Patent Application
20220280580
The present disclosure relates generally to therapeutic compositions including recombinant bacteria, such as recombinant lactic acid bacteria, and methods of using the recombinant bacteria and therapeutic compositions containing them to treat autoimmune and inflammatory disease. Further taught herein are dietary supplements and food additive compositions including the herein described recombinant bacteria.
Autoimmune diseases are characterized by the body's immune responses being directed against its own tissues, causing prolonged inflammation and subsequent tissue destruction. For instance, autoimmune disorders can cause immune-responsive cells to attack the linings of the joints or trigger immune cells to attack the insulin-producing islet cells of the pancreas, leading to rheumatoid arthritis and insulin-dependent diabetes mellitus respectively.
In contrast, a healthy immune system recognizes, identifies, remembers, attacks, and destroys bacteria, viruses, fungi, parasites, cancer cells, or any health-damaging agents not normally present in the body. A defective immune system, on the other hand, wreaks havoc throughout the host by directing antibodies against its own tissues as well as cell-mediated immune responses.
Generally, a disease in which cytotoxic cells and antibodies are directed against self-antigens in the body's tissues is considered autoimmune in nature. Such diseases include celiac disease, Crohn's disease, pancreatitis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto's thyroiditis, and other endocrinopathies. Allergies and multiple sclerosis are also the result of disordered immune functioning.
Rheumatoid arthritis (RA) is a chronic, inflammatory, systemic autoimmune disease that affects about 0.24% of the worldwide population, and 1% of the general population in Western countries. RA is two to three times more common in women than in men (Hunter et al., Rheumatol Int. 37(9):1551-1557, 2017; Cross et al., Ann. Rheum. Dis. 1316-1322, 2014; Barbour et al., MMWR Morb. Mortal. Wkly. Rep. 66: 246-253, 2017; Carmona et al., Best Pract. Res. Clin. Rheumatol. 24: 733-745. 2010; Scott et al., Lancet 376: 1094-108, 2012). RA manifests as a chronic synovitis and progressive destruction of the joints, leukocyte infiltrates, and cartilage destruction and bone erosion. Approximately half of the afflicted patients become disabled over the progression of this disease (de Lange-Brokaar et al., Osteoarthritis Cartilage 12: 1484-499, 2012). RA is mediated predominantly by CD4+ T cells overexpressing proinflammatory cytokines, particularly in the joints (Jung et al., J Immunol Res. 2014:263625. doi: 10.1155/2014/263625).
In order to test the efficacy of potential RA therapeutics and understand mechanisms of disease, the collagen-induced arthritis (CIA) model is often used (Hegen et al., Ann Rheum Dis. 67(11):1505-15, 2008). CIA is typically induced by immunizing rodents with bovine or chick type II collagen together with an adjuvant. This combination leads to immune attack of the host's native collagen involving components of both the innate and adaptive immune systems. Emphasis on regulating proinflammatory cytokines, particularly TNF-α, is key to minimizing disease since TNF-α can be detected in joints of RA patients (Saxne et al., Arithitis Rheum. 31:1041-1045, 2018; Firestein et al., J. Immunol. 144:3347-3353, 1990). Mouse CIA shares several clinical, histopathological and immunological features with human RA. Clinical features include erythema, edema, synovitis, pannus formation, and CD4+ T cell-mediated inflammation with extensive cartilage and bone damage, resulting in joint deformities (Brand et al., Methods Mol. Med. 102: 295-312, 2004; Kannan et al., Pathophysiology 12: 167-181, 2005; Hu et al., Clin. Rheumatol. 32: 161-165, 2013). These similarities are commonly exploited to use CIA as a model for RA and as a tool to investigate novel approaches to prevent and treat RA. Current treatments focus on neutralizing TNF-α action via anti-TNF-α mAbs and TNF-α receptor antagonists (Kourbeti et al., Clin Infect Dis. 58(12):1649-57, 2014; Rosenblum & Amita, Autoimmun. Rev. 10: 563-568, 2011); however, such interventions have been problematic, making patients more susceptible to opportunistic infections (Kourbeti et al., Clin Infect Dis. 58(12):1649-57, 2014; Rosenblum & Amita, Autoimmun. Rev. 10: 563-568, 20111 Keyser Curr. Rheumatol. Rev. 7: 77-87, 2011). Hence, alternatives that can restore tolerance need to be sought.
In view of reducing autoimmunity, the use of probiotics can restore immune homeostasis to reduce autoimmunity (Yamashita et al., Front Microbiol. 8:2596, 2018; Schorpion & Kolasinski, Curr Rheumatol Rep. 19(11):73, 2017; Richards et al., Clin Transl Immunology 5(5):e82, 2016). Historically, lactic acid bacteria (LAB) represented the core of probiotic-based interventions, although more recently nonpathogenic E. coli (Kandasamy et al., Front Immunol. 8:334, 2017; Sonnenborn, FEMS Microbiol Lett. 363(19), 2016; et al., Appl Microbiol Biotechnol. 100(20):8693-9, 2016), attenuated Salmonella (Galen & Curtiss, Vaccine 32(35):4376-85, 2014; Clark-Curtiss & Curtiss, J Immunol. 200(1):39-48, 2018), Bifidobacterium spp. (Kirmiz et al., Annu Rev Food Sci Technol. 9:429-450, 2018), and some yeasts like Saccharomyces boulardii (More & Vandenplas, Clin Med Insights Gastroenterol. 11:1179552217752679, 2018) also proved to be valuable tools as novel therapeutic and prophylactic interventions. Traditional molecular genetics, coupled with synthetic biology, provides an ample selection of promoters and terminators resulting in dynamic expression levels. Protein synthesis can be induced in vitro under nisin controlled expression (NICE), or use a promoter that is silent during in vitro culture, and only active in vivo subsequent infection of the host (Mays & Nair, Curr Opin Biotechnol. 53:224-231, 2018; Kong et al., Nucleic Acids Res. 45(2):1005-1014, 2017; Desmond et al., Appl Environ Microbiol. 70(10):5929-36, 2004). LABs are considered ideal vectors for oral or mucosal delivery since they are inherently nonpathogenic, and they can survive the harsh conditions of the gastric environment. LABs are amenable to recombinant expression of passenger antigens (Ags) to stimulate immunity against a number of pathogens (Mansour & Abdelaziz, Microbiol Immunol. 60(8):527-32, 2016; O'Flaherty & Klaenhammer, Appl Environ Microbiol. 82(20):6091-6101, 2016; Li et al., Oncol Lett. 7(2):576-582, 201), to curb the effects of inflammatory bowel disease (Carvalho et al., Front Microbiol. 8:800, 2017; Del Carmen et al., Appl Environ Microbiol. 80(3):869-77, 2014), to control the proliferation of cancer cells (Zhang et al., Microb Cell Fact. 15(1):102, 2016), and to use for enzyme replacement therapy (Durrer et al., PLoS One. 12(5):e0176286, 2017) among other applications (Mays & Nair, Curr Opin Biotechnol. 53:224-231, 2018; Shigemori & Shimosato, Front Immunol. 8:22, 2017; Cano-Garrido et al., Microb Cell Fact. 14:137, 2015). Currently, the only microbiota-based therapy that is FDA-approved and commercially available is fecal microbiota transplant (FMT) to treat Clostridium difficile infections. However, close to 200 microbiome-based therapeutics and diagnostics are currently in development (Microbiome Therapeutics and Diagnostics Market, 2017-2030. Available online at researchandmarkets.com/research/ls8jkv/microbiome).
The delivery of oral therapeutics represents a significant advantage of adapting LABs. In this context, recombinant Lactococcus lactis (LL) has been developed for oral delivery to treat autoimmune disease (Maddaloni et al., PLoS One. 10(1):e0117825, 2015; U.S. Pat. No. 9,452,205). In a similar fashion, the studies described here focus on the expression of the immunosuppressive cytokine, IL-35. Oral administration of probiotic-based therapeutics is considered ideal because the gastrointestinal (GI) tract is home to T cells that can be stimulated to become Tregs and to seed other mucosal and systemic immune compartments. Another advantage of using genetically modified (GM) probiotics is that these have been shown to be both effective and safe (Shigemori & Shimosato, Front Immunol. 8:22, 2017; McLean et al., Inflamm Bowel Dis. 23(11):1983-1995, 2017; Braat et al., Clin Gastroenterol Hepatol. 4(6):754-9, 2006; Steidler et al., Nat Biotechnol. 21(7):785-9, 2003). Previous work has shown that an engineered LL derived from an industrial dairy strain can ferment commercial milk to a yogurt-like product, and when applied for treatment of CIA, can maintain the same therapeutic properties as when grown on a synthetic medium (Maddaloni et al., PLoS One. 10(1):e0117825, 2015). See also U.S. Pat. No. 9,452,205.
IL-35 belongs to the IL-12 cytokine family (reviewed in Huang et al., J Inflamm (Lond). 14:16, 2017). This heterodimeric cytokine is composed of IL-12p35 and IL-27EBI3 and, in contrast to most members of the IL-12 family, has potent anti-inflammatory attributes. This property is mediated via IL-35 binding both IL-12Rβ2 chain and gp130, which results in specific triggering of STAT1 and STAT4 on T cells (Collison et al., Nat Immunol. 13(3):290-9, 2012) and IL-12Rβ2 and IL-27Rβ on B cells (Wang et al., Nat Med. 20(6):633-41, 2014). IL-35 is immunosuppressive for a number of autoimmune disease models including CIA (Niedbala et al., Eur J Immunol. 37(11):3021-9, 2007; Kochetkova et al., J Immunol. 184(12):7144-53, 2010), experimental autoimmune encephalomyelitis (Collison et al., Nat Immunol. 11(12):1093-101, 2010; Tirotta et al., J Neuroimmunol. 254(1-2):110-6, 2013), uveitis (Wang et al., Nat Med. 20(6):633-41, 2014), type 1 diabetes (Singh et al., Sci Rep. 5:12633, 2015), inflammatory bowel disease (IBD0, and psoriasis (Wang et al., J Cell Mol Med. 22(2):1014-1025, 2018).
