FIG. 19. Inhaled IFNβ alleviated HDM-induced chronic asthma. A. Experimental protocol for treating HDM-induced chronic asthma with IFNβ. (n=4 mice/group) B. Haematoxylin and eosin (H&E) staining of lung sections from asthma mice in (A). Data are representative of three independent experiments. C. Serum levels of HDM-specific IgG1 from asthma mice in (A). Data are representative of three independent experiments. D. Absolute numbers of eosinophils in the BALF from the asthma mice in (A). E. Flow cytometry analysis of lung IL-4-producing CD4⁺ T cells re-stimulated with HDM ex vivo from asthma mice in (A). Data are representative of three independent experiments. F. Serum levels of HDM-specific IgE from asthma mice in (A). Data are representative of three independent experiments. G. Absolute numbers of neutrophils in the BALF from the asthma mice in (A). H. Flow cytometry analysis of lung IL-17-producing CD4⁺ T cells re-stimulated with HDM ex vivo from asthma mice in (A). Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by unpaired student t-test. *P<0.05.

FIG. 20. Inhaled IFNβ prevented HDM-induced asthma attack. A. Experimental protocol for treating HDM-induced acute asthma with IFNβ. (n=4 mice/group) B. Haematoxylin and eosin (H&E) staining of lung sections from asthma mice in (A). Data are representative of three independent experiments. C-D. Serum levels of HDM-specific IgG1 (C) and IgE (D) from asthma mice in (A). Data are representative of three independent experiments. D. Absolute numbers of eosinophils in the BALF from the asthma mice in (A). E. Flow cytometry analysis of lung IL-4-producing CD4⁺ T cells re-stimulated with HDM ex vivo from asthma mice in (A). Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by unpaired student t-test. *P<0.05.

FIG. 21. Inhaled IFNβ generated lung regulatory T cells in HDM-induced asthma mice. A. Absolute number of Tregs in the lungs from HDM-induced asthma mice in FIGS. 1&2&3. (n=4 mice/group) Data are representative of three independent experiments. B. Experimental protocol for R2D2 cell depletion in HDM-induced asthma mice treated with IFNβ/isotype control or IFNβ and anti-TNFR2 antibody (20 μg). (n=4 mice/group). C. Flow cytometry plot of TNFR2⁺ cDC2 on day 33 from mice in (B). Data are representative of three independent experiments. D. Flow cytometry plot of Foxp3⁺ CD4⁺ T cells on day 33 from mice in (B). Data are representative of three independent experiments. E. Serum levels of HDM-specific IgG1 from asthma mice in (B). Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by unpaired student t-test. *P<0.05.

FIG. 22. Inhaled IFNβ reprogramed T_H2-promoting pathogenic lung R2D2 cells from asthma mice to generate regulation T cells in the lung. A. Experimental protocol for adoptively transfer (i.n.) lung R2D2 cells from HDM-induced asthma mice (CD45.1+) into a naïve C57BL/6J or IFNAR1^−/− mouse (CD45.2+). 24 hrs later, the recipient mice were treated with IFNβ (200 ng, i.n.). Lung R2D2 cells were analyzed 24 hours later. Lung CD4+ T cells were analyzed 14 days later. (n=4mice/group) B. Flow cytometry analysis of IL-4+ or Foxp3+CD4+ T cells in the lung from the C57BL/6J recipient mice in (A). Data are representative of three independent experiments. C. Flow cytometry analysis of lung CD45.1+R2D2 cells in the lung from the C57BL/6J recipient mice in (A). Data are representative of three independent. D. Flow cytometry analysis of IL-4+ or Foxp3+CD4+ T cells in the lung from the IFNAR1^−/− recipient mice in (A). Data are representative of three independent experiments. E. Flow cytometry analysis of lung CD45.1+R2D2 cells in the lung from the IFNAR1^−/− recipient mice in (A). Data are representative of three independent. F. Total recovered CD45.1 R2D2 from the recipient mice. in (A). Data are representative of three independent. Graphs represent the mean with error bars indication s.e.m. P values determined by unpaired student t-test. *P<0.05.

FIG. 23. Inhaled IFNβ prevents HDM-induced asthma. A. Experimental protocol for prevent HDM-induced asthma with IFNβ. (n=4 mice/group) B. Lung function were determined by an ex vivo lung perfusion system (EVLP) (Hugo Sachs Elektronik, March-Huggstetten, Germany) from mice in (A). Data are representative of three independent experiments. Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by unpaired student t-test. *P<0.05.

FIG. 24. Inhaled IFNβ-TNF_mutfusion protein prevents HDM-induced asthma attack. A. Proposed mode of action for IFNβ-TNF_mutfusion protein. B. Experimental protocol for prevent HDM-induced asthma with IFNβ-TNF_mut. (n=4 mice/group) C. Haematoxylin and eosin (H&E) staining of lung sections from asthma mice in (B). Data are representative of three independent experiments. D. Lung function were determined by an ex vivo lung perfusion system (EVLP) (Hugo Sachs Elektronik, March-Huggstetten, Germany) from mice in (B). Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by unpaired student t-test. *P<0.05.

FIG. 25. Inhaled IFNβ-TNF_mutinduces long-lasting remission in asthma. A. Experimental protocol for treat HDM-induced asthma with IFNβ-TNF_mut. (n=4 mice/group) B. Haematoxylin and eosin (H&E) staining of lung sections from asthma mice in (A). Data are representative of three independent experiments.C. Lung function were determined by an ex vivo lung perfusion system (EVLP) (Hugo Sachs Elektronik, March-Huggstetten, Germany) from mice in (A). Data are representative of three independent experiments. Graphs represent the mean with error bars indication s.e.m. P values determined by unpaired student t-test. *P<0.05.

FIG. 26. IFNβ reprogramed pathogenic lung R2D2 cells from human lung disease patients. A,C. Flow cytometry analysis of TNFR2+ cDC2 in healthy, emphysema human lungs (A) or chronic lung rejection patients (C). Data were compiled from multiple independent experiments.B,D. Flow cytometry analysis of TNFR2+ cDC2 in human lung cells from emphysema (B) or chronic lung rejection patients (D) treated with human recombinant IFNβ. Data were compiled from multiple independent experiments.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DETAILED DESCRIPTION
Overview

Asthma is a chronic disease that inflames and narrows the airway of lungs and makes breathing difficult. There are more than 3 million cases of asthma each year in U.S. Here, disclosed is a new therapy to treat asthma based on a novel in vivo mechanism controlling lung T-regulatory cells (Tregs) induction. Specifically, the disclosure is based on two new discoveries. First, it was found that the new lung-resident Mgl2+IDO-1+Arginase-1+pRelB+TNFR2+ conventional DC2 subpopulation (R2D2, see FIG. 15) controls lung T-regulatory cell production. Second, it was found that IFNβ-IFNAR1 signaling in R2D2 controls its ability to induce lung T regulatory cells. See Mansouri S, Katikaneni D S, Gogoi H, Pipkin M, Machuca T N, Emtiazjoo A M and Jin I, Mucosal Immunol. 2020 Jan. 20. doi: 10.1038/s41385-020-0254-1. Based on these mechanistic insights, a new strategy was developed involving the use of intranasal administrated IFNβ and R2D2-targeted IFNβ-TNFmut fusion protein to promote lung T-regulatory cells induction and prevent and treat asthma.

As described herein, the inventors identified a lung IFNAR1^hiTNFR2⁺ cDC2 population (iR2D2) as the tolerogenic DC generating antigen-specific lung T-regs and preventing lung inflammation at steady-state. Our conclusion is based exclusively on studies in the lung in vivo. Our conclusion is different from previous reports suggesting pDCs (de Heer H J, Hammad H, Soullie T, Hijdra D, Vos N, Willart M A et al. Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen. J Exp Med 2004; 200(1): 89-98), cDC1 (Khare A, Krishnamoorthy N, Oriss T B, Fei M, Ray P, Ray A. Cutting edge: inhaled antigen upregulates retinaldehyde dehydrogenase in lung CD103+ but not plasmacytoid dendritic cells to induce Foxp3 de novo in CD4+ T cells and promote airway tolerance. J Immunol 2013; 191(1): 25-29) or macrophage (Soroosh P, Doherty T A, Duan W, Mehta A K, Choi H, Adams Y F et al. Lung-resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance. J Exp Med 2013; 210(4): 775-788) induces T-regs in the lung. We showed that mice lacking the iR2D2 population by gene ablation or antibody depletion failed to generate lung T-regs while intranasal administration of IFNβ induced R2D2-dependent lung T-regs. It is noteworthy that lung R2D2 cells, including iR2D2 depend on the constitutive TNFR2 signaling. R2D2 can't survive in the ex vivo cells co-culture system, which was used by the previous investigations.

The lung iR2D2 population is plastic. iR2D2 is a subpopulation of cDC2. Previous studies established that cDC2 mediates T_H2, T_H17 responses. We also showed that R2D2 promotes T_Hresponses of the mucosal adjuvant cyclic di-GMP in vivo. We propose that DCs at the barrier surface, influenced by their microenvironment, are plastic inducing T-regs, maintaining peripheral tolerance at the steady-state but promoting immunogenic responses during pathogenic conditions.

