. For instance, this can be the amount or dose of a disclosed MHC molecule required to treat or inhibit a disorder, such as an inflammatory and/or autoimmune disorder. In one embodiment, an effective amount is the amount that alone, or together with one or more additional therapeutic agents, induces the desired response in a subject, such as treating or inhibiting an inflammatory or autoimmune disorder or other disease or disorder.
Inflammation: A localized protective response elicited by injury to tissue that serves to sequester the inflammatory agent. Inflammation is orchestrated by a complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. An inflammatory response is characterized by an accumulation of white blood cells, either systemically or locally at the site of inflammation. The inflammatory response may be measured by many methods, such as the number of white blood cells, the number of polymorphonuclear neutrophils (PMN), a measure of the degree of PMN activation, such as luminal enhanced chemiluminescence, or a measure of the amount of cytokines present.
A primary inflammation disorder is a disorder that is caused by the inflammation itself. A secondary inflammation disorder is inflammation that is the result of another disorder. Inflammation can lead to a host of inflammatory diseases, including, but not limited to rheumatoid arthritis, osteoarthritis, inflammatory lung disease (including chronic obstructive pulmonary lung disease), inflammatory bowel disease (including ulcerative colitis and Crohn's Disease), periodontal disease, polymyalgia rheumatica, atherosclerosis, systemic lupus erythematosus, systemic sclerosis, Sjogren's Syndrome, asthma, allergic rhinitis, and skin disorders (including dermatomyositis and psoriasis) and the like. Auto-immune disorders which include an inflammatory component (including, but not limited to multiple sclerosis) are also considered to be inflammatory disorders.
Inhibiting or treating a disease: “Inhibiting” a disease refers to inhibiting the full development of a disease, for example in a person who is known to have a predisposition to a disease such as an inflammatory or autoimmune disorder. Inhibition of a disease can span the spectrum from partial inhibition to substantially complete inhibition of the disease, for example in a subject who has a disease or disorder or is at risk of developing a disease or disorder. In some examples, the term “inhibiting” refers to reducing or delaying the onset or progression of a disease. “Treating” a disease refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such as a sign or symptom of an inflammatory or autoimmune disorder. A subject to be administered an effective amount of the pharmaceutical compound to inhibit or treat the disease or disorder can be identified by standard diagnosing techniques for such a disorder, for example, symptoms, basis of family history, or risk factor to develop the disease or disorder.
Linker: A molecule that covalently links two molecules (such as two polypeptides). Linkers (such as a peptide linker or a chemical linker) may be included in the recombinant MHC polypeptides of the present disclosure for example between an α1 domain and an antigenic peptide. Peptide linker sequences, which are generally between 2 and 25 amino acids in length (such as 5-10, 10-15, 15-20, or 20-25 amino acids), include, but are not limited to, the glycine(4)-serine spacer described by Chaudhary et al. (Nature 339:394-397, 1989). Similarly, chemical linkers (such as thiol bonds or crosslinking agents) can also be used.
MHC Class II: MHC Class II molecules are formed from two noncovalently associated proteins, the α chain and the β chain. The α chain comprises α1 and α2 domains, and the β chain comprises β1 and β2 domains. The cleft into which the antigen fits is formed by the interaction of the α1 and β1 domains. The α2 and β2 domains are transmembrane Ig-fold like domains that anchor the α and β chains into the cell membrane of the APC. MHC Class II complexes, when associated with antigen (and in the presence of appropriate co-stimulatory signals) stimulate CD4 T-cells. The primary functions of CD4 T-cells are to initiate the inflammatory response, to regulate other cells in the immune system, and to provide help to B cells for antibody synthesis.
Pharmaceutically acceptable carriers: Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st 30 Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the proteins herein disclosed.
Recombinant: A recombinant nucleic acid or polypeptide is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
Sequence identity: The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Polypeptides or domains thereof that have a significant amount of sequence identity and function the same or similarly to one another—for example, the same protein in different species—can be called ‘homologs.’ Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch, J. Mol. Biol. 48: 443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins & Sharp, Gene, 73: 237-244, 1988; Higgins & Sharp, Comput. Appl. Biosci. 5: 151-153, 1989; Corpet et al., Nucl. Acids Res. 16, 10881-90, 1988; Huang et al., Comput. Appl. Biosci. 8, 155-65, 1992; and Pearson, Methods Mol. Biol. 24:307-331, 1994. Altschul et al. (J. Mol. Biol. 215:403-410, 1990) presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
Nucleic acid sequences that do not show a high degree of sequence identity may nevertheless encode similar amino acid sequences, due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein.
Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals.
Disclosed herein are isolated recombinant polypeptides which include an MHC class II DRα1 domain or fragment thereof and do not include MHC class II α2, β1, or β2 domains, and include a substitution of glutamine (Q) for the leucine (L) present at the amino acid position corresponding to amino acid 14 of SEQ ID NO: 1. The amino acid sequences of mammalian MHC class II DRα chain proteins, as well as nucleic acids encoding these proteins, are well known in the art and available from numerous sources including GenBank. Exemplary sequences are provided in Das et al. (Proc. Natl. Acad. Sci. USA 80:3543-3547, 1983) (human HLA DRα) which is incorporated by reference herein. An exemplary human DRα polypeptide is GenBank Accession No. NP_061984 (incorporated herein by reference as present in GenBank on Oct. 5, 2018). Additional MHC class II DRα polypeptides can be identified by one of ordinary skill in the art, for example, from public databases, such as the IMGT/HLA database (available on the world wide web at ebi.ac.uk/imgt/hla/).
In a particular embodiment, the MHC class II α1 domain is a human HLA-DRA polypeptide. The α1 domain is well defined in mammalian MHC class II α chain proteins. In some examples, MHC class II α chains include a leader sequence involved in trafficking the polypeptide that is proteolytically removed to produce the mature α polypeptide.