CCR6 was previously shown to be expressed by Tregs (Kleinewietfeld et al., Blood 105(7):2877-86, 2005), particularly those expressing RoRyt (Rivino et al., J Exp Med. 207(3):565-77, 2010; Li et al., Int J Clin Exp Med. 8(9):15043-53. 2015). These Tregs have been shown involved in suppressing autoimmune diseases (Smelt et al., PLoS One 7(10):e47244, 2012; Huibregtse et al., Gastroenterology 133(2):517-28, 2007; Borsellino et al., Blood 110: 1225-1232, 2007). CCR6+ Tregs have been found more commonly associated with human Tregs (Li et al., Int J Clin Exp Med. 8(9):15043-53. 2015; Godefroy et al., Gastroenterology pii: S0016-5085(18)34720-6, 2018), but CCR6 has also been found to be induced in mice subjected to CIA (Li et al., Int J Clin Exp Med. 8(9):15043-53, 2015).
Therapeutic agents capable of preventing and/or reversing chronic inflammation is of particular interest to the medical community. Thus, the development of such a therapeutic is urgently needed in the art.
Furthermore, there is a need in the art for dietary supplements and food additives including elements that are beneficial to a subject's immune response.
Described herein is the surprising discovery that mammalian IL-35 can be expressed in Gram-positive, Lactococcal bacteria. These live bacteria provide an effective vector for delivery of functional IL-35, for instance for treatment of diseases such as autoimmune diseases and disorders.
The disclosure therefore presents therapeutic compositions including recombinant Gram-positive bacteria expressing mammalian IL-35 that are beneficial for treating an autoimmune disease or disorder.
Furthermore, the present therapeutic compositions including the recombinant Gram-positive bacteria expressing mammalian IL-35 are beneficial for treating an inflammatory disease or disorder.
Products produced by the recombinant bacteria taught herein provide beneficial properties for the treatment of autoimmune and inflammatory diseases. That is, the protein sequences expressed by the described recombinant bacteria are demonstrated to be beneficial for the prevention and/or treatment of autoimmune and inflammatory diseases, and the amelioration of symptoms linked to such disease.
In particular embodiments, the recombinant Gram-positive bacteria expressing the mammalian IL-35 belong to the lactic acid bacterial clade. Some embodiments utilize members of the Order Lactobacillales as the recombinant bacterial host for the engineered, single-chain mammalian IL-35 gene. In some embodiments, the order of the subunits is IL-12α (p35) then IL-27β (EBI3); in others, the order is reversed—IL-27β (EBI3) then IL-12α (p35). Optionally, there is a rigid or a flexible linker between the two subunits.
Yet other embodiments employ members of the Family Streptococcaceae as the recombinant bacterial host for the engineered, single-chain mammalian IL-35 gene. Yet still other embodiments use bacteria from the Genus Lactococcus to host the engineered, single-chain mammalian IL-35 gene. One particular embodiment utilizes the bacterial species Lactococcus lactis to express and optionally secrete the engineered, single-chain mammalian IL-35 gene.
The current disclosure provides a composition, including: a recombinant lactic acid bacterial cell (for instance, from the genus Lactococcus) including a nucleotide sequence encoding an engineered, single-chain mammalian IL-35. By way of example, the composition may be formulated for treatment of an autoimmune or inflammatory disease.
In example compositions, the recombinant lactic acid bacterial cell expresses at least one heterologous IL-35 nucleic acid encoding the engineered, single-chain mammalian polypeptide that includes the amino acid sequence shown in SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), SEQ ID NO: 7 (human), or a functionally equivalent variant of one of SEQ ID NO: 2, 11, 5, or 6 that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence.
In example compositions, the recombination lactic acid bacterial cell expresses an engineered, single-chain mammalian IL-35 protein including the amino acid sequence shown in SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), or SEQ ID NO: 7 (human) from an engineered, single-chain mammalian IL-35 nucleic acid.
The engineered, single-chain mammalian IL-35 protein in any of the provided composition examples may be expressed from a transgene including the nucleotide sequence of positions 31-1371 of SEQ ID NO: 1, positions 25-1332 of SEQ ID NO: 10, positions 25-1332 of SEQ ID NO: 4, positions 31-1395 of SEQ ID NO: 6, or a functionally equivalent variant thereof that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence. Optionally, in any of the composition embodiments, the nucleotide sequence encoding the engineered, single-chain mammalian IL-35 is integrated into the genome of the recombinant lactic acid bacterial cell.
The compositions provided herein may be used to induce an anti-inflammatory response in a subject treated with the composition.
Examples of the various composition embodiments are a milk-based nutraceutical, for instance a fermented milk product. By way of example, the fermented milk product may include a yogurt, a buttermilk, a kefir, a cheese, a crème fraiche, or a cultured sour cream.
Also provided in another embodiment is use of any of the provided compositions for treating an inflammatory disease or condition, such as one or more of an allergy, asthma, autoimmune arthritis, celiac disease, Crohn's disease, diabetes, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto's thyroiditis, and/or another endocrinopathy.
Another embodiment is use of any one of the composition embodiments for stimulating IL-10 and TGF-β-producing CD39+ regulatory T cells.
Yet another embodiment is use of any one of the composition embodiments for stimulating one or more regulatory cell subsets including Foxp3 (FOXP3)+ and Foxp3 (FOXP3)− T cells, CD25+ and CD25− T cells, regulatory B cells, and regulatory innate cells involved in producing a tolerogenic condition.
Provided in another embodiment is a recombinant lactic acid bacterial cell (such as a cell from the genus Lactococcus), which cell includes: a nucleotide sequence encoding an engineered, single-chain mammalian IL-35. IN examples of this embodiment, the cell expresses at least one heterologous IL-35 nucleic acid encoding the engineered, single-chain mammalian polypeptide that includes the amino acid sequence shown in SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), SEQ ID NO: 7 (human), or a functionally equivalent variant of one of SEQ ID NO: 2, 5, or 6 that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence. In alternative and overlapping examples, the recombinant lactic acid bacterial cell expresses SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), or SEQ ID NO: 7 (human) from an engineered, single-chain mammalian IL-35 nucleic acid.
In yet more examples of the recombinant lactic acid bacterial cell embodiments, the nucleotide sequence encoding the engineered, single-chain mammalian IL-35 includes the sequence of positions 31-1371 of SEQ ID NO: 1, positions 25-1332 of SEQ ID NO: 10, positions 25-1332 of SEQ ID NO: 4, positions 31-1395 of SEQ ID NO: 6, or a functionally equivalent variant thereof that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence. Optionally, in any of the recombinant lactic acid bacterial cell embodiments, the nucleotide sequence encoding the engineered, single-chain mammalian IL-35 may be integrated into the genome of the recombinant lactic acid bacterial cell.
Another embodiment is a milk-based nutraceutical composition including: a recombinant lactic acid bacterial cell including a nucleotide sequence encoding an engineered, single-chain mammalian IL-35. In examples of this embodiment, the recombinant lactic acid bacterial cell expresses at least one heterologous nucleic acid encoding the engineered, single-chain mammalian IL-35 polypeptide that includes the amino acid sequence shown in SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), SEQ ID NO: 7 (human), or a functionally equivalent variant of one of SEQ ID NO: 2, 5, or 6 that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence.
Also provided are milk-based nutraceutical compositions, wherein the engineered, single-chain mammalian IL-35 includes the amino acid sequence of SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), or SEQ ID NO: 7 (human). Optionally, in provided examples of the milk-based nutraceutical composition embodiments, the nucleotide sequence includes the sequence of positions 31-1371 of SEQ ID NO: 1, positions 25-1332 of SEQ ID NO: 10, positions 25-1332 of SEQ ID NO: 4, positions 31-1395 of SEQ ID NO: 6, or a functionally equivalent variant thereof that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence. For instance, the nucleotide sequence may include the sequence of SEQ ID NO: 1 (murine), SEQ ID NO: 4 (canine), or SEQ ID NO: 6 (human).
Yet another embodiment is use of any of the provided milk-based nutraceutical compositions for treating an inflammatory disease or condition. By way of example, the inflammatory disease or condition may include one or more of an allergy, asthma, autoimmune arthritis, celiac disease, Crohn's disease, diabetes, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto's thyroiditis, and/or another endocrinopathy.
Also provided is use any of the provided milk-based nutraceutical compositions for stimulating IL-10 and TGF-β-producing CD39+ regulatory T cells.
Another provided use is use of any of the provided milk-based nutraceutical compositions for stimulating one or more regulatory cell subsets including Foxp3 (FOXP3)+ and Foxp3 (FOXP3)− T cells, CD25+ and CD25− T cells, regulatory B cells, and regulatory innate cells involved in producing a tolerogenic condition.
Provided herein in another embodiment is a method for treating or preventing an autoimmune or inflammatory disease in a subject, the method including: administering to the subject any one of the composition embodiments provided herein, or a composition including any one of the recombinant lactic acid bacterial cells provided herein. In examples of such methods of treating or preventing, the level of IL-10 in the subject is increased upon the administering, as compared to the level of the IL-10 present in the subject before the administering; and/or the level of TGF-β in the subject is increased upon the administering, as compared to the level of the TGF-β present in the subject before the administering; and/or the level of at least one of IFN-γ or IL-17 is decreased upon the administering, as compared to the level of at least one of IFN-γ or IL-17 present in the subject before the administering; and/or the level of TNF-α is decreased upon the administering, as compared to the level of TNF-α present in the subject before the administering.
Yet another embodiment is a method for producing a composition for the treatment of an autoimmune or inflammatory disease, including: introducing a nucleotide sequence encoding an engineered, single-chain mammalian IL-35 into a recipient lactic acid bacterial cell. In examples of this method embodiment, the engineered, single-chain mammalian IL-35 expresses at least one engineered, single-chain mammalian IL-35 including the amino acid sequence of SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), SEQ ID NO: 7 (human) or a functionally equivalent variant of one of SEQ ID NO: 2, 11, 5, or 6 that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence. In alternative and overlapping examples, the nucleotide sequence includes the sequence of positions 31-1371 of SEQ ID NO: 1, positions 25-1332 of SEQ ID NO: 10, positions 25-1332 of SEQ ID NO: 4, positions 31-1395 of SEQ ID NO: 6, or a functionally equivalent variant thereof that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence. For instance, in some specific examples the nucleotide sequence includes the sequence of SEQ ID NO: 1 (murine), SEQ ID NO: 10 (murine), SEQ ID NO: 4 (canine), or SEQ ID NO: 6 (human).
Also provided are method of treating or preventing embodiments, which further include: culturing the lactic acid bacterial cell under conditions which allow for expression of the engineered, single-chain mammalian IL-35.
In examples of any of the method embodiments, wherein introducing the nucleotide sequence encoding the engineered, single-chain mammalian IL-35 includes integrating the encoding sequence into the genome of the recipient lactic acid bacterial cell.