The tolerogenic iR2D2 population is a product of two constitutive signals from its microenvironment: IFNβ-IFNAR1 and tmTNF-TNFR2. Using a TNFR2 agonist, we showed that TNFR2 signaling promoted R2D2 proliferation. TNFR2 expression defines a highly immunosuppressive T-regs found in tumor microenvironment promoting cancer cell survival and tumor growth ^{37, 38}. Similar to our R2D2 cells, TNFR2 activation stimulates the proliferation of TNFR2⁺ Tregs ³⁹. Antagonistic antibodies against human TNFR2 inhibited TNFR2⁺ T-regs proliferation³⁹. The underlying molecular mechanism of TNFR2 signaling induced cell proliferation is unknown. Elucidating the TNFR2 signaling responsible for maintaining this R2D2 population in the lung is a future priority.

R2D2 cells with active IFNAR1 signaling become iR2D2. iR2D2 intrinsic IFNβ signaling is necessary and sufficient for lung T-regs induction via the production of TGFβ1. However, the molecular pathway by which IFNβ-IFNAR1 signaling leading to TGFβ1 production is unknown. IFNβ has an immunomodulatory effect (Kasper L H, Reder A T. Immunomodulatory activity of interferon-beta. Ann Clin Transl Neurol 2014; 1(8): 622-631). In the clinic, IFNβ (Avonex®, Rebif®) has been used to treat relapsed Multiple Sclerosis for over 20 years. Nevertheless, the in vivo mechanism and targeted cells of IFNβ treatment remain poorly defined (Kasper et al. 2014, supra). We propose that lung R2D2 or R2D2-like cells in other peripheral organs are the effector cells for the immunomodulatory function of IFNβ in vivo. Noteworthy, IFNα is not approved for Multiple Sclerosis. Future studies should also determine if IFNβ activates a unique IFNAR1 signaling in iR2D2 cells to control the tolerogenic program at steady-state.

Lung epithelial cells are a major source of tmTNF and the sole producer of lung IFNβ at the steady-state. Impaired bronchial epithelial cells (BECs) IFNβ production has been well-documented in asthmatic patients (Baraldo S, Saetta M, Barbato A, Contoli M, Papi A. Rhinovirus-induced interferon production in asthma. Thorax 2014; 69(8): 772). We thus introduce a new paradigm that at steady-state, lung epithelial cells express tmTNF to keep R2D2 alive and continuously secret IFNβ to condition R2D2 to generate lung T-regs and prevent lung inflammation (FIG. 8). Different from the current lung-epithelial cells-DCs-T_H2 axis during inflammation (Deckers J, De Bosscher K, Lambrecht B N, Hammad H. Interplay between barrier epithelial cells and dendritic cells in allergic sensitization through the lung and the skin. Immunol Rev 2017; 278(1): 131-144), the lung epithelial cells IFNβ-iR2D2-T-regs axis here is tolerogenic and needed to be constitutively active. Loss of tmTNF or IFNβ will result in the loss of iR2D2 and lung mucosal tolerance. Indeed, BECs from asthma patients are biased towards higher TSLP and lower IFNβ production (Uller L, Leino M, Bedke N, Sammut D, Green B, Lau L et al. Double-stranded RNA induces disproportionate expression of thymic stromal lymphopoietin versus interferon-beta in bronchial epithelial cells from donors with asthma. Thorax 2010; 65(7): 626-632).

The plasticity of R2D2 cells makes them an ideal immunotherapy target for chronic inflammatory diseases. We showed that IFNβ administration could enhance lung T-regs induction. A phase II clinical trial of inhaled IFNβ for virus-induced asthma exacerbation showed promising results (clinicaltrials.gov, identifier NCT 01126177) (Djukanovic R, Harrison T, Johnston S L, Gabbay F, Wark P, Thomson N C et al. The effect of inhaled IFN-beta on worsening of asthma symptoms caused by viral infections. A randomized trial. Am J Respir Crit Care Med 2014; 190(2): 145-154. Jackson D J. Inhaled interferon: a novel treatment for virus-induced asthma? Am J Respir Crit Care Med 2014; 190(2): 123-124). Translationally, targeting IFNβ directly to R2D2 will limit the toxicity of IFNβ and improve its efficacy. On the other hand, for studies exploring the therapeutic potential of ex vivo generated tolerogenic DCs, caution should be taken. Due to the plasticity of tolerogenic DCs, these ex vivo generated tolerogenic DCs maybe plastic and could be re-conditioned by the inflammatory milieu and become immunogenic in vivo.

In summary, we identified iR2D2 as the tolerogenic lung DC population and surprisingly found it is plastic. This newly discovered DC plasticity can lead to novel anti-inflammatory therapy for lung diseases.

Definitions

Unless otherwise defined, 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The term “asthma” as used herein may pertain to an exacerbation of asthma, intermittent asthma or chronic asthma.

The term “inflammatory respiratory disorder” as used herein includes asthma and chronic obstructive pulmonary disease (COPD).

The term “interferon-beta”, “interferonβ,” “IFNB” and “IFN-β” as used interchangeably herein to refer to any form or analog of IFN-β that retains the biological activity of native IFN-β and preferably retains the activity of IFN-β that is present in the lung and, in particular, the bronchial and/or alveolar epithelium. The IFN-β may be identical to or comprise the sequence of human IFN-β1a (SEQ ID NO: 2) or human IFN-β1b (SEQ ID NO: 3), or fragments thereof.

The term “interferon-beta related agent” “IFN-β related agent” or “IFNB related agent” are used interchangeable herein and are construed to include IFNB, IFNB conjugates and fusion proteins, and agents that increase IFN-β expression as further described herein.

The term “IFN-TNF fusion protein” as used herein refers to a IFNB fusion protein comprising an amino acid sequence encoding IFN-β, a fragment thereof, or a variant thereof having at least 90% or 95% identity with IFN-β or fragment thereof, linked to an amino acid sequence encoding TNF, a fragment thereof or a variant thereof having at least 90% or 95% identity with TNF or fragment thereof. In a specific embodiment, the IFN-β portion is linked to the TNF portion via an amino acid sequence comprising at least 5 amino acids. Typically, the linker is 5-30 amino acids long. FIG. 17 sets forth examples of fusion proteins that pertain to human and mouse IFN-β and TNF sequences. SEQ ID NO. 4 pertains to the highlighted segment of the human IFN-β sequence. SEQ ID NO. 5 pertains to the highlighted segment of the human TNF sequence. Thus, a specific example of an IFN-TNF fusion protein refers to SEQ ID NO. 4, fragment thereof, or variant having at least 90% or 95% identity therewith linked to SEQ ID NO. 5, or fragment thereof, or variant having at least 90% or 95% identity therewith. In an even more specific embodiment, the fusion protein pertains to SEQ ID NO. 4 linked with SEQ ID NO. 5. An example of a linker sequence is provided in FIG. 17 and pertains to a repeat of GGGGS (e.g. 3×GGGGS). FIG. 17 also sets forth a mouse IFN-TNF fusion protein example. SEQ ID NO. 6 pertains to the highlighted segment of the mouse IFN-β sequence and SEQ ID NO. 7 pertains to the highlighted segment of the mouse TNF sequence. A specific example of a mouse IFN-TNF fusion protein is SEQ ID NO. 6, or fragment thereof, or variant comprising at least 90% or 95% identity with SEQ ID NO. 6 or fragment thereof, linked with SEQ ID NO. 7 or fragment thereof, or a variant comprising at least 90% or 95% identity therewith.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide. Preferred “peptides”, “polypeptides”, and “proteins” are chains of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term “amino terminus” (abbreviated N-terminus) refers to the free α-amino group on an amino acid at the amino terminal of a peptide or to the α-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term “carboxy terminus” refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to peptide mimetics such as amino acids joined by an ether as opposed to an amide bond.

As used herein, an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these regions of the light and heavy chains respectively.

Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases or expressed de novo. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′₂, a dimer of Fab which itself is a light chain joined to V_H-C_H1 by a disulfide bond. The F(ab)′₂may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab′)₂dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab′ fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies, including, but are not limited to, Fab′2, IgG, IgM, IgA, IgE, scFv, dAb, nanobodies, unibodies, and diabodies. In various embodiments preferred antibodies include, but are not limited to Fab′2, IgG, IgM, IgA, IgE, and single chain antibodies, more preferably single chain Fv (scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.

The term “variant” as used herein in the context of an nucleic acid sequence or amino acid sequence that has at least 80% identity to that sequence based on amino acid identity. More preferably, the polypeptide has at least 85% or 90% and more preferably at least 95%, 97% or 99% identity based on amino acid identity to the amino acid sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 40 or more, for example 60, 80, 100, 120, 140 or 160 or more, contiguous amino acids (“hard homology”).

The term “fragment” as used in the context of amino acid sequences refers to seqgment of contiguous amino acid residues of a given amino acid sequence. Fragments may be at least from 80, 100, 120, 130, or 140 amino acids in length, or greater. Such fragments may be used to produce chimeric agents as described in more detail below.

The term “a subject in need” refers to a human or non-human mammal that exhibits one or more symptoms of an inflammatory respiratory disorder.