An α1 domain may include about amino acid residues 1-90 of the mature α chain, but one of ordinary skill in the art will recognize that the C-terminal cut-off of this domain is not necessarily precisely defined, and, for example, might occur at any point between amino acid residues 70-100 of the mature α chain. In some examples, the α1 domain includes amino acids 1-70, 1-71, 1-72, 1-73, 1-74, 1-75, 1-76, 1-77, 1-78, 1-79, 1-80, 1-81, 1-82, 1-83, 1-84, 1-85, 1-86, 1-87, 1-88, 1-89, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99, or 1-100 of a mature MHC class II DRα domain. In other examples, an α1 domain includes about residues 20-120 (such as about residues 20-110, 24-110, 24-109, 25-100, 25-109, 26-110, 26-109, 30-120, 32-120, 32-115, 26-90, 26-85, 26-84, or other overlapping regions) of a full length DR α1 polypeptide. In some examples, the DRα1 domain does not include an N-terminal methionine; however, an N-terminal methionine can be present, for example as a result of expression in a bacterial, yeast, or mammalian system.
In further examples, the DRα1 domain can include deletion or addition of a few amino acids at the 5′- and/or 3′-end, such as addition or deletion of about 1-25 amino acids, such as addition or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids from the 5′- or 3′-end, or combinations thereof (such as a deletion from one end and an addition to the other end). The composition of the α1 domain may also vary outside of these parameters depending on the mammalian species and the particular α chain in question. One of ordinary skill in the art will appreciate that the precise numerical parameters of the amino acid sequence are less important than the maintenance of domain function (for example, CD74 binding or down-regulation).
In some embodiments, disclosed are recombinant polypeptides that include a DRα1 domain including a glutamine residue at a position corresponding to amino acid position 14 of SEQ ID NO: 1. The recombinant polypeptides do not include MHC class II α2, β1, or β2 domains. In some examples, the recombinant DRα1 polypeptide includes, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1. In additional embodiments, the recombinant polypeptide has a sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 or a fragment thereof. For example, the DRα1 polypeptide can be a homolog or ortholog of SEQ ID NO: 1 with an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 or a fragment thereof. Exemplary sequences can be obtained using computer programs that are readily available on the world wide web and the amino acid sequences set forth herein. In some examples, the homolog retains a function of the polypeptide of SEQ ID NO: 1 such as an altered secondary structure, binding to CD74, and/or inhibiting EAE in mice.
In other embodiments, disclosed are recombinant polypeptides that include a DRα1 domain or a fragment thereof including a glutamine residue at a position corresponding to amino acid position 14 of SEQ ID NO: 1 and an antigenic peptide. The recombinant polypeptides do not include MHC class II α2, β1, or β2 domains. In some examples, the recombinant polypeptide further includes a linker (for example, a linker between the DRα1 polypeptide and the antigenic peptide). The linker may be a peptide linker or a chemical linker (such as a chemical cross-linker).
In some embodiments, the antigenic peptide is human or mouse myelin oligodendrocyte glycoprotein (MOG) 35-55. Thus, in some embodiments, the recombinant polypeptide includes, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In additional embodiments, the recombinant polypeptide has a sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2 or SEQ ID NO: 3 or a fragment thereof. For example, the DRα1 portion of the polypeptide can be a homolog or ortholog of SEQ ID NO: 1 with an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 or a fragment thereof. Exemplary sequences can be obtained using computer programs that are readily available on the world wide web and the amino acid sequences set forth herein. In some examples, the polypeptide retains a function of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3 such as an altered secondary structure, binding to CD74, and/or inhibiting EAE in mice. Additional antigenic peptides that can be included in the polypeptide are discussed below.
Minor modifications of the recombinant polypeptides (e.g., SEQ ID NOs: 1-3) may result in polypeptides which have substantially equivalent activity (for example, as compared to SEQ ID NOs: 1-3). Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the polypeptides produced by these modifications are included herein. Thus, a specific, non-limiting example of an MHC class II DRα1 polypeptide is a conservative variant of any one of SEQ ID NOs: 1-3 (such as a conservative amino acid substitution, for example, one or more conservative amino acid substitutions, for example 1-10 conservative substitutions, 2-5 conservative substitutions, 4-9 conservative substitutions, such as 1, 2, 5 or 10 conservative substitutions).
Nucleic acid molecules encoding the disclosed recombinant DRα1 polypeptides (e.g., encoding any one of SEQ ID NOs: 1-3) and/or any homologs or variants thereof can be produced by standard approaches, such as amplification by the polymerase chain reaction (PCR). In some examples, the recombinant polypeptide is encoded by a nucleic acid including or consisting of the nucleic acid sequence of any one of SEQ ID NOs: 4-6. In additional embodiments, the nucleic acid has a sequence at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 4-6 or a fragment thereof.
In some embodiments, the recombinant polypeptide (e.g., SEQ ID NOs: 1-3) can be expressed in prokaryotic or eukaryotic cells from a nucleic acid construct encoding the recombinant polypeptide (such as a nucleic acid construct including any one of SEQ ID NOs: 4-6). Nucleic acid constructs (such as expression constructs) encoding the recombinant polypeptides may also include regulatory elements such as promoters, enhancers, and/or 3′ regulatory regions, the selection of which will be determined based upon the type of cell in which the protein is to be expressed. The constructs are introduced into a vector suitable for expressing the recombinant polypeptide in the selected cell type.
Numerous prokaryotic and eukaryotic systems are known for the expression and purification of polypeptides. For example, heterologous polypeptides can be produced in prokaryotic cells by placing a strong, regulated promoter and an efficient ribosome binding site upstream of the polypeptide-encoding construct. Suitable promoter sequences include the beta-lactamase, tryptophan (trp), phage T7, and lambda PL promoters. Methods and plasmid vectors for producing heterologous proteins in bacteria or mammalian cells are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); and Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999.