Also taught herein are methods for producing a composition for the treatment of an autoimmune or inflammatory disease, including: introducing a nucleotide sequence encoding single-chain mammalian IL-35 into a recipient Gram-positive bacterial cell, e.g. a lactic acid bacterial cell, and culturing the recipient bacterial cell under conditions which allow for expression of the mammalian IL-35 protein.
The methods may further include packaging the recombinant bacterial cells with any pharmaceutically acceptable carrier, buffer, excipient, adjuvant, or mixture thereof.
The methods may further include packaging the recombinant bacterial cells with any foodstuff, such as a: beverage, dairy product, yogurt, fermented food, or the like.
The methods may further include packaging the recombinant bacterial cells with a food supplement, such as a: powdered composition, encapsulated composition, or any liquid formulation.
The recombinant bacterial cells taught herein may be live upon administration or may not. Further, the therapeutic compositions disclosed herein may include mixtures of both live and non-living recombinant bacterial cells.
In any of the provided embodiments, the engineered, single-chain mammalian polypeptide may include an EBI3/IL-27β subunit; a p35/IL-12α subunit; and a linker therebetween. For instance, the order of the components in some instances EBI3/IL-27β subunit-linker-p35/IL-12α subunit; in some instances, it is p35/IL-12α subunit-linker-EBI3/IL-27β subunit. In various exemplary engineered, single-chain mammalian polypeptides, the EBI3/IL-27β subunit is from human IL-35, from murine IL-35, or from canine IL-35; and/or the p35/IL-12α subunit is from human IL-35, from murine IL-35, or from canine IL-35; and/or the linker is a rigid peptide linker or a flexible peptide linker.
The nucleic acid and amino acid sequences described herein and provided in the accompanying Sequence Listing are shown using standard letter abbreviations, as defined in 37 C.F.R. § 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included in embodiments where it would be appropriate. A computer readable text file, entitled “2ND7462.txt (Sequence Listing.txt)” created on Feb. 22, 2022, with a file size of 28 KB, contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
SEQ ID NO: 1 is the nucleic acid sequence encoding an exemplary engineered, single-chain murine IL-35, the sequence of which has been optimized for expression in lactic acid bacteria. The codon usage was hand-optimized for Lactococcus but it has been designed to fit most Lactic acid bacteria except, perhaps, Lactobacillus delbruekii ssp. bulgaricus which has a peculiar codon usage. Within SEQ ID NO: 1:
SEQ ID NO: 2 is the amino acid sequence of the single-chain murine IL-35 encoded by SEQ ID NO: 1. Within SEQ ID NO: 2:
SEQ ID NO: 3 is the nucleic acid sequence of a nisin-inducible promoter, provided in intuitive 5′-3′ orientation; it is provided in GenBank FJ5555208 in 3′-5′ orientation. The full sequence of expression vector pMSP3535H3 is available at NCBI GenBank locus FJ555208.
SEQ ID NO: 4 is the nucleic acid sequence encoding an exemplary engineered, single-chain canine IL-35, the sequence of which has been optimized for expression in lactic acid bacteria (pGnMM54). Within SEQ ID NO: 4:
SEQ ID NO: 5 is the amino acid sequence of the single-chain canine IL-35 encoded by SEQ ID NO: 4. Within SEQ ID NO: 5:
SEQ ID NO: 6 is a nucleic acid sequence encoding an exemplary engineered, single-chain human IL-35, the sequence of which has been optimized for expression in lactic acid bacteria (pGnMM107). Within SEQ ID NO: 6:
SEQ ID NO: 7 is the amino acid sequence of the single-chain human IL-35 encoded by SEQ ID NO: 6. Within SEQ ID NO: 7:
SEQ ID NO: 8 is the amino acid sequence of a synthetic peptide linker.
SEQ ID NO: 9 is the amino acid sequence of a single repeat of a flexible synthetic peptide linker (GGGGS).
SEQ ID NO: 10 is the nucleic acid sequence encoding an exemplary engineered, single-chain murine IL-35, the sequence of which has been optimized for expression in lactic acid bacteria (pGnMM119); this construct has the reverse order of EBI3 and IL-12 p35 subunits compared to that in SEQ ID NOs: 1 and 2. Within SEQ ID NO: 10:
SEQ ID NO: 11 is the amino acid sequence of the single-chain murine IL-35 encoded by SEQ ID NO: 10. Within SEQ ID NO: 11
Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present disclosure may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present disclosure in any appropriate manner.
The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art, or that any publication specifically or implicitly referenced is prior art.
Unless defined otherwise, 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.
As used herein, the term “adjuvant” refers to a substance sometimes included in a vaccine or therapeutic formulation to enhance or modify the immune-stimulating properties of the vaccine or therapeutic formulation.
As used herein, the term “antigen” refers to a substance that triggers an immune system response, resulting in production of an antibody specific for the antigen. Thus, an antigen is a substance that binds specifically to a respective antibody.
As used herein, the term “operon” refers to a cluster or series of adjacent structural genes that are transcribed as a unit into a single mRNA molecule. The cluster or series of adjacent structural genes can be under the control of a single promoter and also under the control of a composite tandem promoter.
As used herein, the term “autoimmune disease” refers to a physiological condition in a subject that is resultant from the subject's own body producing an inappropriate immune response that targets and damages the subject's own cells.
As used herein, the term “inflammatory disease” or “inflammatory condition” encompasses any disease or condition characterized by inflammation. Inflammation is a basic physiological response to a variety of external or internal insults, such as infectious agents, physical injury, hypoxia, or disease processes. Therefore, diseases or conditions falling within “inflammatory disease” do not have to share a common genetic or physiological basis, so long as the disease or condition results in inflammation. Representative conditions that are encompassed in the term inflammatory disease include allergies, alopecia areata, anemia, aphthous ulcer, arthritis (such as rheumatoid, juvenile rheumatoid, psoriatic, and gouty arthritis, and osteoarthritis), asthma (including allergic asthma), celiac disease, Crohn's disease, dermatitis (including atopic dermatitis and eczematous dermatitis), diabetes, diabetes mellitus, Hashimoto's thyroiditis, irritable bowel disease, cutaneous and systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, osteoporosis, psoriasis, scleroderma, Sjogren's syndrome (including keratoconjunctivitis sicca secondary to Sjogren's syndrome), other endocrinopathies, ulcerative colitis, vaginitis and Wegener's granulomatosis.
As used herein, the term “recombinant bacteria” refers to bacteria that have been genetically modified from their native state. For instance, recombinant bacteria may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into the bacterial DNA. Further, recombinant bacteria may include exogenous nucleotide sequences on plasmids or exogenous nucleotide sequences stably incorporated into the chromosomal DNA.
As used herein, and in light of the previous definition, the term “recombinant lactic acid bacterial cell” refers to lactic acid bacterial cells that have been genetically modified from their native state. In some aspects of the disclosure, for example, a “recombinant lactic acid bacterial cell” includes exogenous nucleotide sequences from a mammal or other animal.
As used herein, the term “probiotic microorganism” is a microorganism which has a beneficial effect on a host's intestinal microflora ecology, presumably by promoting the growth of so-called “good” microorganisms, inhibiting the growth of so-called “bad” microorganisms, or by performing metabolic activities that are beneficial to the host. In particular embodiments herein, the disclosed recombinant bacteria perform metabolic functions that are beneficial to a host. In certain embodiments, the recombinant bacteria are lactic acid bacteria, a common probiotic bacterial clade.
There is an ongoing need in the art for the development of safe, bacterial based therapeutics that are not reliant upon attenuated invasive bacterial strains and therefore do not suffer from the aforementioned drawbacks. Specifically, the development of a Gram-positive delivery system for expressing engineered, single-chain mammalian IL-35, in a bacterial species that has been accorded a Generally Recognized as Safe (GRAS) status, would offer consumers suffering from autoimmune and inflammatory disease a superior delivery system for expressing engineered, single-chain mammalian IL-35.
Many mammalian pathogens invade the host through a mucosal surface and arm the mucosa to ultimately prevent pathogens from initiating infection. The mucosa is also the site where many environmental and food antigens are processed. Mucosal immunity, be it to combat pathogens or to maintain tolerance, is accomplished by facilitating vaccine uptake to mucosal inductive tissues. Similarly, non-pathogen or “good” bacteria (such as probiotics) release proteins that are then taken up by the mucosal tissues to induced tolerance, or to down-modulate the immune response, allowing these beneficial bacterial to reside in the intestinal tract without immune activation.
Mucosal inductive sites are present in the gut known as Peyer's patches, and in the upper respiratory tract referred to as nasal-associated lymphoid tissues (NALT) or in humans, referred to as Waldeyer's ring (tonsils and adenoids). At such inductive sites, foreign proteins or materials (e.g., antigens), are sampled and used to trigger a host immune response.
Once proteins are sampled and processed, they will induce memory lymphocyte responses in mucosal tissues, which are the various mucosal surfaces of the gut, respiratory tract, and genitourinary tract. These mucosal effector sites contain naïve and memory B and T lymphocytes, antigen presenting cells (APCs), as well as a plethora of other cell types with different functions in the mucosal network that ultimately determines the outcome of the immune response.
Without wishing to be bound to a particular theory, the present inventors hypothesize that some, but not all, of the molecules that pathogens transfer to target cells may at the same time down-regulate the immune system. In the present disclosure, the inventors have shown that this is correct, and when applied appropriately this discovery leads to the development of novel therapeutic compositions that are useful for the treatment of autoimmune disorders and inflammatory disease.
As described herein, the inventors have exploited a recombinant Lactococcus lactis to express an engineered, single-chain mammalian IL-35, that when orally delivered to mice, is able to prevent the symptoms and to block the progression of collagen-induced arthritis (CIA). CIA is a recognized model of rheumatoid arthritis and therefore implicates the ability of the recombinant bacteria taught herein to be an effective treatment for this highly pervasive autoimmune disease.
Thus, the present inventors have illustrated that the recombinant Lactococcus lactis expressing an engineered, single-chain mammalian IL-35 protein have strong potential to act as multi-purpose modulators of pathological immune response in absence of an autoantigen.
These discoveries have profound implications for the treatment and prevention of autoimmune diseases like rheumatoid arthritis and other inflammatory diseases.