Sequences

Sequence information is provided in FIG. 16 and FIG. 17, as well as the attendant sequence listing. For the purposes of comparing two closely-related polypeptide or polynucleotide sequences, the “% sequence identity” (or “% identity”, used interchangeably herein) between a first sequence and a second sequence may be calculated. Polypeptide or polynucleotide sequences are said to be the same as or identical to other polypeptide or polynucleotide sequences, if they share 100% sequence identity over their entire length. Residues in sequences are numbered from left to right, i.e. from N- to C-terminus for polypeptides; from 5′ to 3′ terminus for polynucleotides. The terms “identical” or percentage “identity”, in the context of two or more polypeptide sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of amino acid residues that are the same (i.e., 70% identity, optionally 75%, 80%, 85%, 90%, 95%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the compliment of a test sequence. Optionally, the identity exists over a region that is at least 250 amino acids in length, such as 300 amino acids or 350 amino acids. Suitably, the comparison is performed over a window corresponding to the entire length of the reference sequence (as opposed to the derivative sequence).

For sequence comparison, one sequence acts as the reference sequence, to which the test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percentage sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, refers to a segment in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman 1981, by the homology alignment algorithm of Needleman & Wunsch 1970, by the search for similarity method of Pearson & Lipman 1988, by computerised implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al. 1995).

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle 1987. The method used is similar to the method described by Higgins & Sharp 1989. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid coordinates for regions of sequence comparison and by designating the program parameters. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al. 1984).

Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. 1977 and Altschul et al. 1990, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (website at www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al., supra). These initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff 1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

A “difference” between sequences refers to an insertion, deletion or substitution of a single residue in a position of the second sequence, compared to the first sequence. Two sequences can contain one, two or more such differences. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity. For example, if the identical sequences are 9 residues long, one substitution in the second sequence results in a sequence identity of 88.9%. If the identical sequences are 17 amino acid residues long, two substitutions in the second sequence results in a sequence identity of 88.2%.

Alternatively, for the purposes of comparing a first, reference sequence to a second, comparison sequence, the number of additions, substitutions and/or deletions made to the first sequence to produce the second sequence may be ascertained. An addition is the addition of one residue into the first sequence (including addition at either terminus of the first sequence). A substitution is the substitution of one residue in the first sequence with one different residue. A deletion is the deletion of one residue from the first sequence (including deletion at either terminus of the first sequence). Suitably, a substitution may be conservative. A ‘conservative’ substitution is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which is expected to have little influence on the function, activity or other biological properties of the polypeptide. Such conservative substitutions suitably are substitutions in which one amino acid within the following groups is substituted by another amino acid residue from within the same group:

Amino acid

Group
residue

Non-polar aliphatic
Glycine

Alanine

Valine

Leucine

Isoleucine

Aromatic
Phenylalanine

Tyrosine

Tryptophan

Polar uncharged
Serine

Methionine

Cysteine

Threonine

Asparagine

Glutamine

Negatively charged
Aspartate

Glutamate

Positively charged
Lysine

Arginine

Histidine

Interferon-Beta

IFNB as defined in SEQ ID NOs: 2 and 3 may be administered in therapeutically effective amounts via an appropriate mode of administration. In addition to the native sequences, variants retaining the biological function of IFNB may be administered as well. Accordingly, IFN-β also refers to a variant polypeptide having an amino acid sequence which varies from that of SEQ ID NO: 2 or 3. Alternatively, IFN-β may be chemically-modified.

A variant of IFN-β may be a naturally occurring variant, for example a variant which is expressed by a non-human species. Also, variants of IFN-β include sequences which vary from SEQ ID NO: 2 or 3 but are not necessarily naturally occurring. Over the entire length of the amino acid sequence of SEQ ID NO: 2 or 3, a variant will preferably comprise at least 80% identity to that sequence based on amino acid identity. More preferably, the polypeptide has at least 85% or 90% and more preferably at least 95%, 97% or 99% identity to the amino acid sequence of SEQ ID NO: 2 or 3 over the entire sequence, or to a fragment thereof. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 40 or more, for example 60, 80, 100, 120, 140 or 160 or more, contiguous amino acids (“hard homology”).

One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 or 3 may alternatively or additionally be deleted. From 1, 2, 3, 4 or 5 to 10, 20 or 30 residues may be deleted, or more. IFN-β also includes fragments of the above-mentioned sequences. Such fragments retain IFN-β activity. Fragments may be at least from 120, 130, or 140 amino acids in length. Such fragments may be used to produce chimeric agents as described in more detail below.

IFN-β includes chimeric proteins comprising fragments or portions of SEQ ID NO: 2 or 3. One or more amino acids may be alternatively or additionally added to the polypeptides described above. An extension may be provided at the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 2 or 3, or polypeptide variant or fragment thereof. The extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer. A carrier protein may be fused to an amino acid sequence described above. A fusion protein incorporating one of the polypeptides described above can thus be used in the invention.

IFN-β also includes SEQ ID NO: 2 or 3 or variants thereof that have been chemically-modified. A number of side chain modifications are known in the art and may be made to the side chains of the proteins or peptides discussed above. Such modifications include, for example, glycosylation, phosphorylation, modifications of amino acids by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄, amidination with methylacetimidate or acylation with acetic anhydride. The modification is preferably glycosylation. The IFN-β may be made synthetically or by recombinant means using methods known in the art. The amino acid sequence of proteins and polypeptides may be modified to include non-naturally occurring amino acids or to increase the stability of the compound. When the proteins or peptides are produced by synthetic means, such amino acids may be introduced during production. The proteins or peptides may also be modified following either synthetic or recombinant production. The IFN-β may also be produced using D-amino acids. In such cases the amino acids will be linked in reverse sequence in the C to N orientation. This is conventional in the art for producing such proteins or peptides.

The IFN-β may be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. The IFN-β or analog thereof may be produced in large scale following purification by any protein liquid chromatography system after recombinant expression. Preferred protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-RadBioLogic system and the Gilson HPLC system. Commercially available forms of IFN-β or analogs thereof may be used in the invention. Examples include Betaseron® and Avonex®.

IFNB Fusion Proteins

In various embodiments, this disclosure pertains to the discovery that attaching an interferon to a targeting moiety (e.g., a molecule that specifically and/or preferentially binds a marker on or associated with a cell) substantially improves the therapeutic efficacy of the interferon and appears to reduce systemic toxicity. Accordingly, certain embodiments pertain to constructs comprising IFNB attached to a targeting moiety and uses of such constructs to specifically and/or preferentially target R2D2 cells. Accordingly, in certain embodiments, a chimeric construct is provided where the construct comprises IFNB attached to a targeting moiety that binds to TNFR2, where the construct when contacted to a R2D2 cells promotes induction of lung T-regulatory cells. In certain embodiments the targeting moiety is chemically coupled to the IFNB. In certain embodiments the targeting moiety is joined to IFNB with a peptide linker. In certain embodiments the peptide linker is fewer than 15, fewer than 14, fewer than 12, fewer than 11, fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, fewer than 3, or fewer than 2 amino acids in length. In certain embodiments the linker is 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in length. In certain embodiments the linker is not (Gly₄Ser)₃(See U.S. Pat. No. 8,258,263 ('263 patent) incorporated herein in its entirety). In certain embodiments the linker is a linker that is resistant or substantially resistant to proteolysis. In certain embodiments the peptide linker is Gly₄Ser. In certain embodiments the linker comprises or consists of an amino acid sequence found in Table 4 of the '263 patent. In certain embodiments the construct is a recombinantly expressed fusion protein. In certain embodiments the targeting moiety is an Anti-TNFR2 blocking antibody or an Anti-Mgl2 antibody.

Generally speaking, the targeting moiety (e.g., Anti-TNFR2 or anti-Mgl2) can be joined together in any order. Thus, for example, the antibody can be joined to either the amino or carboxy terminal of IFNB. The antibody can also be joined to an internal region of IFNB, or conversely, IFNB can be joined to an internal location or to any terminus of the antibody, as long as the attachment does not interfere with binding of the antibody to its target.

The antibody and IFNB can be attached by any of a number of means well known to those of skill in the art. In certain embodiments, IFNB is conjugated, either directly or through a linker (spacer), to the antibody. In certain embodiments, however, it is preferable to recombinantly express the chimeric moiety as a fusion protein.

In certain embodiments, the targeting moiety (e.g., Anti-TNFR2 or anti-Mgl2) is chemically conjugated to IFNB. Means of chemically conjugating molecules are well known to those of skill. The procedure for conjugating two molecules varies according to the chemical structure of the agent. Polypeptides typically contain variety of functional groups; e.g., carboxylic acid (COOH) or free amine (—NH₂) groups, that are available for reaction with a suitable functional group on the other peptide, or on a linker to join the molecules thereto. Alternatively, the antibody and/or IFNB can be derivatized to expose or attach additional reactive functional groups. The derivatization can involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Ill.

A “linker”, as used herein, typically refers to a molecule that is used to join the antibody to the interferon. In various embodiments, the linker is capable of forming covalent bonds to both the antibody and to the interferon. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In certain embodiments, the linker(s) can be joined to the constituent amino acids of the antibody and/or the interferon through their side groups (e.g., through a disulfide linkage to cysteine). In certain preferred embodiments, the linkers are joined to the alpha carbon amino and/or carboxyl groups of the terminal amino acids of the antibody and/or the interferon.

A bifunctional linker having one functional group reactive with a group on the antibody and another group reactive on the interferon, can be used to form the desired conjugate. Alternatively, derivatization can involve chemical treatment of the targeting moiety. Procedures for generation of, for example, free sulfhydryl groups on polypeptides, such as antibodies or antibody fragments, are known (See U.S. Pat. No. 4,659,839).