Suitable prokaryotic cells for expression of large amounts of proteins include Escherichia coli and Bacillus subtilis. Often, proteins expressed at high levels are found in insoluble inclusion bodies; methods for extracting proteins from these aggregates are described for example, by Sambrook et al. (2001, see chapter 15). Recombinant expression of recombinant polypeptides in prokaryotic cells may alternatively be conveniently obtained using commercial systems designed for optimal expression and purification of fusion proteins. Such fusion proteins typically include a tag that facilitates purification. Examples of such systems include: the pMAL protein fusion and purification system (New England Biolabs, Inc., Beverly, MA); the GST gene fusion system (Amersham Pharmacia Biotech, Inc., Piscataway, NJ); and the pTrcHis expression vector system (Invitrogen, Carlsbad, CA). Additional systems include the His6-tag (e.g., Roche Applied Science, Mannheim, Germany) or streptavidin binding peptide (e.g., Sigma-Aldrich, St. Louis, MO). For example, the pMAL expression system utilizes a vector that adds a maltose binding protein to the expressed protein. The fusion protein is expressed in E. coli. and the fusion protein is purified from a crude cell extract using an amylose column. If necessary, the maltose binding protein domain can be cleaved from the fusion protein by treatment with a suitable protease, such as Factor Xa. The maltose binding fragment can then be removed from the preparation by passage over a second amylose column.
The recombinant polypeptides can also be expressed in eukaryotic expression systems, including Pichia pastoris, Drosophila, Baculovirus and/or Sindbis expression systems produced by Invitrogen (Carlsbad, CA). Eukaryotic cells such as Chinese Hamster ovary (CHO), monkey kidney (COS), HeLa, Spodoptera frugiperda, and Saccharomyces cerevisiae may also be used to express recombinant polypeptides. Regulatory regions suitable for use in these cells include, for mammalian cells, viral promoters such as those from CMV, adenovirus or SV40, and for yeast cells, the promoter for 3-phosphoglycerate kinase or alcohol dehydrogenase.
The vectors can be introduced into recipient cells (such as eukaryotic cells) as pure DNA (transfection) by, for example, precipitation with calcium phosphate or strontium phosphate, electroporation, lipofection, DEAE dextran, microinjection, protoplast fusion, or microprojectile guns. Alternatively, the nucleic acid molecules can be introduced by infection with virus vectors. Systems are developed that use, for example, retroviruses, adenoviruses, or Herpes virus.
A DRα1 polypeptide (such as those described herein) produced in mammalian cells may be extracted following release of the protein into the supernatant and may be purified using an immunoaffinity column prepared using anti-MHC or other antibodies. Alternatively, the polypeptide may be expressed as a chimeric protein with, for example, β-globin. Antibody to β-globin is thereafter used to purify the chimeric protein. Corresponding protease cleavage sites engineered between the β-globin gene and the nucleic acid sequence encoding the recombinant polypeptide are then used to separate the two polypeptide fragments from one another after translation. One useful expression vector for generating β-globin chimeric proteins is pSG5 (Stratagene, La Jolla, CA).
Expression of the recombinant polypeptides in prokaryotic cells will result in polypeptides that are not glycosylated. Glycosylation of the polypeptides at naturally occurring glycosylation target sites may be achieved by expression of the polypeptides in suitable eukaryotic expression systems, such as mammalian cells. In other examples, the recombinant polypeptide can be modified (for example, by site-directed mutagenesis) to include desired post-translational modification sites such as one or more sites for N-linked glycosylation, phosphorylation, or other modifications.
Purification of the expressed protein is generally performed in a basic solution (typically around pH 10) containing 6M urea. Folding of the purified protein is then achieved by dialysis against a buffered solution at neutral pH (typically phosphate buffered saline at around pH 7.4). Other methods of protein purification are also known and can be used with the recombinant polypeptides disclosed herein.
In some embodiments, the recombinant polypeptides disclosed herein comprise antigenic peptides covalently linked to the DRα1 domain—either directly or via a peptide or chemical linker. The presentation of antigen in MHC complexes on the surface of APCs generally does not involve a whole antigenic peptide (see for example U.S. Pat. No. 5,468,481). Rather, a peptide located in the groove between the β1 and α1 domains in the case of MHC II or the α1 and α2 domains in the case of MHC1 is typically a small linear fragment of a whole polypeptide antigen. As discussed in Janeway & Travers (Immunobiology: The Immune System in Health and Disease, 1997), peptides located in the peptide groove of MHC class I molecules are constrained by the size of the binding pocket and are typically 8-15 amino acids long (such as 8, 9, 10, 11, 12, 13, 14, or 15 amino acids), more typically 8-10 amino acids in length (but see Collins et al., Nature 371:626-629, 1994 for possible exceptions). In contrast, peptides located in the peptide groove of MHC class II molecules are not constrained in this way and are often larger, typically at least 3-50 amino acids in length (such as 8-30, 10-25, or 15-23 amino acids in length). In some examples, the peptide located in the peptide groove of an MHC class II molecule is about 15-23 amino acids in length. Peptide fragments can be prepared by standard approaches, such as use of synthetic peptide synthesis machines or they can be expressed as part of a recombinant polypeptide.