Lactic Acid Bacteria
The disclosure herein demonstrates that engineered, single-chain mammalian IL-35 can be successfully expressed in a Gram-positive bacterial vector system. That is, the disclosure presents a lactic acid bacterium, Lactococcus lactis, which includes an engineered plasmid containing a nucleotide sequence encoding an engineered, single-chain mammalian IL-35. The nucleotide sequence is shown to be expressed in the Lactococcus lactis system.
Lactic Acid Bacteria Classification
The lactic acid bacteria include a clade of Gram-positive bacteria associated by their common metabolic and physiological characteristics. For instance, these bacteria have low-GC, are acid-tolerant, are generally non-sporulating and non-respiring, rod-shaped bacilli or cocci phenotypes. As their name implies, lactic acid bacteria produce lactic acid as the major metabolic end-product of carbohydrate fermentation.
The order Lactobacillales includes the lactic acid bacteria. Families present in the Lactobacillales include: Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae. A representative genus of Streptococcaceae is Lactococcus. A representative species of Lactococcus is Lactococcus lactis.
The aforementioned is not an exhaustive list of the members of the lactic acid bacteria but is merely illustrative of the structuring of the group. One of skill in the art would be able to ascertain the members of the lactic acid bacteria.
The present disclosure utilizes Lactococcus bacteria in the exemplary embodiments, but it would be within the skill of one in the art to utilize the taught methods for expression of the provided engineered, single-chain mammalian IL-35 in other lactic acid bacteria. For example, the disclosed promoter sequences along with the taught signal sequence coding regions (encoding the signal sequence peptides) are engineered for lactic acid bacteria and would be useful for deployment in other lactic acid bacterial species.
One of skill in the art would be able to utilize the disclosed methods to insert the engineered, single-chain mammalian IL-35 structural gene sequence, with appropriate signal sequence coding regions for the particular lactic acid bacterial recipient, into a recipient lactic acid bacterial cell and obtain expression of an engineered, single-chain mammalian IL-35. The ascertainment of the appropriate signal sequence coding regions would be ascertainable based upon the particular lactic acid bacterial species that would be receiving the engineered, single-chain IL-35 structural gene sequence.
Also appropriate for use in the methods and compositions described herein are probiotic Bacilli, such as strains of Bacillus subtilis. B. subtilis is a Gram-positive, nonpathogenic, spore-forming organism; the robustness of its spores is thought to enable passage across the gastric barrier, whereby the spores germinate in the small intestine and transiently populate it. Probiotic strains of B. subtilis have long been used for oral bacteriotherapy and bacterioprophylaxis for gastrointestinal disorders (Mazza, Boll Chim Farm. 133(1):3-18, 1994; Tompkins et al., Benef Microbes. 1(1):93-106, 2010). Spores of B. subtilis are extremely resistant and stable, easy to manipulate and are known to interact with immune cells inducing protective, antigen-specific immune responses. Furthermore, B. subtilis has outstanding biotechnological potentials including ease of manipulation, high-protein production and secretion (Song et al., J Microbiol Biotechnol. 25(7):963-77, 2015).
Lactic Acid Bacteria Benefits
There are several benefits to utilizing lactic acid bacteria to host and deliver the engineered, single-chain mammalian IL-35.
First, lactic acid bacteria do not present lipopolysaccharide complex (LPS) that is associated with the outer membrane of, for instance, a Salmonella based vector.
Second, lactic acid bacteria do not present a problem with reversion to a virulent state, because lactic acid bacteria are not virulent. In fact, they have a Generally Recognized as Safe (GRAS) status. GRAS status is a Food and Drug Administration designation that a chemical or substance added to food is considered safe by experts, and thus is exempted from the usual Federal Food, Drug, and Cosmetic Act food additive tolerance requirements.
Third, there is an insignificant risk of horizontal gene transfer to other invasive bacteria as would be more prevalent with a Gram-negative vector (such as Salmonella).
Furthermore, the taught lactic acid bacteria vectors offer several distinct advantages over a Salmonella vector.
For instance, lactic acid bacteria have a long history of beneficial association with human intestinal microflora. Thus, the use of lactic acid bacteria offers the opportunity to create synergistic effects between recombinant lactic acid bacteria expressing an engineered, single-chain mammalian IL-35 and other resident intestinal microbial flora.
Consider that probiotics are products aimed at delivering living, potentially beneficial, bacterial cells to the intestinal ecosystem of humans and other animals. Strains of lactic acid bacteria are the most common microbes employed as probiotics (Sonomoto & Yokota, Current Progress in Advanced Research. Caister Academic Press. ISBN 978-1-904455-82-0 2011). This presents the opportunity to utilize the herein described lactic acid bacteria expressing engineered, single-chain mammalian IL-35 in compositions that also include probiotic bacterial strains such as those from the genus Lactobacillus and Bifidobacterium.
As will be illustrated in the disclosed Examples, the present recombinant Lactococcus lactis strains expressing engineered, single-chain mammalian IL-35 also demonstrate potency.
IL-35
Produced by Tregs (Kochetkova et al., J. Immunol. 184:7144-7153, 2010; Collison, et al., Nature 450:566-571, 2007; Niedbala et al., Eur. J. Immunol. 37: 3021-3029, 2007), IL-35 can potently suppress arthritis (Kochetkova et al., J. Immunol. 184:7144-7153, 2010; Niedbala et al., Eur. J. Immunol. 37: 3021-3029, 2007; Nakano et al., Rheumatology (Oxford). 54(8):1498-506, 2015), colitis (Collison, et al., Nature 450:566-571, 2007; Wirtz et al., J. Gastroenterol. 141:1875-1886, 2011), Sjögren's syndrome (Guo et al., Scand J Immunol. 88(5):e12718, 2018), atherosclerosis (Park et al., J Biol Chem. 291(7):3359-70, 2016), type 1 diabetes (Bettini et al., Diabetes. 61(6):1519-26, 2012; Manzoor et al., Eur J Immunol. 47(1):144-154, 2017; Manzoor et al., Eur J Immunol. 47(1):144-154, 2017) and EAE (Collison, et al., Nat. Immunol. 11:1093-1101, 2010; Duffy et al., J Neurosci. 39(12):2326-2346, 2019). IL-35 is also important for maintaining peripheral immune tolerance during pregnancy (Liu et al., Exp Cell Res. 111513, 2019).
Humans produce IL-35, and its inhibitory effect is contact-independent (Chaturvedi et al., J. Immunol. 186:6661-6666, 2011). Since IL-35 can mediate its effects via the stimulation of IL-10 (Kochetkova et al., J. Immunol. 184:7144-7153, 2010; Niedbala et al., Eur. J. Immunol. 37: 3021-3029, 2007; Collison et al., J. Immunol. 182:6121-6128, 2009) and TGF-β (Kochetkova et al., J Immunol. 192(2):804-16, 2014), as well as perpetuating itself, it has profound capacity for immunoregulation dampening innate and adaptive immune responses. Thus, limited use of IL-35 offers an attractive alternative to remedy autoimmunity.
Prior to the current work, IL-35 has proved difficult to work with. For instance, Huang et al. (J Inflamm (Lond). 14:16, 2017. doi: 10.1186/s12950-017-0164-5) describes difficulties of producing recombinant IL-35. Expression in E. coli leads to non-functional protein (Aparicio-Siegmund et al., PLoS One. 9(9):e107990, 2014). Experience of the present inventors, gained in part while generating data for Kochetkova et al. (J Immunol. 184:7144-53, 2010), confirms that IL-35 is laborious and costly to produce in yeast. The clear difficulty in working with IL-35 is also indirectly supported when considering the tremendous anti-inflammatory properties of IL-35, coupled with the scarcity of papers describing related translational research.
Functional IL-35 has been produced in insect cells (Wang et al., Nat Med. 20:633-41, 2014), but their approach is hampered by the need to purify the recombinant protein. The herein-described approach, which uses Lactococcus both as a microbial cell factory and a delivery agent, is more effective and reduces production costs by 95-99%.
Producing the two subunits of IL-35, p35 and EBI3, as separate proteins introduces the hurdle of the physical interaction of the two subunits. One of the mechanisms believed to regulate the low in vivo concentration of heterodimeric IL-35 is that the EBI3 sub-unit is sequestered and degraded in the endoplasmic reticulum (Devergne et al., J Virol. 70(2):1143-53, 1996). The herein-described system for producing IL-35 as a holoprotein rather than a bicistronic construct has effectively overcome this issue.
The anti-inflammatory role of IL-35 has been demonstrated in rodents and humans. Among mammals, p35 and EBI3 show some sizeable, but not stringent, amino acid identity. As an example, mouse and human p35 (GenBank Accession Nos. AAL01442.1 and NP_000873.2, respectively) and EBI3 (GenBank Accession Nos. NP_056581.1 and ABK41923.1, respectively) have a 58% and 64% identity, respectively. It is therefore expected that IL-35 may function better when it is species-matched, or used in closely related species.
Thus, provided herein are engineered murine-derived IL-35 constructs (e.g., SEQ ID NOs: 1 and 2, 10 and 11) that are particularly useful in embodiments involving rodent subjects. Similarly, there are provided engineered canine-derived IL-35 constructs (e.g., SEQ ID NOs: 4 and 5), which are particularly useful in embodiments involving canine subjects. Finally, there are provided herein engineered human-derived IL-35 constructs (e.g., SEQ ID NOs: 6 and 7), which are particularly useful in embodiments involving human and other primate subjects.
Also specifically contemplated are constructs in which the order of the p35 and EBI3 subunits in the construct is reversed—that is, though SEQ ID NOs: 2, 6, and 7 are provided with the subunits in the order P35-then-EBI3, also completed are single chain IL-35 constructs in which the subunits are in the order EBI3-then-p35 (exemplified herein in SEQ ID NO: 11). Thus, also specifically contemplated are engineered human and canine single-chain IL-35 fusions in which the subunits are in the order EBI3-then-p35. Though usually the provided single-chain IL-35 fusion proteins will originate from the same mammal (e.g., both from human, canine, or murine origin), also contemplated are single-chain fusions in which the p35 (IL-12α) subunit is of human original while the EBI3 (IL-27β) subunit is of canine or murine origin—or vice versa, in all of the available combinations.
Also contemplated are functional variants of the provided nucleic acid and amino acid sequences. Such functional variants include nucleic acids (e.g., gene, pre-mRNA, mRNA) and polypeptides, polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of over a region of at least 25, 50, 100, 200, 300, 400, or more amino acids, to a polypeptide encoded by a respectively referenced nucleic acid or an amino acid sequence; which variant maintains at least one biological function of the single-chain engineered IL-35 as described herein.