Many procedures and linker molecules for attachment of various compounds including radionuclide metal chelates, toxins and drugs to proteins such as antibodies are known. See, for example, European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987) Cancer Res. 47: 4071-4075. In particular, production of various immunotoxins is well-known within the art and can be found, for example in “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,” Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982); Waldmann (1991) Science, 252: 1657; U.S. Pat. Nos. 4,545,985 and 4,894,443, and the like.

In certain embodiments, a chimeric targeting moiety-interferon fusion protein is synthesized using recombinant DNA methodology. Generally this involves creating a DNA sequence that encodes the fusion protein, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein. DNA encoding the fusion proteins (e.g., Anti-TNFR2 or anti-Mgl2) described herein can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862); the solid support method of U.S. Pat. No. 4,458,066, and the like.

Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

Alternatively, subsequences can be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments can then be ligated to produce the desired DNA sequence.

In certain embodiments, DNA encoding fusion proteins of the present invention can be cloned using DNA amplification methods such as polymerase chain reaction (PCR). Thus, for example, the gene for IFNB is PCR amplified, using a sense primer containing the restriction site for, e.g., Ndel and an antisense primer containing the restriction site for HindIII. This can produce a nucleic acid encoding the mature interferon sequence and having terminal restriction sites. An antibody having “complementary” restriction sites can similarly be cloned and then ligated to the interferon and/or to a linker attached to the interferon. Ligation of the nucleic acid sequences and insertion into a vector produces a vector encoding the interferon joined to the antibody.

While the two molecules can be directly joined together, one of skill will appreciate that the molecules can be separated by a peptide spacer consisting of one or more amino acids. Generally, the spacer will have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. In certain embodiments, however, the constituent amino acids of the spacer can be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.

According to a specific example, the IFNB fusion protein comprises an IFN-TNF fusion protein. In a specific embodiment, the fusion protein includes SEQ ID NO. 4, fragment thereof, or variant having at least 90% or 95% identity therewith linked to SEQ ID NO. 5, or fragment thereof, or variant having at least 90% or 95% identity therewith. In an event more specific embodiment, the fusion protein pertains to SEQ ID NO. 4 linked with SEQ ID NO. 5. An example of a linker sequence is provided in FIG. 17 and pertains to a repeat of GGGGS (e.g. 3×GGGGS). A specific example of a mouse IFN-TNF fusion protein is SEQ ID NO. 6, or fragment thereof, or variant comprising at least 90% or 95% identity with SEQ ID NO. 6 or fragment thereof, linked with SEQ ID NO. 7 or fragment thereof, or a variant comprising at least 90% or 95% identity therewith.

Agents that Increase IFN-β Expression

In addition to administration of IFBN, other related embodiments involve administrating an agent that increases endogenous expression of IFN-β in the lung or preferably the bronchial and/or alveolar epithelium, these include agents that induce expression of endogenous IFNB, expression of heterologous sequence (gene therapy agents), and vectors that harbor IFNB expression constructs. As such, agents that increase IFN-β expression are included as INFB related agents.

The agents may act directly on the promoter or other regulatory sequences of the IFN-β gene. Such agents may act to reduce the constitutive silencing of the IFN-β promoter. Alternatively, the agent may stimulate cells to produce endogenous IFN-β by acting at receptors at the cell surface. Agents that increase endogenous expression of IFN-β of interest in relation to the present invention include, but are not limited to, poly(inosinic acid)-poly(cytidylic acid) (poly(IC)), ANA773, perindopril, BL-20803, Tilorone, ABMP, DRB, Atabrine, 10-carboxy-9acridone, CP-28888, Bropirimine, and Imiquimod.

The embodiments may also involve using a polynucleotide which is capable of expressing IFN-β or an agent that increases endogenous expression of IFN-β in lung airways. Such a polynucleotide may preferably be in the form of a vector capable of directing expression of IFN-β or an agent that induces IFN-β in the bronchial and/or alveolar epithelium. The resulting IFN-β or agent may then have a therapeutic effect (“gene therapy”). The polynucleotide may encode any of the forms of IFN-β discussed above including the variants, fragments and chimeric proteins thereof. The polynucleotide encoding IFN-β may comprise a polynucleotide encoding a human sequence (SEQ ID NO: 2 or 3) or a naturally occurring sequence variant, for example a variant which is expressed by a non-human species. Also, a polynucleotide encoding IFN-β includes sequences that encode variant of SEQ ID NO: 2 or 3 but are not necessarily naturally occurring. Over the entire length of the amino acid sequence of SEQ ID NO: 2 or 3, a variant will preferably have at least 80% identity to that sequence. More preferably, the variant has at least 85% or 90% and more preferably at least 95%, 97% or 99% identity to the amino acid sequences of SEQ ID NO: 2 or 3 over the entire sequence.

The polynucleotides may comprise DNA or RNA but preferably comprise DNA. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to polynucleotides are known in the art. These include methylphosphate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3′ and/or 5′ ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Polynucleotides such as a DNA polynucleotide may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. The polynucleotides are typically provided in isolated and/or purified form. Polynucleotides will generally be produced using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the required gene which it is desired to clone, bringing the primers into contact with DNA obtained from a suitable cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al, 1989. As hereinbefore indicated, preferably the polynucleotide is used in an expression vector wherein it is operably linked to a control sequence which is capable of providing for the expression of the coding sequence in the airways of human lung.

Expression vectors for use in accordance with the invention may be any type of vector conventionally employed for gene therapy. It may be a plasmid expression vector administered as naked DNA or complexed with one or more cationic amphiphiles, e.g. one or more cationic lipids, e.g. in the form of DNA/liposomes. A viral vector may alternatively be employed. Vectors for expression of therapeutic proteins in the airways of human lung have previously been described. For example, Published International Application WO 01/91800 (Isis Innovation Limited) describes for such purpose expression vectors including the human ubiquitin C promoter or functional analogues thereof. The human ubiquitin C promoter has been shown to be capable of producing high level protein expression in the airways of mice over many weeks and hence has been proposed as a favoured promoter for use in airway gene therapy for a variety of respiratory diseases. Examples of expression vectors for use in directing transgene expression in airway epithelia have also been described in Chow et al. Proc. Natl. Acad. Sci. USA 1997; 94: 14695-14700. Such expression vectors can be administered via the airways, e.g. into the nasal cavity or trachea.

Compositions and Therapeutic Methods

The IFN-β related agent may be administered in a medicament or pharmaceutical composition suitable for airway delivery which will typically also include a pharmaceutically acceptable excipient. Such an “excipient” generally refers to a substantially inert material that is nontoxic and does not interact with other components of the composition in a deleterious manner. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. It is also preferred, although not required, that a composition or medicament comprising the therapeutic agent will contain a pharmaceutically acceptable carrier that serves as a stabilizer, particularly for peptide, protein, polynucleotide or other like agents. Examples of suitable carriers that also act as stabilizers for peptides include, without limitation, pharmaceutical grades of dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, and the like. Other suitable carriers include, again without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEGs), and combination thereof. It may also be useful to employ a charged lipid and/or detergent. Suitable charged lipids include, without limitation, phosphatidylcholines (lecithin), and the like. Detergents will typically be a nonionic, anionic, cationic or amphoteric surfactant. Examples of suitable surfactants include, for example, Tergitol® durfactant and Triton® surfactants (available from Union Carbide Chemicals anaplastics, Danbury, Conn., Tergitol® and Triton® are registered trademarks of Union Carbide Corporation, New York), polyoxyethylenesorbitans, for example, TWEEN® surfactants (available from Atlas Chemical Industries, Wilmington, Del. Tween® is a registered trademark of Uniqema Americas LLC, Delaware), polyoxyethylene ethers, for example Brij® (Brij is a registered trademark of Uniqema Americas LLC), pharmaceutically acceptable fatty acid esters, for example, lauryl sulfate and salts thereof (SDS), and like materials. A thorough discussion of pharmaceutically acceptable excipients, carriers, stabilizers and other auxiliary substances is available in Remingtons Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

A suitable composition for airway delivery of IFN-β related agent may, for example, be formulated as described in U.S. Pat. No. 6,030,609 by dissolving lyophilized IFN-β in a pharmaceutically acceptable vehicle such as sterile distilled water or sterile physiological saline, optionally with addition of one or more carriers, stabilizers, surfactants or other agents in order to enhance effectiveness of the IFN-β related agent. A suitable composition has a pH of from 5 to 8, more preferably 5.5 to 7.5. Preferably, the composition is buffered, e.g. using a citrate buffer.

The Rentschler composition, which is based on formulations disclosed in U.S. Pat. No. 6,030,609, has a pH of 6.5 and osmolarity of 290 mOsm/kg. The composition is preferably provided as a sterile, clear and colorless, ready-to-use aqueous nebulizer solution presented in a disposable glass syringe.

In addition to the active ingredient, the composition preferably comprises a buffering system to maintain the pH at between 5 and 8, more preferably 5.5 and 7.5, especially 6.5. The composition also preferably comprises an antioxidant, for example, DL-methionine.

A composition comprising a therapeutically effective amount of the IFN-β related agent described herein may conveniently be delivered to the lung airways by means of an inhalation device that is capable of delivering fine particles of the active ingredient to the lower respiratory tract or airways. Typically, particles of the active ingredient will have a mass median diameter of 1-10 microns. Suitable inhalation devices include dry powder inhalation (DPI) devices, pressurized metered dose inhalers (pMDI) and aerosol nebulizers.