In some examples, an antigenic peptide includes a peptide from a neuronal or central nervous system protein, such as a myelin protein (for example, myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), or proteolipid protein (PLP)). In specific examples the antigenic peptide includes a human or mouse MOG-35-55 peptide, exemplified by amino acids 1-21 of SEQ ID NO: 2 or 1-21 of SEQ ID NO: 3, respectively. In other examples, an antigenic peptide is a peptide from a retinal protein, such as interphotoreceptor retinoid binding protein (IRBP), arrestin, phosducin, or recoverin. Additional antigenic peptides include peptides from type II collagen (collagen II), fibrinogen-α, vimentin, α-enolase, human cartilage glycoprotein-39, a2 gliadin, or insulin. In some examples, an antigenic peptide includes a post-translational modification, such as phosphorylation, glycosylation, or citrullination. Sequences of exemplary antigenic peptides are provided in Table 1 and in International Pat. Publ. No. WO 2012/103365 and U.S. Pat. Publ. No. 2012/0276127, both of which are incorporated herein by reference in their entirety. One of ordinary skill in the art can identify additional antigenic peptides relevant to a particular disease or disorder.
In some examples, the antigenic peptide is covalently linked to the MHC class II DRα1 polypeptide by operably linking a nucleic acid sequence encoding the selected antigenic peptide to the 5′ end of the construct encoding the DRα1 polypeptide such that, in the expressed peptide, the antigenic peptide is linked to the amino-terminus of the DRα1 domain. In other examples, the antigenic peptide is covalently linked to the DRα1 polypeptide by operably linking a nucleic acid sequence encoding the selected antigen to the 3′ end of the construct encoding the DRα1 polypeptide such that, in the expressed peptide, the antigenic peptide is linked to the carboxy-terminus of the DRα1 domain. One convenient way of obtaining this result is to incorporate a sequence encoding the antigenic peptide into the PCR primers used to amplify the DRα1 domain coding region. In some examples, a sequence encoding a linker peptide sequence is included between the antigenic peptide and the DRα1 polypeptide. However, it is not necessary that the antigenic peptide be ligated exactly at the 5′ end (or 3′ end) of the MHC class II α1 domain coding region. For example, the antigenic peptide coding region may be inserted into the α1 domain within the first few (typically within the first 10) codons of the 5′ or 3′ end of the DRα1 domain coding sequence.
In some embodiments, a genetic system for linkage of the antigenic peptide to the DRα1 domain is particularly useful where a number of DRα1 domains with differing antigenic peptides are to be produced. The described system permits the construction of an expression vector in which a unique restriction site is included in the DRα1 domain (e.g., at the 5′ or 3′ end of the α1 domain). In conjunction with such a construct, a library of antigenic peptide encoding sequences is made, with each antigen-coding region flanked by sites for the selected restriction enzyme. The inclusion of a particular antigen into the DRα1 domain is then performed simply by (a) releasing the antigen-coding region with the selected restriction enzyme, (b) cleaving the DRα1 domain construct with the same restriction enzyme, and (c) ligating the antigen coding region into the DRα1 domain construct. In this manner, a large number of DRα1 domain-peptide antigen constructs can be made and expressed in a short period of time.
In some examples, the antigen is covalently linked to the DRα1 domain polypeptide by a peptide linker. In some examples, the linker includes one or more (for example, 1, 2, 3, 4, or more) glycine-serine spacers, for example GGGGS (SEQ ID NO: 52). In some examples, the linker includes two glycine-serine spacers. In a non-limiting example, the recombinant polypeptide may include a first glycine-serine spacer, a thrombin cleavage site, and a second glycine-serine spacer between the antigenic peptide and the DRα1 polypeptide.
In some examples, the antigen is covalently linked to the DRα1 domain polypeptide by a disulfide bond. In some examples, the disulfide linkage is utilizing a naturally occurring cysteine residue in the DRα1 domain polypeptide (such as a cysteine residue in the DRα1 domain). One of ordinary skill in the art can identify a suitable cysteine residue in a DRα1 domain polypeptide. In other examples, the disulfide linkage is formed utilizing a non-naturally occurring cysteine residue in the DRα1 domain polypeptide, such as a cysteine residue introduced in the DRα1 domain polypeptide by mutagenesis. In further examples, the disulfide linkage is formed utilizing a naturally occurring cysteine residue in the peptide antigen. In still further examples, the disulfide linkage is formed utilizing a non-naturally occurring cysteine residue in the peptide antigen, such as a cysteine residue introduced in the peptide antigen by mutagenesis.
Disclosed herein are methods of treating or inhibiting disorders in a subject, including but not limited to inflammatory and/or autoimmune disorders. The disclosed methods include administering to a subject a recombinant polypeptide comprising an MHC class II DRα1 domain polypeptide with a glutamine residue at a position corresponding to amino acid position 14 of SEQ ID NO: 1 (exemplified by SEQ ID NO: 1) or the recombinant polypeptide comprising MHC class II DRα1 domain polypeptide with a glutamine residue at a position corresponding to amino acid position 14 of SEQ ID NO: 1 covalently linked to an antigenic peptide (exemplified by SEQ ID NOs: 2 and 3) or a nucleic acid encoding the recombinant polypeptide. In one non-limiting example, the subject has multiple sclerosis and is administered an MHC class II DRα1 domain polypeptide with a glutamine residue at a position corresponding to amino acid position 14 of SEQ ID NO: 1 linked to a MOG-35-55 peptide (such as SEQ ID NO: 2 or SEQ ID NO: 3).
In some embodiments, the methods include selecting a subject with a disorder for treatment and administering an effective amount of the recombinant polypeptide described herein, a nucleic acid encoding the recombinant polypeptide described herein, or a pharmaceutical composition including a disclosed recombinant polypeptide or nucleic acid to the subject.
In some embodiments, the subject has an inflammatory and/or autoimmune disease or disorder, including but not limited to, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, type I diabetes mellitus, Wegener's granulomatosis, inflammatory bowel disease, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, celiac disease, Addison's disease, adrenalitis, Graves' disease, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, multiple sclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, myasthenia gravis, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult onset diabetes mellitus (Type II diabetes), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, Crohn's disease, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, juvenile onset rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, allergic disease, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, glomerulonephritis, graft versus host disease, transplantation rejection, human immunodeficiency virus infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post vaccination syndromes, congenital rubella infection, Hodgkin's and Non-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenstrom's macroglobulemia, Epstein-Barr virus infection, rubulavirus, and Evan's syndrome.