The phrase conservatively modified variant(s) applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, the phrase ‘conservatively modified variants’ refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein or protein domain. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one type of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant”, where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain amino acids that are considered conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
Described herein are working embodiments that employ a minimalist GPG linker between the two IL-35 subunits; this linker was selected because it presents the low risk of disturbing protein function. The GPG linker includes two glycines, which allow for unhindered swiveling; and a proline that acts as a “circuit-breaker”, preventing secondary structures to spill into neighboring protein domains. Alternative configurations and modification(s) of the linker are contemplated as useful in the current invention. There are three categories of linkers: 1) flexible, 2) rigid, 3) cleavable. Each design has pros and cons (reviewed in Chen et al., Adv Drug Deliv Rev. 65(10):1357-69, 2013, for instance), and linker optimization is within the skill of the ordinary artisan. Such optimization can readily be carried out on a case-by-case basis. Additional contemplated linkers included, for instance, (GGGGS)3 (corresponding to positions 230-245 of SEQ ID NO: 7; three serial repetitions of SEQ ID NO: 9), the rigid linker corresponding to positions 240-264 of SEQ ID NO: 11, and another rigid peptide linker AEAAAKEAAAKA (SEQ ID NO: 8). Contemplated herein are engineered mammalian single chain IL-35 proteins that use a rigid linker or a flexible linker. Optionally, the linker may be short or long.
Expression of Mammalian IL-35 in Gram-Positive Bacteria
The present disclosure teaches that Gram-positive bacteria expressing engineered, single-chain mammalian IL-35 are therapeutically effective at treating or preventing autoimmune and/or inflammatory disease.
The method of expressing engineered, single-chain mammalian IL-35 in Gram-positive bacteria may include inserting a plasmid carrying an engineered, single-chain IL-35 encoding sequence into a recipient Gram-positive bacterial host.
Further, in some embodiments, the method of expressing an engineered, single-chain mammalian IL-35 in Gram-positive bacteria may include stably integrating the engineered, single-chain IL-35 encoding sequence into the chromosome of a recipient Gram-positive bacterial host.
The term “plasmid” is used to refer to a molecule capable of autonomous replication that is suitable for transformation of a recipient bacterial strain and contains DNA sequences that direct and/or control the expression of inserted heterologous DNA sequences. Various types of plasmids may be used such as low and high copy number plasmids, narrow and broad-host range plasmids, expression plasmids, and cosmids.
In order to prevent loss of the plasmid expressing the engineered, single-chain mammalian IL-35, an element may be added to the plasmid which enhances its stability. Methods for improvement of plasmid stability are known to those in the art.
In general, heterologous gene expression is achieved by cloning of the heterologous gene into a plasmid (such as a representative plasmid described herein), which is replicated within the recipient in multiple copies thus leading to high expression of foreign gene product. Expression of the herein described heterologous sequences encoding an engineered, single-chain mammalian IL-35 are achievable by application of known genetic engineering techniques such as those described in, e.g. Sambrook and Russell (2001) “Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York. It is demonstrated herein that engineered, single-chain mammalian IL-35 may be expressed in Gram-negative bacteria.
The disclosed engineered DNA construct, i.e. a DNA construct including the encoding sequence of any one of SEQ ID NOs: 1, 10, 4, or 6, including a promoter operably linked to DNA encoding the engineered, single-chain mammalian IL-35 may be made and transformed into the Gram-positive bacteria using conventional techniques. Transformants containing the DNA construct may be selected, for example, by screening for a selectable marker on the construct. Bacteria containing the construct may be grown in vitro before being formulated for administration to the host. Selectable markers suitable for the herein described recombinant bacteria would be known to those of skill in the art.
The recombinant bacteria taught herein may include a nucleotide sequence encoding engineered, single-chain mammalian IL-35 that have been codon optimized. “Codon optimization” is defined as modifying a nucleic acid sequence for enhanced expression in the cells of the host bacterial species of interest, e.g. Gram-positive bacteria, by replacing at least one, more than one, or a significant number of codons of the native mammalian IL-35 sequence with codons that are more frequently or most frequently used in the genes of the recipient Gram-positive bacteria.
Various bacterial species exhibit particular bias for certain codons of a particular amino acid and thus codon optimization can ensure optimal expression of the engineered, single-chain mammalian IL-35 in the recipient bacterial host cell being transformed. Codon preference or codon bias is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, the presently disclosed engineered, single-chain mammalian IL-35 encoding sequences can be tailored for optimal gene expression in a given Gram-positive bacteria based on codon optimization.
Working embodiments herein use the Usp45 signal peptide to ensure the single-chain, engineered IL-35 is secreted. However, it is recognized that alternative signal peptides, both natural as well as engineered and/or optimized signal peptides, can be useful in expression constructs of the current invention. The art recognizes many signal peptides that are functional for secreted expression from Lactococcus lactis and the like. See, for instance, the teachings in: Ng & Sarkar (App Environ Microbiol., 79(1):347-356, 2013), Le Loir et al. (App Environ Microbiol., 67(9):4119-4127, 2001), Ravn et al. (Microbiology, 149(Pt 8):2193-2201, 2003), Morello et al. (J Mol Microbiol Biotechnol. 14:48-58, 2008), and Mahmud et al. (J Biotechnol. 296:22-31, 2018).
Optionally, the engineered IL-35 provided herein can be further modified to include a molecular anchor for surface-display. Anchoring and surface-display may directly expose IL-35 to cell-surface receptor(s) either during the sampling process by GI DCs through the mucous layer, or during subsequent trafficking of bacteria. Also, mucosal immunization other than oral (nasal, vaginal, anal, sublingual) may benefit from a facilitated contact between the surface-displayed IL-35 and relevant cell types. (Leenhouts et al., Antonie Van Leeuwenhoek. 76(1-4):367-76, 1999; Michon et al., Microb Cell Fact. 15:70, 2016; Cortes-Perez et al., FEMS Microbiol Lett. 229(1):37-42, 2003).
Furthermore, this approach may lead to deployment of the GEM (Gram-positive enhanced matrix) technology, whereby the recombinantly-produced protein of interest is engrafted onto a non-viable LAB scaffold, thus bypassing the recombinant regulatory hurdles (van Roosmalen et al., Methods. 38(2):144-9, 2006; Sirec et al., FEMS Microbiol Lett 358(2):194-201, 2014). The GEM tech also allows for loading more than one functional protein onto the same particles.
Additional components of the herein-described IL-35 expression constructs can be modified or varied, including for instance: the promoter used to express the engineered polypeptide, the construct from which the polypeptide is expressed, whether the construct remains exosomal or integrates into the genome of the host bacteria, and so forth. Representative systems for expressing polypeptides, including therapeutic proteins, in Lactococcus bacteria are described for instance, in: Morello et al., J Mol Microbiol Biotechnol. 14:48-58, 2008; Plavec & Berlec, Appl. Microbiol Biotechnol., 103(5):2053-2066, 2019; Mierau & Kleerebezem, Appl. Microbiol Biotechnol. 68(6):705-717, 2005; Ng & Sarkar, App Environ Microbiol., 79(1):347-356, 2013; Lim et al., Microb Cell Fact 16(1):221, 2017; and other references cited herein.
Formulation of Compositions
The compositions presented herein are suitable for combination with any known pharmaceutically acceptable carrier, buffer, excipient, adjuvant, or mixture thereof.
Pharmaceutically acceptable carriers are well known and are usually liquids, in which an active therapeutic agent is formulated. In the present case, the active therapeutic agent is the disclosed recombinant bacteria expressing an engineered, single-chain mammalian IL-35. The carrier generally does not provide any pharmacological activity to the formulation, though it may provide chemical and/or biological stability, release characteristics, and the like. Exemplary formulations can be found, for example, in Alfonso R. Gennaro. Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000, the entire contents of which are hereby incorporated by reference, and include saline, water, buffered water, dextrose and the like. Optionally, the compositions provided herein may be lyophilized, with or without stabilizers (such as trehalose), before it is used to produce a formulation.
The compositions presented herein may in some embodiments be placed within foodstuffs, such as: beverages, dairy products, yogurts, fermented food products, and the like, as feasible and consumer friendly delivery vehicles.
The compositions taught herein may also be delivered in food supplements, such as: powdered compositions including the herein described recombinant bacterial cells, encapsulated compositions including the herein described recombinant bacterial cells, or any liquid formulation including the herein described recombinant bacterial cells.
The compositions may include other therapeutically effective agents such as anti-inflammatory cytokines.
The compositions may include other bacterial species, such as those bacterial species commonly referred to as “probiotics.” Furthermore, “prebiotics” may also be present in the herein described compositions.
Probiotics are often defined as live microorganisms that when administered in adequate amounts confer health benefits to the host. The compositions of the present disclosure may include probiotic microorganisms in an amount sufficient to at least partially produce a health benefit.
Prebiotics are often defined as food substances that promote the growth of probiotics in the intestines. They are not broken down in the stomach and/or upper intestine or absorbed in the GI tract of the person ingesting them, but they are fermented by the gastrointestinal microflora and/or by probiotics. The prebiotics that may be used in accordance with the present disclosure are not particularly limited and include all food substances that promote the growth of probiotics in the intestines.
Thus, disclosed herein are probiotic compositions including recombinant lactic acid bacteria expressing an engineered, single-chain mammalian IL-35. In certain aspects, the herein described probiotic compositions support a healthy immune system. The herein described probiotic compositions may also be used to supplement an individual's normal dietary regime.
Furthermore, in certain embodiments, the present disclosure teaches dietary supplements that include recombinant bacteria including a nucleotide sequence encoding an engineered, single-chain mammalian IL-35. In particular embodiments, the recombinant bacteria express the engineered IL-35 protein. In certain aspects, the herein described dietary supplements support a healthy immune system. The dietary supplements may also be used to supplement an individual's normal dietary regime.
The present disclosure also teaches food additive compositions that include recombinant bacteria containing a nucleotide sequence encoding an engineered, single-chain mammalian IL-35 protein. In particular embodiments, the recombinant Gram-positive bacteria expresses the engineered, single-chain mammalian IL-35.
In certain aspects, the herein described food additives support a healthy immune system.
The herein described food additive compositions of the disclosure may be directly ingested or used as an additive in conjunction with foods.
It will be appreciated that the disclosed food additives may be incorporated into a variety of foods and beverages including: yogurt, ice cream, cheese, baked products such as bread, biscuits and cakes, dairy and dairy substitute foods, confectionery products, edible oil compositions, spreads, breakfast cereals, juices, and the like.