Typically, the inhalation device will produce an aerosol with a particle size, as determined using a Malvern Masterizer S, with a mass median diameter of 1-10 micron, preferably 3-8 micron, in which mass percent have a diameter below 5 micron is from 25-80%, preferably 30-65%. A suitable nebulizer is the I-neb device, a CE-marked nebulizer manufactured by Philips Respironics.

An appropriate effective amount may be determined by appropriate clinical testing and will vary with for example the activity of the IFN-β related agent administered or induced. The IFN-β related agent may for example, be administered in microgram amounts. They are administered to the subject to be treated in a manner compatible with the dosage formulation, and in an amount that will be effective to bring about the desired effect. The amount to be delivered per dose may be 0.1 μg to 500 μg, for example 1 to 50 μg, depending on the subject to be treated. The treatment normally lasts from 5-7 days and may continue to 14 days if symptoms persist. Treatment may be given from every other day to several times a day. Preferably, treatment is given once per day.

The IFN-β related agent may be administered on its own or simultaneously, sequentially or separately in combination with another therapeutic compound. In particular, the IFN-β, agent or polynucleotide may be administered in conjunction with a therapeutic compound used to treat the respiratory disease or antiviral to the individual. The IFN-β, agent or polynucleotide and additional therapeutic compound may be formulated in the same or different compositions.

In one embodiment, disclosed is a pharmaceutical formulation comprising an effective amount of a drug for treatment of a respiratory or sinus system, and an additive that enhances absorption of the drug into tissue of the respiratory system. In one aspect of this embodiment, the additive comprises a hydrophilic part and a hydrophobic part. In another aspect of this embodiment, the drug is not enclosed in micelles or encapsulated in polymer particles. In another aspect of this embodiment, the formulation does not include oil, a lipid, or a polymer. In yet another aspect of this embodiment, the formulation is an aqueous aerosol formulation, a dry powder aerosol formulation, or a propellant-based formulation.

Blocking T-Req Production

Embodiments are discussed above that involve inducing Treg production via administration of IFNB and IFNB-related agents. In conducting the studies concerning how T-reg production can be induced, it was discovered that administration of certain antibodies can actually inhibit T-reg production. In particular, the Examples below show that administration of anti-TNFR2 blocking antibody or anti-IFNAR1 blocking antibody reduced production of T-reg production. Intentional inhibition of T-reg production would be beneficial in scenarios where immune responses are desired, such as in the instance of cancer and cancer immunotherapies. Anti-TNFR2 and/or anti-IFNAR1 blocking antibodies that are commercially available such as those provided by Invitrogen (Cat #AHR3022, Cat #MA5-31263, Cat #MA1-24723, Cat #MA5-23630) or Biolegend (Cat #BE0247, Cat #113202, Cat #127303) or newly developed antibodies may be used in accord with the teachings herein.

Accordingly, one embodiment involves administering an amount of anti-TNFR2 blocking antibody or anti-IFNAR1 blocking antibody, or both, sufficient to block or reduce production of Treg cells. A related embodiment pertains to a method of enhancing efficacy of a cancer immunotherapy involving co-administering an amount of anti-TNFR2 or anti-IFNAR1 blocking antibody, or both, with a cancer immunotherapy.

The anti-TNFR2 and/or anti-IFNAR1 blocking antibodies may be used in combination with a cancer immunotherapy such that the cancer immunotherapy is enhanced and tumor cytotoxicity is increased. When the anti-TNFR2 and/or anti-IFNAR1 blocking antibodies are used in combination with cancer immunotherapy, the dosage of the drug for cancer immunotherapy can be reduced, which may lead to reduced side effects. Moreover, the reduction in the dosage of the drug for cancer immunotherapy meets social needs including healthcare cost reduction.

Examples of the cancer immunotherapy include cancer vaccine therapy, immune cell infusion therapy, a therapy for reversal of immunosuppression and a therapy for inducing the depletion of regulatory T cells. In some embodiments, the cancer immunotherapy may be a therapy for reversal of immunosuppression. The immune checkpoint inhibitor used in the therapy for reversal of immunosuppression is an anti-CTLA-4 antibody, a PD-1 blocker, an anti-PD-1 antibody, a PD-L1 blocker, an anti-PD-L1 antibody, or the like. Examples of the immune cell infusion therapy include chimeric antigen receptor-modified T-cell therapy. The administration of the agent of the present invention after depletion of regulatory

T cells is expected to produce the same effect as produced by a combination of the agent of the present invention with an immune checkpoint inhibitor because regulatory T cells play a role in immunological tolerance. Examples of the drug that induces the depletion of regulatory T cells include alkylating agents, an IL-2-diphtheria toxin fusion protein, an anti-CD25 antibody, an anti-KIR antibody, an IDO inhibitor and a BRAF inhibitor.

Examples of the drug for cancer immunotherapy include Picibanil, Krestin, sizofiran, lentinan, ubenimex, interferons, interleukins, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, erythropoietin, lymphotoxins, BCG vaccine, Corynebacteriumparvum, levamisole, polysaccharide K, procodazole, ipilimumab, nivolumab, ramucirumab, ofatumumab, panitumumab, pembrolizumab, obinutuzumab, trastuzumab emtansine, tocilizumab, bevacizumab, trastuzumab, siltuximab, cetuximab, infliximab, rituximab and metformin.

When anti-TNFR2 and/or anti-IFNAR1 blocking antibodies are used in combination with cancer vaccine, efficient infiltration of cancer vaccine-stimulated T cells into a tumor can be achieved. In addition, the agent of the present invention can enhance the efficacy of immune cell infusion therapy using immune cells such as T cells from a patient or a non-patient.

The anti-TNFR2 and/or anti-IFNAR1 blocking antibodies can be embodied in the form of a medicament. That is, the anti-TNFR2 and/or anti-IFNAR1 blocking antibodies can be produced in a dosage form by blending the anti-TNFR2 and/or anti-IFNAR1 blocking antibodies with a pharmaceutically acceptable carrier or additive as appropriate according to a known production method for pharmaceutical preparations (e.g., the methods described in the Japanese Pharmacopoeia, etc.). Specifically, the anti-TNFR2 and/or anti-IFNAR1 blocking antibodies containing medicament can be, for example, an oral preparation or a parenteral preparation, including tablets (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, and buccal tablets), pills, powders, granules, capsules (including soft capsules and microcapsules), troches, syrups, liquids, emulsions, suspensions, controlled-release preparations (e.g., fast-release preparations, sustained release preparations, sustained release microcapsules, etc.), aerosols, films (e.g., orally disintegrating films, oral mucosal adhesive films, etc.), injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.), intravenous infusions, transdermal preparations, ointments, lotions, patches, suppositories (e.g., rectal suppositories, vaginal suppositories, etc.), pellets, transnasal preparations, transpulmonary preparations (inhalants), and eye drops. The amount of the carrier or the additive to be added is determined as appropriate based on the range of amount conventionally used in the pharmaceutical field. The carrier or the additive that can be added is not particularly limited, and examples include various carriers such as water, physiological saline, other aqueous solvents, and aqueous or oily bases; and various additives such as fillers, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, corrigents and flavors.

Examples
Example 1: Lung TNFR2⁺ cDC2 (R2D2) Population Maintains Lung Mucosal Tolerance at Steady-State

It was reasoned that lung mucosal tolerance is actively maintained by a specialized lung DC population, and mice lacking this tolerogenic lung DC population will spontaneously lose lung mucosal tolerance. We first examined lung CD4⁺ T cells in mice lacking different DC subsets, Batf3^−/− (cDC1), IRF4^fl/flCD11c^cre(cDC2) and CCR2^−/− (moDCs). Only the IRF4^fl/flCD11c^cremice had spontaneously increased CD4⁺ T cells in the lung (FIG. 1A). Furthermore, IRF4^fl/flCD11c^cremice had enlarged mediastinal lymph nodes (medLNs), but relatively normal spleens (FIG. 1C-1D) suggesting a selective loss-of-lung tolerance by the lack of cDC2.

cDC2 is a heterogeneous population (Tussiwand R, Everts B, Grajales-Reyes G E, Kretzer N M, Iwata A, Bagaitkar J et al. KIf4 expression in conventional dendritic cells is required for T helper 2 cell responses. Immunity 2015; 42(5): 916-928. Mansouri S, Patel S, Katikaneni D S, Blaauboer S M, Wang W, Schattgen S et al. Immature lung TNFR2(−) conventional DC 2 subpopulation activates moDCs to promote cyclic di-GMP mucosal adjuvant responses in vivo. Mucosal Immunol 2019; 12(1): 277-289.). We reasoned that to actively maintain lung tolerance, the tolerogenic cDC2 subset may be constitutively activated. A subpopulation of lung cDC2, marked by expression of TNFR2, has constitutively activated RelB (pRelB) (FIG. 9A-9B) (Mansouri, 2019, supra). Notably, the TNFR2⁺ cDC2 (R2D2) population is the only lung DC subset that has constitutively activated RelB (FIG. 9C). TNFR2^−/−mice lack the pRelB⁺ cDC2 subpopulation²¹. Furthermore, the R2D2 population expresses tolerogenic DC markers of PD-L1, PD-L2, Arg-1, BTLA (Mansouri 2019, supra). Last, the lack of RelB expression in DCs promotes the development of spontaneous allergic airway inflammation³⁰. We thus generated RelB^fl/flCD11c^creand TNFR2^fl/flCD11c^cremice to examine their lung mucosal tolerance at the steady-state.