Additional inflammatory diseases include osteoarthritis, inflammatory lung disease (including chronic obstructive pulmonary lung disease), periodontal disease, polymyalgia rheumatica, atherosclerosis, systemic sclerosis, allergic rhinitis, and skin disorders (including dermatomyositis and psoriasis) and the like.
In other embodiments, the subject has a retinal disorder, such as a retinal degeneration, such as retinitis pigmentosa, cone-rod dystrophy, Leber congenital amaurosis, or a maculopathy (for example, age-related macular degeneration, Stargardt-like macular degeneration, vitelliform macular dystrophy (Best disease), Malattia Leventinese (Doyne's honeycomb retinal dystrophy), diabetic maculopathy, occult macular dystrophy, and cellophane maculopathy). In other examples, a retinal disorder includes a retinopathy, such as autoimmune retinopathy, diabetic retinopathy, or vascular retinopathy. In still further examples, a retinal disorder includes retinal detachment or glaucoma. Retinal disorders may be progressive (for example, retinal degeneration or glaucoma) or acute (for example, retinal detachment). In additional examples, the subject is a subject with uveitis or optic neuritis. In other embodiments, the subject has had a stroke (such as ischemic stroke or hemorrhagic stroke). In still further examples, the subject is a subject with substance addiction, for example, a subject with cognitive or neuropsychiatric impairment induced by substance addiction including methamphetamine and alcohol abuse.
In some embodiments, a subject is administered an effective amount of a composition including an DRα1 domain or a portion thereof (such as a portion of an α1 domain which is capable of binding CD74 or decreasing expression and/or activity of CD74) that includes a substitution of glutamine for leucine at a position corresponding to amino acid position 14 of SEQ ID NO: 1. In one example, the composition includes modified DRα1-MOG-35-55 (for example, SEQ ID NO: 2 or SEQ ID NO: 3).
Pharmaceutical compositions that include a recombinant polypeptide or nucleic acid disclosed herein (such as an effective amount of a disclosed recombinant polypeptide or nucleic acid) can be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. The pharmaceutically acceptable carriers and excipients useful in this disclosure include those known to one of ordinary skill in the art. See, e.g., Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005). For instance, parenteral formulations usually include injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate. Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations. The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, topical, inhalation, oral and suppository formulations can be employed. Topical preparations can include eye drops, ointments, sprays, patches and the like. Inhalation preparations can be liquid (e.g., solutions or suspensions) and include mists, sprays and the like. Oral formulations can be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powders, pills, tablets, or capsules). Suppository preparations can also be solid, gel, or in a suspension form. For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.
In some examples, the pharmaceutical composition may be administered by any mode that achieves its intended purpose. Amounts and regimens for the administration of the recombinant polypeptides or portion thereof (or a nucleic acid encoding such polypeptides) can be determined by the attending clinician. Effective doses for therapeutic application will vary depending on the nature and severity of the condition to be treated, the particular DRα1 domain or portion thereof and/or antigenic peptide selected, the age and condition of the patient, and other clinical factors. Typically, the dose range will be from about 0.1 μg/kg body weight to about 100 mg/kg body weight. Other suitable ranges include doses of from about 100 μg/kg to about 50 mg/kg body weight, about 500 μg/kg to about 10 mg/kg body weight, or about 1 mg/kg to about 5 mg/kg body weight, for example, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, or about 5 mg/kg. The dosing schedule may vary from once a month to daily depending on a number of clinical factors, such as the subject's sensitivity to the protein. Examples of dosing schedules are about 1 mg/kg administered once a month, bi-weekly, once a week, twice a week, three times a week or daily; a dose of about 2.5 mg/kg once a week, twice a week, three times a week or daily; a dose of about 5 mg/kg once a week, twice a week, three times a week or daily; a dose of about 10 mg/kg once a week, twice a week, three times a week or daily; or a dose of about 30 mg/kg once a week, twice a week, three times a week or daily.
The pharmaceutical compositions that include one or more of the disclosed recombinant polypeptides can be formulated in unit dosage form, suitable for individual administration of precise dosages. In one specific, non-limiting example, a unit dosage can contain from about 1 ng to about 5 g of the recombinant polypeptide (such as about 10 μg to 1 g, about 100 mg to 500 mg, or about 10 mg to 100 mg). The amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated.
The recombinant polypeptides can be administered to humans or other animals on whose tissues they are effective in various manners such as topically, orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, subcutaneously, intraocularly, via inhalation, or via suppository. In one example, the compounds are administered to the subject subcutaneously. In another example, the compounds are administered to the subject intravenously.
In some embodiments, the recombinant polypeptides can be included in an inert matrix for topical or local application. In some examples, the formulation can be injected into the eye, for example for intravitreal injection. As one example of an inert matrix, liposomes may be prepared from dipalmitoyl phosphatidylcholine (DPPC), such as egg phosphatidylcholine (PC). Liposomes, including cationic and anionic liposomes, can be made using standard procedures as known to one skilled in the art. Liposomes including one or more recombinant polypeptides can be applied topically, either in the form of drops or as an aqueous based cream, or can be injected intraocularly. In a formulation for topical application, the recombinant polypeptide is slowly released over time as the liposome capsule degrades due to wear and tear from the eye surface. In a formulation for intraocular injection, the liposome capsule degrades due to cellular digestion. Both of these formulations provide advantages of a slow release drug delivery system, allowing the subject to be exposed to a substantially constant concentration of the recombinant polypeptide over time. In one example, the DRα1 polypeptide can be dissolved in an organic solvent such as DMSO or alcohol and contain a polyanhydride, poly(glycolic) acid, poly(lactic) acid, or polycaprolactone polymer. The recombinant polypeptide can be included in a delivery system that can be implanted at various sites in the eye, depending on the size, shape and formulation of the implant, and the type of transplant procedure. Suitable sites include but are not limited to the anterior chamber, anterior segment, posterior chamber, posterior segment, vitreous cavity, suprachoroidal space, subconjunctiva, episcleral, intracorneal, epicorneal and sclera.