Heating (e.g., cooking) a product containing gram-positive bacteria expressing IL-35, or simply containing the expressed IL-35, will likely destroy significant IL-35 activity. More specifically, heating is expected to denature the IL-35 polypeptide.
It is believed that lyophilization, with or without a stabilizer (such as trehalose or another disaccharide) is a viable option for processing of IL-35-containing compositions produced using the herein-described technology. The art recognizes that freezing and freeze-drying can be used in the preparation of pharmaceutical formulations. Optionally, the IL-35 is stabilized during such processing; see, for instance, Olsson et al. (J Phys Chem B. 120(20):4723-4731, 2016) and Izutsu (Adv Exp Med Biol. 1081:371-383, 2018).
Routes of Administration
The herein described compositions may be used for mucosal administration, such as oral, nasal, vaginal, and rectal delivery; as well as topical administration. Particular embodiments of administration include oral administration, intranasal administration, and topical administration (e.g., for treatment of psoriasis or eczema).
In some oral administration embodiments, the compositions include the disclosed recombinant bacteria expressing an engineered, single-chain mammalian IL-35 and optionally other molecules that are dissolved or suspended in a pharmaceutically acceptable, preferably an aqueous carrier. In addition, the composition may contain excipients, such as buffers, binding agents, diluents, flavors, lubricants, etc.
Consumption of LL-IL35 is an effective means to deliver the recombinant IL-35 to the oral cavity and gastrointestinal tract. Hence, such delivery can be applied to treat diseases of the oral cavity as well as the gastrointestinal tract. Once absorbed from the oral cavity or GI tract, the IL-35 may also enter circulation to treat peripheral autoimmune diseases. An advantage of using Lactococcus lactis is that it can be used to ferment milk into yogurt, in which the Lactococcus lactis are viable and stable for at least two weeks. Such fermentation preserves the Lactococcus lactis and extends the lifespan of the therapeutic product.
The engineered lactic acid bacteria provided herein are not only delivery agents, but they are also beneficial because they can be used to produce a palatable, yogurt-like therapeutic composition/formulation. This provides a remarkable innovation in terms of ease and cost of production. More importantly, the redressed therapeutic agent is predicted to improve acceptance by sections of the market that may be uncomfortable with or wary of “pill” medications, particularly for long-terms ailments. Some consumers do not like to be administered pills, but they may perceive an enhanced probiotic product in their refrigerators as more acceptable. However, pills, capsules, and the like are also still contemplated as formulation formats for the therapeutic compounds and compositions provided herein.
Quantitative Administration
The compositions taught herein may include varying amounts of the recombinant bacteria expressing an engineered, single-chain mammalian IL-35. The particular amount of therapeutic bacterial vector present in the composition may depend upon the disease being treated and/or the subject being administered the therapeutic composition.
For instance, factors such as age, gender, ethnicity, genetic disposition to disease, health, weight, etc. may govern the amount/number of recombinant bacteria present in a composition.
The type of disease or condition being treated may also be taken into consideration when determining the optimal amount of recombinant bacterial vector that should be in a given composition.
In some embodiments, a particular amount of the disclosed therapeutic composition including recombinant bacterial cells expressing an engineered, single-chain mammalian IL-35 is defined as “a therapeutically effective amount” or “therapeutically effective dose.” This amount represents a quantity of the disclosed compositions that is capable of eliciting an immune response in the recipient. For example, a “therapeutically effective dose” may be capable of increasing the level of an anti-inflammatory cytokine in the recipient. Furthermore, a “therapeutically effective does” may be able to suppress the level of an inflammatory cytokine in the recipient.
In some particular embodiments, a “therapeutically effective dose” increases the level of the regulatory cytokine IL-10 in a subject upon administration of the herein described composition, as compared to the level of IL-10 present in the subject before administration of the herein described composition.
In other embodiments, a “therapeutically effective dose” suppresses the level of at least one of IFN-γ or IL-17 upon administration of the herein described composition, as compared to the level of at least one of IFN-γ or IL-17 present in the subject before administration of the herein described composition.
Compositions of the present disclosure may include: 1×108 CFU, 2×108 CFU, 3×108 CFU, 4×108 CFU, 5×108 CFU, 6×108 CFU, 7×108 CFU, 8×108 CFUs, 9×108 CFU, 10×108 CFU, 11×108 CFU, 12×108 CFU, 13×108 CFU, 14×108 CFU, 15×108 CFU, 16×108 CFU, 17×108 CFU, 18×108 CFUs, 19×108 CFU, 20×108 CFU, 30×108 CFU, 40×108 CFU, 50×108 CFU, or more of a recombinant bacteria expressing an engineered, single-chain mammalian IL-35.
Further, the compositions may include any range of CFU that is achievable based upon the aforementioned individual concentrations. For example, the compositions may include from 1×108 CFU to 50×108 CFU per treatment.
Further, the compositions may include ranges of: 1×106 to 1×1010 CFU, or 1×106 to 2×1010 CFU, or 1×106 to 3×1010 CFU, or 1×106 to 4×1010 CFU, or 1×106 to 5×1010 CFU, or 1×106 to 6×1010 CFU, or 1×106 to 7×1010 CFU, or 1×106 to 8×1010 CFU, or 1×106 to 9×1010 CFU, or 1×106 to 10×1011 CFU of a recombinant bacteria expressing an engineered, single-chain mammalian IL-35.
Embodiments of the compositions may include at least 1×108 CFU, or at least 2×108 CFU, or at least 3×108 CFU, or at least 4×108 CFU, or at least 5×108 CFU, or at least 6×108 CFU, or at least 7×108 CFU, or at least 8×108 CFUs, or at least 9×108 CFU, or at least 10×108 CFU, or at least 11×108 CFU, or at least 12×108 CFU, or at least 13×108 CFU, or at least 14×108 CFU, or at least 15×108 CFU, or at least 16×108 CFU, or at least 17×108 CFU, or at least 18×108 CFUs, or at least 19×108 CFU, or at least 20×108 CFU, or at least 30×108 CFU, or at least 40×108 CFU, or at least 50×108 CFU of a recombinant bacteria expressing an engineered, single-chain mammalian IL-35.
In a particular embodiment, the composition includes 5×108 CFU of a recombinant bacteria expressing an engineered, single-chain mammalian IL-35.
In some embodiments, the compositions include from 1×106 to 10×1011 CFU, or 1×106 to 5×1010 CFU of a recombinant bacteria expressing an engineered, single-chain mammalian IL-35.
The compositions may be administered once a day, twice a day, three times a day, four times a day, or five times a day to a subject in need of such treatment.
The compositions may also be administered at least once a day, at least twice a day, at least three times a day, at least four times a day, or at least five times a day.
Furthermore, the compositions may be administered on an as needed basis based upon a subject's physiological symptoms, such as pain, swelling, irritation, or discomfort.
Some embodiments include administering the herein described compositions including the recombinant bacteria on a prophylactic bases to a subject that does not presently experience physiological symptoms associated with an autoimmune or inflammatory disease.
In particular embodiments, the compositions may be taken daily as part of a food product delivery vehicle, e.g. yogurt, as part of a daily health regimen.
Collagen Induced Arthritis (CIA) Model
Collagen induced arthritis (CIA), a model of rheumatoid arthritis (RA), can be induced upon immunization with heterologous collagen II (CII) in DBA/1 or C57BL/6 mice or by mAbs to CII combined with LPS. CIA shares with RA several critical characteristics of the disease pathogenesis, including CD4+ T cells' mediated inflammation and extensive cartilage and bone damage, resulting in joint deformities. This similarity permits the use of the CIA model as an investigative tool to test novel approaches for prevention and treatment of RA (Courtenay et al., Nature 283:666-668, 1980; Terato et al., J. Immunol., 148: 2103-2108, 1992; Terato et al., Autoimmun, 22: 137-147 1995).
The Exemplary Embodiments and Examples below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
1. A composition, including: a recombinant lactic acid bacterial cell including a nucleotide sequence encoding an engineered, single-chain mammalian IL-35.
2. The composition of embodiment 1, formulated for treatment of an autoimmune or inflammatory disease.
3. The composition of embodiment 1, wherein the recombinant lactic acid bacterial cell expresses at least one heterologous IL-35 nucleic acid encoding the engineered, single-chain mammalian polypeptide that includes the amino acid sequence shown in SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), SEQ ID NO: 7 (human), or a functionally equivalent variant of one of SEQ ID NO: 2, 11, 5, or 6 that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence.
4. The composition of embodiment 1, wherein the recombination lactic acid bacterial cell expresses an engineered, single-chain mammalian IL-35 protein including the amino acid sequence shown in SEQ ID NO: 2 (murine), SEQ ID NO: 5 (canine), or SEQ ID NO: 7 (human) from an engineered, single-chain mammalian IL-35 nucleic acid.
5. The composition of embodiment 4, wherein the engineered, single-chain mammalian IL-35 protein is expressed from a transgene including the nucleotide sequence of positions 31-1371 of SEQ ID NO: 1, positions 25-1332 of SEQ ID NO: 10, positions 25-1332 of SEQ ID NO: 4, positions 31-1395 of SEQ ID NO: 6, or a functionally equivalent variant thereof that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence.
6. The composition of embodiment 1, wherein the recombinant lactic acid bacterial cell is a cell from the genus Lactococcus.
7. The composition of embodiment 1, wherein the composition induces an anti-inflammatory response in a subject treated with the composition.
8. The composition of any of the above embodiments, wherein the nucleotide sequence encoding the engineered, single-chain mammalian IL-35 is integrated into the genome of the recombinant lactic acid bacterial cell.
9. The composition of any one of the above embodiments, which is a milk-based nutraceutical.
10. The composition of embodiment 9, wherein the milk-based nutraceutical is a fermented milk product.
11. The composition of embodiment 10, wherein the fermented milk product includes a yogurt, a buttermilk, a kefir, a cheese, a crème fraiche, or a cultured sour cream.
12. Use of the composition of any one of embodiments 1-11 for treating an inflammatory disease or condition.
13. The use of embodiment 12, wherein the inflammatory disease or condition includes one or more of an allergy, asthma, autoimmune arthritis, celiac disease, Crohn's disease, diabetes, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto's thyroiditis, and/or another endocrinopathy.
14. Use of the composition of any one of embodiments 1-11 for stimulating IL-10 and TGF-β-producing CD39+ regulatory T cells.