Similar to the IRF4^fl/flCD11c^cremice, RelB^fl/flCD11c^creand TNFR2^fl/flCD11c^cremice had increased immune cells in the lung and medLNs, but not the spleen (FIG. 1B-1D). The IRF4^fl/flCD11c^cre, RelB^fl/flCD11c^creand TNFR2^fl/flCD11c^cremice may delete IRF4, RelB and TNFR2 in other lung CD11c⁺ cells. We examined RelB, IRF4, and TNFR2 expression in these mice. We found that while RelB^fl/flCD11c^credeleted RelB in alveolar macrophage (AM), IRF4 and TNFR2 expression in lung AM were intact in the IRF4^fl/flCD11c^creor TNFR2^fl/flCD11c^cremice (FIG. 9D-9F). Nevertheless, to exclude the possibility that the lack of IRF4, RelB or TNFR2 in lung cells other than R2D2 cause spontaneous lung inflammation, we did the adoptive cell transfer experiment. We sorted out lung TNFR2⁺ (R2D2) and TNFR2⁻ cDC2 from C57BL/6 mice and adoptively transfer (i.n.) them into the IRF4^fl/flCD11c^crerecipient mice. The quality of the adoptive cell transfer was confirmed after 24 hrs (FIG. 9G-9H). After two weeks, IRF4^fl/flCD11c^cremice receiving WT R2D2 cells had their lung CD4⁺ T cells numbers reduced while the IRF4^fl/flCD11 C^cremice receiving the WT TNFR2⁻ cDC2 still had elevated numbers of lung CD4⁺ T cells (FIG. 1E). Together, these data strongly suggested that lung R2D2 population maintains lung mucosal tolerance at the steady-state.

Example 2: Lung Mgl2⁺/IDO-1⁺ R2D2 Population Promotes Antigen-Specific Lung T-Regs Induction at Steady-State

DCs promote tolerance via the generation of T-regs (Akbari O, DeKruyff R H, Umetsu D T. Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat Immunol 2001; 2(8): 725-731. de Heer H J, Hammad H, Soullie T, Hijdra D, Vos N, Willart M A et al. Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen. J Exp Med 2004; 200(1): 89-98. Hintzen G, Ohl L, del Rio M L, Rodriguez-Barbosa J I, Pabst O, Kocks J R et al. Induction of tolerance to innocuous inhaled antigen relies on a CCR7-dependent dendritic cell-mediated antigen transport to the bronchial lymph node. J Immunol 2006; 177(10): 7346-7354. Semmrich M, Plantinga M, Svensson-Frej M, Uronen-Hansson H, Gustafsson T, Mowat A M et al. Directed antigen targeting in vivo identifies a role for CD103+ dendritic cells in both tolerogenic and immunogenic T-cell responses. Mucosal Immunol 2012; 5(2): 150-160). We found that lung from IRF4^fl/flCD11c^crehad decreased T-regs at the steady-state (FIG. 1F). We reasoned that R2D2 cells induce T-regs in the lung. Intranasal administration of one dose of 1 μg innocuous protein antigens (e.g., OVA, PspA, H7N7-HA, H1N1-NP) induces lung T-regs (FIG. 10A). These lung T-regs were antigen-specific and neuropilin-negative peripheral T-regs (FIG. 10B-10C). They also produce IL-10 (FIG. 10D). Furthermore, intranasal administration of OVA turned adoptive transferred (i.v) naïve CD45.1⁺ T cells into Foxp3⁺ T-regs in vivo (FIG. 9E). Last, consistent with previous report³, blocking DC migration by anti-CCR7 mAb inhibited OVA-induced lung T-regs induction (FIG. 9F).

The induction of lung T-regs by OVA depended on IRF4, RelB, or TNFR2 expression in CD11 C⁺ cells but not Batf3, TNFR1, or CCR2 (FIG. 2A-2C, 10G). Adoptive transfer of R2D2 cells, but not lung TNFR2⁻ cDC2 or lung tissue macrophage, restored lung T-regs induction in the IRF4^fl/flCD11c^cremice (FIG. 2D, 1H). Notably, the TNF2^fl/flLysM^cremice, which delete TNFR2 in myeloid cells, had unaltered lung T-regs induction (FIG. 101). Thus, the lung R2D2 population generates lung T-regs. R2D2 cells express tolerogenic DC markers of PD-L1, Arg-12¹. Closer examination found that a subpopulation of R2D2 expresses Mgl2 (FIG. 2E). The Mgl2⁺R2D2 population is PD-L1⁺PD-L2⁺ILT3⁺Arg-1⁺ and constitutively express indoleamine 2,3-dioxygenase 1 (IDO-1) (FIG. 2E). Notably, all the IDO-1⁺R2D2 cells are Mgl2⁺ and Arg1⁺ (FIG. 2F). The Mgl2⁺ DC population has been characterized before in the skin to promote T_H2 responses and suppress T_FHresponse (Kumamoto Y, Linehan M, Weinstein J S, Laidlaw B J, Craft J E, Iwasaki A. CD301 b(+) dermal dendritic cells drive T helper 2 cell-mediated immunity. Immunity 2013; 39(4): 733-743. Kumamoto Y, Hirai T, Wong P W, Kaplan D H, Iwasaki A. CD301 b(+) dendritic cells suppress T follicular helper cells and antibody responses to protein antigens. Elife 2016; 5). We then adoptively transferred (i.n.) lung Mgl2⁺R2D2 and Mgl2-R2D2 cells into the IRF4^fl/flCD11 Cc^re mice and examined their ability to induce lung T-regs. Only Mgl2⁺R2D2 generated lung T-regs in the IRF4^fl/flCD11c^cremice (FIG. 2G). Taken together, the lung Mgl2⁺/IDO-1⁺ R2D2 population induces lung T-regs at steady-state.

Example 3: Constitutive TNFR2 Signaling is Required for the Presence of R2D2 Population in the Lung at Steady-State

TNFR2^−/− mice lack the pRelB⁺ population (Mansouri 2019, supra). Here, we found that TNFR2^fl/flCD11c^cre, not the TNFR2^fl/flLysM^cremice, lack the TNFR2⁺pRelB⁺ cDC2 (R2D2) population (FIG. 3A-3B, 11A). Thus, TNFR2 expression in DCs mediates pRelB activation. Notably, at the steady-state, lung cDC2 is the only TNFR2+ DCs population (FIG. 11B). We hypothesized that there was an active TNFR2-pRelB signaling in the lung R2D2 population at steady-state.

Indeed, blocking TNFR2 signaling with anti-TNFR2 blocking mAb inhibited pRelB expression in cDC2 (FIG. 3B) and T-regs induction in the lung (FIG. 11C). Unexpected, blocking TNFR2 dramatically decreased the numbers of lung TNFR2+pRelB⁺ cDC2 (R2D2) at steady-state (FIG. 3A-3C). To exclude the possibility that the anti-TNFR2 blocking mAb may interfere with the TNFR2 detection by Flow cytometry, we used the TNFR2-Fc (human IgG1) fusion protein to disrupt the TNFR2 interaction with its ligand in vivo. Again, TNFR2-Fc (human IgG1) reduced R2D2 population (FIG. 3A-3C). Notably, in these experiments, the numbers of lung R2D2 cells showed a more dramatic reduction (FIG. 3C) than the percentage changes (FIG. 3A). Indeed, the total numbers of lung cDC2 cells decreased in the TNFR2^fl/flCD11c^cremice, mice treated with anti-TNFR2 blocking mAb or TNFR2-Fc likely due to the loss of the R2D2 subpopulation in lung cDC2 subset.

We further examined the Mgl2⁺/IDO-1⁺ R2D2 population and found, as expected, that blocking TNFR2 or TNFR2-Fc treatment reduced the Mgl2⁺ R2D2 number by 90% (FIG. 3D). Thus, the lung R2D2, including the T-reg inducing Mgl2⁺ R2D2 subset, depends on the constitutive TNFR2 signaling.

TNFR2 binds specifically to transmembrane TNF (tmTNF)(Grell M, Douni E, Wajant H, Lohden M, Clauss M, Maxeiner B et al. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 1995; 83(5): 793-802). To identify TNF-expressing lung cells at the steady-state, we first did intracellular TNF staining to detect total cellular TNF including tmTNF and soluble TNF. We found that the CD45⁻ lung cells were the main TNF⁺ lung cells at steady-state (FIG. 3E). Next, we used the TNFR2-Fc (human IgG1) fusion protein to detected TNFR2 ligands, i.e., tmTNF. TNFR2 ligands were mainly on CD45-EP-CAM⁺ lung epithelial cells at the steady-state (FIG. 3F). We reasoned that, at the steady-state, tmTNF on lung epithelial cells engages TNFR2 on R2D2 to maintain its presence in the lung.