In some examples, an effective amount (for example, a therapeutically effective amount) of a disclosed recombinant polypeptide can be the amount of recombinant polypeptide necessary to treat or inhibit a disorder (such as an inflammatory and/or autoimmune disorder) in a subject. In other examples, a therapeutically effective amount of a disclosed recombinant polypeptide can be the amount of recombinant polypeptide necessary to treat or inhibit a retinal disorder, stroke, traumatic brain injury, or disorders associated with substance addiction (such as cognitive or neuropsychiatric impairment resulting from substance addiction).
The present disclosure also includes combinations of one or more of the disclosed recombinant polypeptides with one or more other agents useful in the treatment of a disorder. In some examples, the recombinant polypeptides can be administered with effective doses of one or more therapies for inflammatory or autoimmune disorders, including but not limited to non-steroidal anti-inflammatory drugs, corticosteroids, methotrexate, anti-TNF compounds, mycophenolate, aminosalicylates, antibiotics, interferons, glatiramer acetate, antibody therapies (such as rituximab or milatuzumab), or immunosuppressant or immunomodulator compounds. In another example, the recombinant polypeptides can be administered in combination with effective doses of one or more therapies for retinal disorders, including but not limited to, gene therapy, vitamin or mineral supplements (such as vitamins A, C, and/or E, or zinc and/or copper), anti-angiogenic therapy (such as ranibizumab or bevacizumab), photocoagulation, photodynamic therapy, lutein or zeaxanthin, corticosteroids, or immunosuppressants. Appropriate combination therapy for a particular disease can be selected by one of ordinary skill in the art. The term “administration in combination” or “co-administration” refers to both concurrent and sequential administration of the active agents.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
Fab, antibodies and other reagents. G4 is a human Fab reactive to the DRα1 domain derived from a human IgG library and was a kind donation from Dr. Yoram Reiter, Technion Israel. Anti-human MOG antibody was purchased from Santa Cruz Biotechnology. CHAPS, T20 and bovine serum albumin were purchased from Sigma-Aldrich. Anti-CD74 antibody was purchased from Everest Biotech. UltraPure™ TRIS was purchased from Invitrogen. Vector pET21d(+) was purchased from Novagen. BL21 (DE3) was purchased from New England Biolabs. IPTG was purchased from Inalco.
DRα1 constructs cloning, expression and purification. The DRα1 construct purification has been reported (Vandenbark et al., J. Immunol. 171:127-133, 2003). Briefly, a synthetic DNA fragment containing a sequence encoding the human MOG-35-55 peptide, a flexible linker, and the MHC Class II DRα1 domain from amino acid residues 15 through 97 and a similar synthetic DNA fragment containing the mutation L50Q in the DRα1 domain were cloned into the high level expression vector pET21d(+) (Novagen). These clones were transformed into E. coli strain BL21 (DE3) (New England Biolabs), plated onto LB-agar plates containing 50 μg/ml of the antibiotic carbenicillin, and incubated overnight at 37° C. The next day three individual colonies from each clone were selected and grown in LB broth supplemented with the antibiotic to test IPTG-induced production of the protein of interest. After confirmation, a 100 ml overnight culture was prepared and used to inoculate a 4×1 L flask with LB supplemented with the antibiotic. Induction of the target protein synthesis was performed with the addition at the logarithmic growth point of IPTG to a final concentration of 2 mM. The cultures were incubated for an additional 4 hours at 37° C., harvested by centrifugation, and the bacterial paste was frozen at −80° C. until use.
Pellets were resuspended in sonication buffer (50 mM Tris, 300 mM NaCl, 2 mM EDTA pH 8.0) and sonicated to disrupt the cells and release the inclusion bodies. The protein contained in these inclusion bodies were solubilized in a 20 mM Ethanolamine, 6 M Urea, pH 10 buffer overnight at 4° C. with gentle stirring. Purification was achieved by passing the solubilized protein through an anion exchange column and eluting the protein with a NaCl gradient in solubilization buffer. Fractions were collected and analyzed by SDS-PAGE. Those fractions containing the protein of interest were pooled together, dialyzed against 20 mM Tris, pH 8.5, concentrated to 5 mg/ml, and flash frozen and then stored at −80° C. in 1 ml aliquots until use.
Amino acid sequence alignment. All the different Class II α1 domain sequences of interest were retrieved from the NCBI and aligned using the BLAST Two or More sequences from the BLAST website (NIH) and then optimized manually to show relevant regions.
Structural analysis of the proteins by circular dichroism. Proteins were thawed and analyzed by SDS-PAGE (data not shown) prior to testing for their secondary structure content by absorbance of far UV light (180-260 nm) using an AVIV spectrometer. Proteins were greater than 95% pure. One hundred microliters of each of the polypeptides were prepared in 20 mM Tris buffer pH 8.5 at 1 mg/ml concentration and placed in a CD cuvette. Protein was scanned for absorbance in the far UV spectrum from 180 nm to 260 nm through a 0.1 mm light path length taking measurements at every 0.5 nm intervals. A sample containing only 20 mM Tris buffer was also scanned and the signal was subtracted from the protein readings. At least three scans were averaged for each protein and plotted as molar ellipticity.