15. Use of the composition of any one of embodiments 1-11 for stimulating one or more regulatory cell subsets including Foxp3 (FOXP3)+ and Foxp3 (FOXP3)− T cells, CD25+ and CD25− T cells, regulatory B cells, and regulatory innate cells involved in producing a tolerogenic condition.
16. A recombinant lactic acid bacterial cell, including: a nucleotide sequence encoding an engineered, single-chain mammalian IL-35.
17. The recombinant lactic acid bacterial cell of embodiment 16, wherein the cell expresses at least one heterologous IL-35 nucleic acid encoding the engineered, single-chain mammalian polypeptide that includes the amino acid sequence shown in SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), SEQ ID NO: 7 (human), or a functionally equivalent variant of one of SEQ ID NO: 2, 11, 5, or 6 that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence.
18. The recombinant lactic acid bacterial cell of embodiment 16, wherein the recombinant lactic acid bacterial cell expresses SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), or SEQ ID NO: 7 (human) from an engineered, single-chain mammalian IL-35 nucleic acid.
19. The recombinant lactic acid bacterial cell of embodiment 16, wherein the nucleotide sequence encoding the engineered, single-chain mammalian IL-35 includes the sequence of positions 31-1371 of SEQ ID NO: 1, positions 25-1332 of SEQ ID NO: 10, positions 25-1332 of SEQ ID NO: 4, positions 31-1395 of SEQ ID NO: 6, or a functionally equivalent variant thereof that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence.
20. The recombinant lactic acid bacterial cell of embodiment 16, wherein the recombinant lactic acid bacterial cell is a cell from the genus Lactococcus.
21. The recombinant lactic acid bacterial cell of any of embodiments 16-20, wherein the nucleotide sequence encoding the engineered, single-chain mammalian IL-35 is integrated into the genome of the recombinant lactic acid bacterial cell.
22. A milk-based nutraceutical composition including: a recombinant lactic acid bacterial cell including a nucleotide sequence encoding an engineered, single-chain mammalian IL-35.
23. The composition of embodiment 22, wherein the recombinant lactic acid bacterial cell expresses at least one heterologous nucleic acid encoding the engineered, single-chain mammalian IL-35 polypeptide that includes the amino acid sequence shown in SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), SEQ ID NO: 7 (human), or a functionally equivalent variant of one of SEQ ID NO: 2, 11, 5, or 6 that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence.
24. The milk-based nutraceutical composition of embodiment 22, wherein the engineered, single-chain mammalian IL-35 includes the amino acid sequence of SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), or SEQ ID NO: 7 (human).
25. The milk-based nutraceutical composition of embodiment 22, wherein the nucleotide sequence includes the sequence of positions 31-1371 of SEQ ID NO: 1, positions 25-1332 of SEQ ID NO: 10, positions 25-1332 of SEQ ID NO: 4, positions 31-1395 of SEQ ID NO: 6, or a functionally equivalent variant thereof that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence.
26. The milk-based nutraceutical composition of embodiment 25, wherein the nucleotide sequence includes the sequence of SEQ ID NO: 1 (murine), SEQ ID NO: 10 (murine) SEQ ID NO: 4 (canine), or SEQ ID NO: 6 (human).
27. Use of the milk-based nutraceutical composition of embodiment 22-26 for treating an inflammatory disease or condition.
28. The use of embodiment 27, wherein an inflammatory disease or condition includes one or more of an allergy, asthma, autoimmune arthritis, celiac disease, Crohn's disease, diabetes, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto's thyroiditis, and/or another endocrinopathy.
29. Use of the milk-based nutraceutical composition of embodiment 22-26 for stimulating IL-10 and TGF-β-producing CD39+ regulatory T cells.
30. Use of the milk-based nutraceutical composition of embodiment 22-26 for stimulating one or more regulatory cell subsets including Foxp3 (FOXP3)+ and Foxp3 (FOXP3)− T cells, CD25+ and CD25− T cells, regulatory B cells, and regulatory innate cells involved in producing a tolerogenic condition.
31. A method for treating or preventing an autoimmune or inflammatory disease in a subject, including: administering to the subject the composition of any one of embodiments 1-11 or a composition including the recombinant lactic acid bacterial cell of any one of embodiments 16-21.
32. The method of embodiment 31, wherein the level of IL-10 in the subject is increased upon the administering, as compared to the level of the IL-10 present in the subject before the administering.
33. The method of embodiment 31, wherein the level of TGF-β in the subject is increased upon the administering, as compared to the level of the TGF-β present in the subject before the administering.
34. The method of embodiment 31, wherein the level of at least one of IFN-γ or IL-17 is decreased upon the administering, as compared to the level of at least one of IFN-γ or IL-17 present in the subject before the administering.
35. The method of embodiment 1, wherein the level of TNF-α is decreased upon the administering, as compared to the level of TNF-α present in the subject before the administering.
36. A method for producing a composition for the treatment of an autoimmune or inflammatory disease, including: introducing a nucleotide sequence encoding an engineered, single-chain mammalian IL-35 into a recipient lactic acid bacterial cell.
37. The method of embodiment 36, wherein the engineered, single-chain mammalian IL-35 expresses at least one engineered, single-chain mammalian IL-35 including the amino acid sequence of SEQ ID NO: 2 (murine), SEQ ID NO: 11 (murine), SEQ ID NO: 5 (canine), SEQ ID NO: 7 (human) or a functionally equivalent variant of one of SEQ ID NO: 2, 11, 5, or 6 that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence.
38. The method of embodiment 36, wherein the nucleotide sequence includes the sequence of positions 31-1371 of SEQ ID NO: 1, positions 25-1332 of SEQ ID NO: 10, positions 25-1332 of SEQ ID NO: 4, positions 31-1395 of SEQ ID NO: 6, or a functionally equivalent variant thereof that has at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to that sequence.
39. The method of embodiment 38, wherein nucleotide sequence includes the sequence of SEQ ID NO: 1 (murine), SEQ ID NO: 10 (murine), SEQ ID NO: 4 (canine), or SEQ ID NO: 6 (human).
40. The method of embodiment 36, further including: culturing the lactic acid bacterial cell under conditions which allow for expression of the engineered, single-chain mammalian IL-35.
41. The method of embodiment 36, wherein introducing the nucleotide sequence encoding the engineered, single-chain mammalian IL-35 includes integrating the encoding sequence into the genome of the recipient lactic acid bacterial cell.
IL-35, a relatively newly discovered cytokine belonging to the larger IL-12 family, shows unique anti-inflammatory properties, believed to be associated with dedicated receptors and signaling pathways. IL-35 plays a pivotal role in the development and the function of both regulatory B (Bregs) and T cells (Tregs). In order to further its therapeutic potential, a dairy Lactococcus lactis strain was engineered to express an engineered, single-chain murine IL-35 (LL-IL35), and this recombinant strain was applied to suppress collagen-induced arthritis (CIA). Oral administration of LL-IL35 effectively reduced the incidence and disease severity of CIA. When administered therapeutically, LL-IL35 abruptly halted CIA progression with no increase in disease severity by reducing neutrophil influx into the joints. LL-IL35 treatment reduced IFN-γ and IL-17 3.7- and 8.5-fold, respectively, and increased IL-10 production compared to diseased mice. Foxp3+ and Foxp3− CD39+ CD4+ T cells were previously shown to be the Tregs responsible for conferring protection against CIA. Inquiry into their induction revealed that both CCR6+ and CCR6− Foxp3+ or − CD39+ CD4+ T cells act as the source of the IL-10 induced by LL-IL35. Thus, this example demonstrates the feasibility and benefits of engineered probiotics, and in particular Lactococcus expressing an engineered, single-chain mammalian IL-35 protein, for treating autoimmune diseases. At least some of the material contained in this Example was published as Maddaloni et al. (Frontiers in Immunology, 9: Article 2691, 2018; doi:10.3389/fimmu.2018.02691).
Materials and Methods
Bacterial Strain Engineering and Maintenance: Lactococcus lactis subsp. lactis IL1403 (IL1403) was grown on M17 plus 0.5% glucose (M17G). Initial attempts to express IL-35 under the control of the constitutive p23 promoter yielded only rearranged, nonfunctional clones which confirmed the notion that IL-35 is difficult to express and to stabilize in a wide panel of hosts (unpublished observations; Huang et al., J Inflamm (Lond). 14:16, 2017; Kochetkova et al., J Immunol. 184(12):7144-53, 2010).
To express IL-35, a synthetic gene (SEQ ID NO: 1) codon-optimized for LL was designed in-house and then synthesized by GenScript (Piscataway, N.J.). The fragment contains an optimal Shine-Dalgarno sequence properly spaced from the ATG start codon, the usp45 secretion signal, the p35 coding region, a short flexible linker, the EBI3 coding region, and Agel and Smal sites at both ends. The fragment was excised with Agel, gel-purified and cloned into pMSP3535H3 (Kleinewietfeld et al., Blood 105(7):2877-86, 2005; a kind gift of Dr. DA Mills, University of California, Davis) yielding a construct named pBzMM150 (LL-IL35). Expression was achieved under the control of the nisin-inducible promoter (SEQ ID NO: 3) borne on the vector. The expressed single-chain IL-35 protein has the sequence shown in SEQ ID NO: 2.
Collagen-induced Arthritis (CIA): All the animal experiments described in the present study were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal studies were conducted under protocols approved by Montana State University's and the University of Florida's Institutional Animal Care and Use Committee.
C57BL/6 males (B6; 8- to 10-weeks of age; Charles River Laboratories, Horsham, Pa. USA) were maintained at Montana State University Animal Resources Center or the University of Florida Animal Center Services. Groups of B6 males were induced with CIA using 100 μg of chicken collagen II (CII; Chondrex, Redmond, Wash. USA) emulsified in complete Freund's adjuvant (CFA) and administered s.c. as previously described (Kochetkova et al., J Immunol. 184(12):7144-53, 2010; Kochetkova et al., J. Immunol. 187: 4654-4666, 2011; Kochetkova et al., J Immunol. 192(2):804-16, 2014). To treat CIA, mice were first orally gavaged with sterile 50% saturated sodium bicarbonate solution to neutralize stomach acidity, followed by 5×108 CFUs of LL vector or LL-IL35, or vehicle only, sterile PBS. Two dosing regimens were tested, three doses administered on days 14, 21, and 28, and two doses given on days 18 and 25 post-CII challenge. Clinical scores were measured in a double-blind fashion after treatment, and mice were monitored to day 40. Each of the four limbs was evaluated using a scale of 0-3 (Kochetkova et al., J Immunol. 184(12):7144-53, 2010; Kochetkova et al., J. Immunol. 187: 4654-4666, 2011; Kochetkova et al., J Immunol. 192(2):804-16, 2014): 0, no clinical signs; 1, mild redness of a paw or swelling of single digits; 2, significant swelling of ankle or wrist with erythema; 3, severe swelling and erythema of multiple joints; maximum score per mouse is 12.