To mimic the effect of tmTNF, we made a TNF_D221N/A223Rmutant that only binds to TNFR2³³. TNF_D221N/A223Ris a monomer. TNF works as a trimer³⁴. We first crosslinked TNFR2 with the non-blocking TNFR2 mAb followed by the addition of TNF_D221N/A223Rto activate TNFR2 signaling. Intranasal administration of a non-blocking anti-TNFR2 mAb (TR75.89) and the TNF_D221N/A223Rincreased R2D2 population (FIG. 3G). Importantly, we found increased Ki67⁺ cells in the R2D2 population in mice treated with tmTNF (FIG. 3H), indicating the enhanced proliferation of R2D2 cells by TNFR2 engagement. As a negative control, intranasal administration of TNF_D221N/A223Rdid not enhance Ki67 expression in the cDC1 population, which do not express TNFR2 (FIG. 11D). In conclusion, the steady-state lung R2D2 population, including the Mgl2⁺/IDO-1⁺R2D2, is a specialized DC subset adapted to the lung microenvironment and needs the constitutive tmTNF-TNFR2 signaling for its existence.

Example 4: Constitutive IFNAR1 Signaling in Mgl2⁺R2D2 Cells Drives Lung T-Regs Induction

IDO-1⁺, not IDO-1⁻, R2D2 cells induce lung T-regs. However, we found that intranasal administration of an anti-IFNAR1 blocking mAb inhibited T-regs induction in the lung (FIG. 4A) but did not affect IDO-1 expression in R2D2 cells (FIG. 12A). Similarly, IFNAR1^−/− mice failed to generated lung T-regs in response to OVA (FIG. 4B) but retained the R2D2 population, including the Mgl2⁺ R2D2 (FIG. 12B) population in the lung. Thus, IDO-1 or Mgl2 expression in R2D2 is not sufficient for T-regs induction. Rather, IFNAR1 signaling is critical.

R2D2 has the highest expression of IFNAR1 among lung DCs subsets (FIG. 12C-12D) at steady-state. In the R2D2 population, all IDO-1⁺ R2D2 cells express high IFNAR1 compared to the IDO-1-R2D2 (FIG. 4C). To establish that R2D2 cell-intrinsic IFNAR1 signaling promotes lung T-regs induction, we adoptively transferred (i.n.) WT R2D2 into IFNAR1^−/− mice and treated the recipient mice with OVA. After 14 days, we found that IFNAR1^−/− mice receiving WT R2D2 cells restored T-regs induction in the lung (FIG. 4D). We concluded that R2D2 cell-intrinsic IFNAR1 signaling is sufficient for lung T-regs induction.

Indeed, intranasal administration of IFNβ enhanced T-regs induction in the lung (FIG. 4E). Importantly, IFNβ did not induce T-regs in IRF4^fl/flCD11c^cremice, suggesting IFNβ acts on cDC2 to induce T-regs (FIG. 4F). Interestingly, the intranasal administration of IFNβ did not increase the total number of lung R2D2 cells (FIG. 4G). Rather, IFNβ treatment tended to increase cell numbers of Mgl2⁺ R2D2 while decrease numbers of Mgl2⁻ R2D2 (FIG. 4G) suggesting IFNβ treatment in vivo may turn Mgl2-into Tregs-inducing Mgl2⁺R2D2. In light of the critical role of R2D2 intrinsic IFANR1 signaling in T-regs induction, we named the T-regs inducing Mgl2⁺/IDO-1⁺ R2D2 as IFNAR1^hiR2D2 (iR2D2).

Last, we wanted to determine the cellular source of IFNβ in the lung that generates iR2D2 at steady-state. Using intracellular IFNβ stain, we found that at steady-state, the Epcam⁺CD45⁻ lung epithelial cells were the sole IFNβ⁺ producing cells (FIG. 4H). Intriguingly, only ~18% EPCAM⁺CD45⁻ lung epithelial cells were IFNβ⁺ suggesting a selected population of lung epithelial cells are responsible for IFNβ production at steady-state (FIG. 4I).

Example 5: IFNβ Activates TGFβ1 in iR2D2 to Drives Lung T-Regs Induction

How does the IFNAR1 signaling activate the tolerogenic program in the iR2D2 cells? pT-regs are generated by TGFβ1^26-28. R2D2 produced TGFβ1 in response to H7N7 influenza HA antigen (FIG. 5A). Neutralizing TGFβ1 inhibited T-regs induction in the lung (FIG. 5B).

The induction of TGFβ1 in R2D2 dependeds on IFNβ-IFNAR1 signaling. Intranasal administration of IFNβ increased TGFβ1 in R2D2 (FIG. 5C) while anti-IFNAR1 blocking mAb inhibited OVA-induced TGFβ1 production in R2D2 (FIG. 5D). Notably, anti-IFNAR1 mAb did not affect OVA-induced TGFβ1 production in cDC1, moDCs or AMs (FIG. 13A). Block IFNAR1 inhibited OVA-induced T-regs induction (FIG. 4A). Thus, TGFβ1 production by cDC1, AM or moDCs were not sufficient for the induction of T-regs in the lung.

To establish that IFNβ acted on R2D2 to induce TGFβ1 expression, we adoptively transferred (i.n.) the lung R2D2 cells from the CD45.1⁺ mice into IFNAR1^−/− mice and treated (i.n.) the recipient IFNAR1^−/− mice with IFNβ (FIG. 5E). We found that IFNβ administration induced TGFβ1 in the transferred CD45.1⁺ cells (FIG. 5F). Furthermore, IFNβ restored T-regs induction in the IFNAR1^−/− mice received CD45.1+R2D2 cells (FIG. 5G), suggesting that IFNβ acted on lung R2D2 cells to generate TGFβ1 for lung T-regs induction. Taken together, IFNAR1 signaling in iR2D2 induces T-regs by producing TGFβ1.

Example 6: R2D2 Cells Promote T_H2 Responses in HDM-Induced Asthmatic Mice

cDC2 mediates HDM-induced asthma (Tussiwand R, Everts B, Grajales-Reyes G E, Kretzer N M, Iwata A, Bagaitkar J et al. KIf4 expression in conventional dendritic cells is required for T helper 2 cell responses. Immunity 2015; 42(5): 916-928. Plantinga M, Guilliams M, Vanheerswynghels M, Deswarte K, Branco-Madeira F, Toussaint W et al. Conventional and monocyte-derived CD11 b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen. Immunity 2013; 38(2): 322-335). Recent studies indicated that a KIf4⁺/Mgl2⁺, RelB⁺ cDC2 subset mediates HDM-induced T_H2 responses. iR2D2 expresses Mgl2, RelB. We suspected that the lung R2D2 population may be plastic and promotes T_H2 response in HDM-induced asthma.

First, we generated the HDM induced asthmatic mice (FIG. 6A). HDM-treated mice generated HDM-specific IgE and IgG1 (FIG. 14A), lung inflammation determined by H&E stain (FIG. 14B), eosinophils infiltration in the lung (FIG. 14C) and T_H2 dominant cytokines in draining lymph nodes (FIG. 14D). Next, we examined the iR2D2 population in the asthmatic mice. We found increased numbers of total R2D2 cells in the asthmatic cells (FIG. 6B). The Mgl2⁺/IDO-1⁺ R2D2 population increased as well in the HDM-induced asthmatic mice (FIG. 6C, 14E). However, no T-regs were generated in HDM mice (FIG. 6D). The R2D2 cells from HDM-induced mice also downregulated tolerogenic markers PD-L1, PD-L2, and, critically, IFNAR1 expression (FIG. 6E). In contrast, R2D2 cells from the HDM mice increased immunogenic markers OX40L, ICOSL, and T1/ST2 (FIG. 6F).

Last, adoptive transfer (i.n.) R2D2 from HDM mice into naïve mice generated T_H2 response in vivo (FIG. 6G), which was different from the T-regs inducing R2D2 at the steady-state (FIG. 2D). Comparing the tolerogenic R2D2 (iR2D2) from OVA treated mice with the T_H2-promoting R2D2 from the HDM mice, the tolerogenic R2D2 expresses TGFβ1 while the immunogenic R2D2 expresses IL-4 and IL-4/TGFβ1 (FIG. 6H). We concluded that the lung R2D2 population is plastic and can promote T-regs or T_H2 responses depending on the environmental cue, e.g., IFNβ or HDM.

Example 7: Human Lungs have a Phenotypically Similar Plastic iR2D2 Population

We reasoned that healthy human lung should have a functionally similar tolerogenic DC subset. Little is known about human lung cDC2 subsets in healthy individuals or patients. We sampled healthy donor lungs that were transplanted into lung patients and identified the same TNFR2⁺pRelB⁺IDO-1⁺PD-L1⁺PD-L2⁺ cDC2 population in the healthy human lungs (FIG. 7A, 15A-15B). The healthy human lung iR2D2 cells constitutively express TGFβ1 and Arg-1 (FIG. 7B) as well.

We also identified IDO-1⁺ R2D2 population in lung explants from emphysema, interstitial lung disease patients (FIG. 7C). However, R2D2 cells from lung patients had decreased TGFβ1, Arg-1, PD-L1, but increased IL-4 expression (FIG. 7B) similar to the R2D2 from the asthmatic mice. Notably, lung explants from COPD patients had very little R2D2 cells (FIG. 7C). We conclude that the phenotypically similar R2D2 cells can be found in healthy human lungs and some lung disease patients.