Binding of DRα1 constructs to CD74 and competition assays. These experiments were performed by ELISA using Maxisorp plates (Nunc) as follows. Prior to the binding and competition experiments the proteins were labeled with Alexa Fluor 488 (Invitrogen) or biotin (Pierce Biotechnology) that targets lysine side chain primary amines. Unconjugated Alexa Fluor 488 or biotin were removed by size exclusion chromatography using a Superdex 75 10/300 column (GE Healthcare). For binding experiments, a recombinant human CD74 (rhCD74) construct was produced (data not shown) and the design, production and purification has already been described (Benedek et al., Proc. Natl. Acad. Sci. USA 114:E8421-E8429, 2017). Plates were coated with rhCD74 (C27S) in TBS at a concentration of 0.1 μg/ml for 2 hours at RT or overnight at 4° C. After blocking with 5% BSA in TBS and 0.0125% Tween 20 (T20), DRα1-MOG constructs prepared in blocking buffer with 0.0125% T20 were captured for 3 hours at room temperature followed by detection with streptavidin conjugated to horseradish peroxidase (HRP). Data were loaded onto Prism software and fitted to a one or two binding site equation in order to determine relative affinity.
For competition experiments, ELISA plates were coated with MIF at room temperature at a concentration of 0.5 μg/ml in TBS. Plates were then blocked overnight with 5% BSA in TBS and 0.0125% T20 followed by the addition of a competitive mix containing rhCD74 with serially diluted DRα1 constructs prepared in 5% BSA/TBS plus 0.0125% T20 for 1.5 hours at 25° C. Bound rhCD74 was detected with a monoclonal antibody that specifically recognizes human CD74 in a region away from the putative MIF/D-DT binding site. Absorbance at 450 nm was determined and data were analyzed with Prism, the parameters were calculated with the competition equation included in the software. For competition between DRα1-hMOG-35-55 and P2 peptide (see next section), mouse CD74 was immunoprecipitated from DR*1501 Tg mouse splenocytes and a competition experiment was set. 2.5 μl of P2 peptide were used with increasing concentrations of DRα1-hMOG-35-55 protein. Bound peptide was eluted from the immunoprecipitation with 2% SDS and analyzed in a 16.5% PAG Tris/Tricine. The gel was scanned for FITC and fluorescence was quantified by densitometry. P2 fluorescence vs. DRα1 competitor was plotted and data analyzed with Prism. The relative density values were fit to a One-binding site equation.
Mapping of the CD74-binding epitope on DRα1-hMOG-35-55. A Fab that is able to bind the DRα1-MOG proteins and their parent molecule, RTL1000, with high affinity was previously described (Meza-Romero et al., J. Immunol. 192:4164-4173, 2014). However, its binding region on the DRα1 domain has not yet been determined. In order to map the epitope for the G4 Fab, a series of 7 overlapping N-terminus-FITC labeled peptides covering the full length of the DRα1 domain were synthesized. These peptides were tested for their ability to bind In1-immunoprecipitated mouse CD74 from splenocytes and immuno-adsorbed G4 to Protein L beads.
Immunoprecipitation and western blot experiments. After immuno-precipitation, individual peptides were analyzed in a binding experiment for 14-16 hours in 0.1% CHAPS/TEN buffer (50 mM Tris, 2 mM EDTA, 150 mM NaCl, pH 7.4). Immune complexes were eluted with 50 μl of 2% SDS/ESB (electrophoresis sample buffer) for 20 minutes and then analyzed by electrophoresis in a 10-20% SDS-PAGE under non-reducing conditions. After electrophoresis, the gel was scanned for FITC-labeled peptides. In a parallel experiment immunoprecipitated mouse H2M was used to test the binding of the peptide set. H2M molecules bind the Class II α1 domain but in different regions, therefore they should show a different selectivity for the α1 domain synthetic peptides during binding. We also tested the possibility of G4 and CD74 recognizing the same peptide set since G4 blocks binding of DRα1 constructs to rhCD74. In that regard, G4 Fab was bound to Protein L beads and then either P2 or P7 peptides were applied to the immune complexes. Bound material was eluted as described above, analyzed by electrophoresis and the gel scanned for FITC-labeled material. Western blot experiments were carried out using standard techniques of protein transfer to PVDF (pore size 0.1 μm), followed by blocking with 5% BSA in TBS and 0.05% T20.
ERK1/2 phosphorylation blockade. Two million splenocytes from EAE mice were treated in vitro at 37° C. with 20 mM Tris pH 8.5, 25 μg of DRhQ or DRα1-hMOG-35-55 for 30 minutes. Cells were then spun down and lysed with RIPA buffer supplemented with protease and phosphate inhibitors. Cell lysis was allowed to proceed for 30 minutes on ice and the debris containing nuclei and organelles removed by centrifugation at 14,000 rpm at 4° C. for 10 minutes. Supernatants were collected and subjected to SDS-PAGE in 10-20% gradient gels under reducing conditions. After electrophoresis, proteins were transferred to PVDF and membrane was probed first for p-ERK1/2, stripped and then probed with anti-total ERK1/2 antibody.