Cytokine ELISA: CD4+ T cells were cell-sorted by negative selection on magnetic beads (Invitrogen, Grand Island, N.Y. USA) from axillary, popliteal, and inguinal lymph nodes (LNs) yielding purity >98%. Purified CD4+ T cells (3×106/ml) were restimulated with 5 μg/ml plate-bound anti-CD3 mAb (eBioscience, San Diego, Calif. USA) plus 5 μg/ml of soluble anti-CD28 mAb (eBioscience) for 48-72 hrs at 37° C. and 5% CO2 similar to that previously described (Kochetkova et al., J Immunol. 184(12):7144-53, 2010). Culture supernatants were collected for cytokine-specific ELISAs (Kochetkova et al., J Immunol. 184(12):7144-53, 2010; Kochetkova et al., J. Immunol. 187: 4654-4666, 2011; Kochetkova et al., J Immunol. 192(2):804-16, 2014).
Flow Cytometry: Splenic and LN cells were stained with fluorochrome-labeled mAbs to CD4, CD39, Ly-6G, Ly-6C, CD11b, and Foxp3 (eBioscience, San Diego, Calif.), TGF-ß (R&D Systems, Minneapolis, Minn. USA), and fluorochrome-conjugated streptavidin (BD Pharmingen, San Jose, Calif. USA). For flow cytometry of Tregs, whole splenic and LN cells (5×106/ml culture) were restimulated overnight with 50 μg/ml of CII (T-Cell Proliferation; Chondrex). The next day, cells were stimulated with 25 ng/ml PMA and 1 μg/ml ionomycin for an additional 3 hrs. Cells were harvested, washed, stained and analyzed as previously described (Kochetkova et al., J Immunol. 184(12):7144-53, 2010; Kochetkova et al., J. Immunol. 187: 4654-4666, 2011; Kochetkova et al., J Immunol. 192(2):804-16, 2014).
To measure inflammatory cells in the arthritic joints, isolated limb joints were digested with 2 mg/ml collagenase (Clostridium histolyticum, Type IV; Sigma-Aldrich, St. Louis, Mo.) for 30 min at 37° C., and cell suspensions passed through a 70 μm cell strainer similar to that previously described (Maddaloni et al., PLoS One. 10(1):e0117825, 2015; Rampersad et al., PLoS One 6(10):e25833, 2011). Leukocytes were stained and analyzed by forward and side-scatter plots for Ly-6G+ Ly-6C+ CD11b+ neutrophils.
Statistics: Mann-Whitney U test was applied to statistically analyze clinical scores. The difference in arthritis incidence between experimental groups was checked with Fisher's exact probability test. One-way ANOVA was performed to analyze ELISA and flow cytometry results. Data were considered statistically significant, if p-value was <0.05.
Results and Discussion
RA is a chronic, systemic autoimmune disorder affecting millions of patients in the US. Treatment of this progressive, degenerative disease demands constant use of anti-inflammatory drugs and often immunosuppressive treatments that increase susceptibility to infections and neoplasia (Carmona et al., Best Pract. Res. Clin. Rheumatol. 24: 733-745, 2010; Rosenblum & Amita, Autoimmun. Rev. 10: 563-568, 2011; Keyser, Curr. Rheumatol. Rev. 7: 77-87, 2011).
Instead, intervention strategies that focus on redirecting or reeducating T cell responses to produce tolerance instead of inflammation have the potential of being a superior treatment for RA.
To address the void for such tolerance induction, it was theorized that a probiotic LAB engineered to express the potent anti-inflammatory cytokine, IL-35 (
Using the three-dose regimen, 50% of the LL-IL35-treated mice showed no symptoms and the remaining 50% developed minor symptoms as opposed to PBS- or LL vector-treated mice, who all developed severe arthritis by day 24 post-CII challenge. Notably, the severity of disease symptoms was significantly less (p<0.001) in the LL-IL35-treated mice exhibiting an average clinical score of 1 in contrast to PBS- or LL vector-treated mice eventually achieving clinical scores of ˜9 (
Analysis of knee joints was performed to determine the extent of neutrophil infiltration. In agreement with these clinical findings, the LL-IL35-treated mice had markedly reduced Ly-6G+ CD11 b+ cells (neutrophils) infiltrating the joints (
To investigate the possible mechanism of protection conferred by LL-IL35, CD4+ T cells purified from draining LNs were anti-CD3+ anti-CD28-restimulated and analyzed for cytokine production. These CD4+ T cells were obtained from mice dosed three times with PBS, LL vector, or LL-IL35 as described in
CD39+ CD4+ T cells are the primary Tregs responsible for resolving CIA (Kochetkova et al., J Immunol. 184(12):7144-53, 2010; Kochetkova et al., J. Immunol. 187: 4654-4666, 2011; Kochetkova et al., J Immunol. 192(2):804-16, 2014). CD39 is an ectonucleoside triphosphate diphosphohydrolase-1 which hydrolyzes ATP into AMP, thus quenching inflammatory signaling by extracellular ATP (Borsellino et al., Blood 110: 1225-1232, 2007; Deaglio et al., J. Exp. Med. 204: 1257-1265, 2007). CD25+ Tregs remained a subset of CD39+ CD4+ T cells, and that CD39 encompassed all of the Treg subsets (Kochetkova et al., J. Immunol. 187: 4654-4666, 2011). In fact, CD39+ Tregs were protective against CIA (Maddaloni et al., PLoS One. 10(1):e0117825, 2015; Kochetkova et al., J. Immunol. 187: 4654-4666, 2011). These Tregs are composed of two subsets, Foxp3+ and Foxp3−, and are interchangeable (Kochetkova et al., J. Immunol. 187: 4654-4666, 2011). Analysis of induction of CD39+ Tregs by the LL vector revealed no increase in the percentage of these Tregs in CIA mice (Maddaloni et al., PLoS One. 10(1):e0117825, 2015), and CIA had only a modest impact upon their induction (Maddaloni et al., PLoS One. 10(1):e0117825, 2015; Kochetkova et al., J Immunol. 192(2):804-16, 2014).
To examine the types of Tregs induced by LL-IL35 treatment, whole splenic and draining LN lymphocytes were cultured overnight with CII, and then pulsed with PMA+ ionomycin to ascertain the type of Tregs induced in PBS-dosed and LL-IL35-treated mice. Lymphocytes were then stained for CD39, Foxp3, and CCR6 to identify the Treg subsets. Since CCR6 has been shown to be expressed by Tregs (Kleinewietfeld et al., Blood 105(7):2877-86, 2005; Rivino et al., J Exp Med. 207(3):565-77, 2010; Li et al., Int J Clin Exp Med. 8(9):15043-53, 2015), it was hypothesized that such Tregs may be induced as a consequence of IL-35 treatment. CD39+ CD4+ T cells were evaluated for expression of Foxp3 and CCR6 (
Additional analyses were performed on both CCR6− Foxp3+ and CCR6− Foxp3− CD39+ CD4+ T cells (
Inquiring into the activity of these LN CD39+ Tregs, analysis for IL-10 production was performed (
The data presented demonstrate the potency of IL-35 as an anti-inflammatory therapeutic. Moreover, this investigation further supports the multifaceted benefits of adapting recombinant L. lactis as a vector to deliver therapeutic doses of IL-35. In fact, previous studies by us (Kochetkova et al., J Immunol. 184(12):7144-53, 2010) or others, using IL-35 to treat type 1 diabetes model (Singh et al., Sci Rep. 5:12633, 2015), IBD (Wang et al., J Cell Mol Med. 22(2):1014-1025, 2018), or psoriasis (Wang et al., J Cell Mol Med. 22(2):1014-1025, 2018), required daily treatments with recombinant protein to control disease. In contrast, only two or three oral doses of LL-IL35 were sufficient to prevent the onset or stop CIA progression. Oral dosing has the substantial advantage of being less invasive circumventing the need for injections. LL-derived IL-35 eliminates the labor-intensive efforts needed to produce and purify the recombinant protein, dramatically reducing the cost of manufacturing this therapeutic. Moreover, IL-35 is a dimeric protein which adds to the difficulty and cost to generate. The L. lactis used for this study is a lab-adapted recombinant, originally derived from an industrial dairy strain capable of fermenting milk into a product that has the same textural and olfactory properties of yogurt. It has been demonstrated that the curative properties of recombinant L. lactis are maintained when grown on a synthetic medium or used to ferment milk into a yogurt-like product (Maddaloni et al., PLoS One. 10(1):e0117825, 2015). These attributes make L. lactis an ideal tolerogen delivery platform for the treatment of autoimmune diseases.
Using methods similar to those described in Example 1, another murine IL-35 fusion protein expression construct was constructed, in which the IL-35 subunits were in the reverse order—that is, EBI3/IL-27β followed by p35/IL-12α (see
The expression cassette sequence borne on pGnMM119 (shown in SEQ ID NOs: 10 & 11) and pGnMM121 (used in the current example) is identical. The difference is the backbone: pGnMM119 is the pUC-based construct/cloning vector bearing the synthetic gene (as it was provided by GenScript). The pGnMM121 bears the same sequence after Agel cloning (ACCGGT) into the pMSP3535H3 vector, which is the shuttle vector that was used for expression in Lactococcus.
In both cases (pBzMM150 and pGnMM121), IL-35 migrates slightly lower than expected. This may be an artifact caused by the folding properties of p35.
As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect, in this context, is a measurable alteration in the biological function of a described engineered, single-chain mammalian IL-35, a bacterial cell expressing that protein, or a composition containing the protein and/or a bacterial cell expressing it.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified.
Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.
It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.
The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).
This is the 371 U.S. National Phase of PCT/US2020/048467, filed Aug. 28, 2020; which claims priority to and the benefit of the earlier filing date of U.S. Provisional Application No. 62/893,074, filed on Aug. 28, 2019, which is incorporated by reference herein in its entirety.
This invention was made with government support under contract AR-058010 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/048467 | 8/28/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62893074 | Aug 2019 | US |