Methods Related Examples 1-7

KEY RESOURCES TABLE

REAGENT or RESOURCE
SOURCE
IDENTIFIER

Antibodies

Anti-mouse CD4-PE/Cy7 (clone: GK1.5)
BioLegend
Cat#100422

Anti-mouse IFNβ-PerCP/Cy5.5
BioLegend
Cat#505822

(clone: XMG1.2)

Anti-mouse IL-4-APC (clone: 11B11)
BioLegend
Cat#504106

Anti-mouse IL-17a-PE (clone: TC11-1810.1)
BioLegend
Cat#506903

Anti-mouse CD44-Alexa Fluor 700 (clone: IM7)
BioLegend
Cat#103025

Anti-mouse CD45-PercP/Cy5.5 (clone: 30-F11)
Biolegend
Cat#103131

Anti-mouse Foxp3-Pacific Blue (clone: MF-14)
BioLegend
Cat#26410

Anti-mouse MHCII(I-A/I-E)-Brilliant Violet 421
BioLegend
Cat#107636

(clone: M5/114.15.2)

Anti-mouse MHCII(I-A/I-E)-Alexa Fluor
BioLegend
Cat#107622

(clone: M5/114.15.2)

Anti-mouse CD11c-APC/Cy7 (clone: N418)
Biolegend
Cat#117323

Anti-mouse/human CD11b-PE/Cy7
BioLegend
Cat#101216

(clone: M1/70)

Anti-mouse/human CD11b-Brilliant Violet 605
BioLegend
Cat#101237

(clone: M1/70)

Anti-mouse CD64-PerCP/Cy5.5
BioLegend
Cat#139307

(clone: X54-5/7.1)

Anti-mouse TNFR2-PE (Clone:TR75-89)
BioLegend
Cat#113405

Anti-mouse TNFR2-APC (Clone: REA228)
Miltenyi Biotec
Cat#130-104-698

Anti-mousPDL1-Brilliant Violet 421
BioLegend
Cat#124315

(clone: 10F.9G2)

Anti-mouse PDL2-APC (clone: TY25)
BioLegend
Cat#107210

Anti-mouse/human Arg1-FITC
RD systems
Cat#IC5868F

Anti-mouse ILT3-PE (clone: H1.1)
BioLegend
Cat#144904

Anti-mouse IDO1-Alexa Fluor
BioLegend
Cat#654003

(clone: 2E2/IDO1)

Anti-mouse/human pReIB-PE (clone: D4169)
Cell Signaling
Cat#:13567

Technology

Anti-mouse EPCAM-PerCP/Cy5.5
BioLegend
Cat#118219

(clone: G8.8)

Anti-mouse/human Ki67-PE (clone: 11F6)
BioLegend
Cat#151209

Anti-mouse IFNAR1-APC (clone: MAR1-5A3)
BioLegend
Cat#127313

Anti-mouse IFNβ-Primary (clone: D2J1D)
Cell Signaling
Cat#:974505

Technology

Anti-mouse TNF-Primary (clone: D2D4)
Cell Signaling
Cat#:11948

Technology

Anti-mouse LAP (TGFβ1)-Brilliant Violet 421
BioLegend
Cat#141407

(clone: TW7-1664)

Anti-mouse LAP (TGFβ1)-FITC
BioLegend
Cat#141413

(clone: TW7-1664)

Anti-mouse CD45.1-APC (clone: A20)
BioLegend
Cat#110713

Anti-mouse OX40L-PE (clone: RM134L)
BioLegend
Cat#108805

Anti-mouse ICOSL-PE (clone: HK5.3)
BioLegend
Cat#107405

Anti-mouse T1ST2-APC (clone: Di H4)
BioLegend
Cat#146605

Anti-mouse IRF4-APC (clone: IRF4.3E4)
BioLegend
Cat#646407

Anti-human TNFR2-APC (clone: 3G7A02)
BioLegend
Cat#358405

Anti-human HLA-DR-APC/Cy7 (clone: L243)
BioLegend
Cat#307617

Anti-human PDL1-Brilliant Violet 421
BioLegend
Cat#329713

(clone: 29E.2A3)

Anti-human PDL2-PE (clone: 24F.10C12)
BioLegend
Cat#329605

Anti-human IDO1-Primary
R&D Systems
Cat#MAB6030

Anti-human TGFβ1-PE (clone : Tw4-2F8)
BioLegend
Cat#349603

Anti-human Arginase 1-PE (clone: 14D2C43)
BioLegend
Cat#369703

Anti-human IL-4-PE (clone: G077F6)
BioLegend
Cat#355003

Anti-mouse F4/80-PerCP/Cy5.5 (clone: BM8)
BioLegend
Cat#123127

I-A(b) chicken ova 325-335 QAVHAAHAEIN
NIH Tetramer

APC-Labeled Tetramer
Core Facility

I-A(b) human CLIP 87-101
NIH Tetramer

PVSKMRMATPLLMQA (Control) APC Labeled
Core Facility

Tetramer

Anti-mouse Neuropilin-APC (clone: 3E12)
BioLegend
Cat#145205

Anti-human CD1c-PE/Cy7 (clone: L161)
BioLegend
Cat#331515

Anti-human CD14-PerCP/Cy5.5 (clone: 63D3)
BioLegend
Cat#367109

Anti-human CD206-FITC (clone: 15-2)
BioLegend
Cat#321103

anti-mouse IgG1-HRP
Southern Biotech
Cat#1030-05

anti-mouse IgE-HRP
Southern Biotech
Cat#1110-05

Chemicals, Peptides, and Recombinant Proteins

Cell Activation Cocktail with Brefreldin A
Biolegend
423303

OVA
Invivogen
cat#vac-pova

Anti-CCR7 monoclonal antibody
R&D Systems
Cat#MAB34477

Anti-TNFR2 monoclonal antibody
BioXcell
Cat#BE0247

(clone: TR75-54.7)

Anti-TNFR2 monoclonal antibody
Biolegend
Cat#113202

(clone: TR75-32.4)

Isotype Control (clone HTK888)
Biolegend
Cat#400931

TNFR2-Fc (human IgG1) fusion protein
SinoBiological
Cat#50128-M02H

TNFR2-agonist (TNF_D221N/A223R)
Creative ® Biolabs
Custom made

Anti-IFNAR1 monoclonal antibody
Biolegend
Cat#127303

(clone: MAR1-5A3)

Recombinant murine IFNβ
R&D
Cat#8234-MB/CF

anti-TGFβ1 neutralizing antibody (19D8)
Biolegend
Cat#521707

House dust mites Dermatophagoides
Greer Laboratories
Cat#XPB82D3A2.5

pteronyssinus (HDM-Der p1)

Dermatophagoides farinae (HDM-Der f 1)
Greer Laboratories
Cat#XPB81D3A2.5

PspA
BEI Resources,
NR-33178

NIAID, NIH

H7N7-HA
BEI Resources,
NR-2633

NIAID, NIH

H1N1-NP
SinoBiological
cat#11675-V08B

Foxp3/Transcription Factor Staining Buffer Set
EBioscience
cat#00-5523-00

Experimental Models: Organisms/Strains

Mouse: IRF4^fl/fl
Jackson
Cat#009380

Laboratory

Mouse: CD11c^Cre
Jackson
Cat#008068

Laboratory

Mouse: Batf3^−/−
Jackson
Cat#013596

Laboratory

Mouse: CCR2^−/−
Jackson
Cat#004999

Laboratory

Mouse: RelB^fl/fl
Jackson
Cat#028719

Laboratory

Mouse: TNFR2^fl/fl[C57BL/6-
EMMA-European
Cat#05925

Tnfrsf1b < tm1c(EUCOMM)Wtsi>/lcs]
Mouse Mutant

Archive

Mouse: IL-10^eGFP
Jackson
Cat#014530

Laboratory

Mouse: IFNAR1^−/−
Jackson
Cat#028288

Laboratory

Mouse: CD45.1
Jackson
Cat#002014

Laboratory

Mouse: Lysm^cre
Jackson
Cat#004781

Laboratory

Software and Algorithms

FlowJo version 10.1r1
FlowJo
http://www.flowjo.com

Prism6
GraphPad
http://www.graphpad.com

Mice. Age- and gender-matched mice (8-18 weeks old) were used for all experiments. C57BL/6, B6.CD45.1, Batf3^−/−CCR2^−/−, IL-10^GFP, IRF4^fl/fl, RelB^fl/fl, IFNAR1^−/−, CD11c^cre, LysM^creand TNFR1^−/− mice on C57BL/6 background were purchased from The Jackson Laboratory. TNFR2^fl/flmice were from the Euorpean Conditional Mouse Mutagenesis Program. Mice were housed and bred under pathogen-free conditions in the Animal Research Facility at the University of Florida. All mouse experiments were performed by the regulations and approval of the Institutional Animal Care and Use Committee at the University of Florida, IACUC number 201909362.

Reagents. Anti-TNFR2 monoclonal antibody (20 μg, TR75-54.7, BioXcell or TR75-32.4, BioLegend), anti-IFNAR1 monoclonal antibody (20 μg, MAR1-5A3, BioLegend), isotype control (20 μg HTK888, BioLegend), anti-TGFβ1 neutralizing antibody (75 μg, 19D8, BioLegend), anti-IFNβ monoclonal antibody (D2J1D, Cell Signaling, cat no. 97450), TNFR2-Fc (human IgG1) fusion protein (2 μg, SinoBiological, cat no. 50128-M02H) or TNF