EAE induction. C57BL/6 WT male mice between 8 and 12 weeks of age were purchased from the Jackson Laboratory. All procedures were approved and performed according to federal, state, and institutional guidelines. Mice were immunized subcutaneously at four sites on the flanks to distribute 0.2 ml of an emulsion of 200 μg mouse MOG-35-55 peptide and complete Freund's adjuvant containing 400 μg of heat-killed Mycobacterium tuberculosis H37RA (Difco) (Meza-Romero et al., J. Immunol. 192:4164-4183, 2014). In addition, mice were injected intraperitoneally with Pertussis toxin (Ptx) from List Biological Laboratories on days 0 and 2 post-immunization (75 and 200 ng per mouse, respectively). DRα1-hMOG-35-55, DRbQ, DRα1-mMOG-35-55 and DRmQ proteins (100 μg in 0.1 ml) were injected s.c. daily for 5 days beginning at an EAE score of ≥2.0 and the mice were scored for clinical signs of EAE graded on a six-point scale of combined hindlimb and forelimb paralysis scores as described before (Meza-Romero et al., J. Immunol. 192:4164-4183, 2014). Mean EAE scores and SDs for mouse groups were calculated for each day from day 8 through day 27 post-immunization and summed for each mouse by numerically integrating the EAE score curve over the entire experiment (CDI, represents total disease load).
Data analysis. Statistical analyses comparing EAE severity data and equation fitting data for binding and competition results were computed using a Prism software package (GraphPad).
The production of DRα1-hMOG-35-55, DRα1-mMOG-35-55, DRhQ, and DRmQ proteins were comparable, indicating that the L50Q substitution did not affect the transcription and the expression rate. The protein yield was consistently ˜90 to 100 mg/liter of LB broth. Fab G4 was used to detect the purified proteins. In previous studies, it was shown that Fab G4 detected the DRα1 domain in different contexts, including as a stand-alone domain, as part of larger constructs (like the ones described here), or as part of a two-domain recombinant protein (like RTL1000).
An analysis of the alignment of several human Class II α1 domains (
In order to explore the functional role of L18 vs. Q18, binding assays in ELISA experiments were carried out using several previously constructed RTLs from mouse and human origin.
Human Fab G4 (that recognizes the DRα1 domain) was used as a tool to determine whether the L50Q substitution affected the immunological recognition of the DRα1 constructs. The Fab G4 cross-reacted with the DRhQ mutant in the same manner as it did with the DRα1-hMOG-35-55 as well as the DRα1-mMOG-35-55 (
Seven overlapping peptides spanning the whole length of the DRα1 protein were designed (
In order to test the usefulness of this strategy the experiment was also run with immuno-precipitated H2M. It was hypothesized that a different peptide (or set of peptides) would bind to H2M. As seen in
In previous publications, it has been demonstrated that the G4 Fab was effective to block DRα1 construct binding to CD74 (Meza-Romero et al., J. Immunol. 192:4164-4173, 2014). Therefore, an experiment aimed to determine whether P2 also interacted with Protein L bound G4 Fab and compare the binding to immunoprecipitated CD74 was carried out. Potentially, the P7 peptide would not bind to G4 or CD74. As expected only the P2 peptide was able to bind G4, demonstrating that G4 and CD74 have the same binding site on DRα1 polypeptides (
DRα1-MOGs, including DRα1-hMOG-35-55, DRhQ, DRα1-mMOG-35-55, and DRmQ were evaluated for binding to their receptor. A recombinant human CD74(C27S) was coated onto the ELISA plate and then, following blocking, DRα1 constructs were captured for 1 to 1.5 hours at RT and detected with an anti-MOG antibody. The results were entered into the Prism software and the KD calculated fitting the curve to a one-site specific binding equation. As shown in
The DRhQ mutant bound with between 8- to 10-fold higher affinity to the receptor when compared to the counterpart wild type version of the constructs (see Table 2 with KD). This confirms that the region containing L50 is likely to be part of the binding site of DRα constructs to CD74, as suggested by the docking model of the DRα/CD74 interaction (Meza-Romero et al., Metab. Brain Dis. 31:249-255, 2016; Wingerchuk et al., Mayo Clin. Proc. 89:225-240, 2014) and the experiments with the P2 peptide discussed above. Competition assays were consistent with the direct binding results (
As shown in
Studies have shown that MIF is the main pro-inflammatory cytokine driving the ERK1/2 phosphorylation through the interaction with CD74/CD44 on the cell surface (Leng et al., J. Exp. Med. 197:1467-1476, 2003). Splenocytes harvested from EAE mice showed upregulated levels of p-ERK1/2, indicating an ongoing active signaling cascade associated with the inflammatory process. Upon treatment with the DRα1-MOG constructs this phosphorylation was downregulated in vitro after a 30 minute incubation. This suggests that treating splenocytes from an animal with an ongoing inflammatory reaction with the constructs resulted in down regulation of ERK1/2 phosphorylation (
In the crystal structures of several human and mouse Class II molecules deposited in the Protein Data Bank, the Q amino acid residue locates at the end of the β-strand 1 in the loop between the β-strand 1 and the β-strand 2 at the N-terminus of the domain (
Earlier experimental studies suggested that in addition to DRα1-MOG-35-55 (Benedek et al., Eur. J. Immunol. 43:1309-1321, 2013), RTL1000 was also able to bind to and block MIF binding to CD74 (Benedek et al., Proc. Natl. Acad. Sci. USA 114:E8421-E8429, 2017). By using a set of overlapping peptides spanning the DRα1 domain amino acid sequence, the major binding site was narrowed down to the N-terminus of the DRα1 domain (
We also tested this novel variant for its binding to a recombinant version of human CD74 and its activity to prevent or outcompete MIF from binding to their receptor. Our results indicate that the Q-harboring variant showed a KD 8-10-fold higher affinity for CD74 in direct binding ELISA assays, in sharp contrast to the parent molecule, suggesting that the Q at this position favorably influenced the interaction (
In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
This application is the § 371 U.S. National Stage of International Application No. PCT/US2019/054850, filed Oct. 4, 2019, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Application No. 62/741,941, filed Oct. 5, 2018, which is incorporated herein by reference in its entirety.
This invention was made with government support under AI122574 awarded by The National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/054850 | 10/4/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2020/072992 | 4/9/2020 | WO | A |
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| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20210380660 A1 | Dec 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62741941 | Oct 2018 | US |
