WO2020033398A1

WO2020033398A1 - Compositions and methods for treating atii cell-dependent lung diseases

Info

Publication number: WO2020033398A1
Application number: PCT/US2019/045294
Authority: WO
Inventors: Nicolino Valerio DORRELLO; Michele Pagano; Gordana Vunjak-Novakovic
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2018-08-06
Filing date: 2019-08-06
Publication date: 2020-02-13

Abstract

Compositions and methods are provided for the treatment of ATII cell-dependent lung diseases. Compositions include a fusion protein which includes a fragment of a Diphtheria toxin fused to Surfactant Protein A for selective uptake and ablation of ATII cells. Methods for using such compositions to treat ATII cell-dependent lung diseases are also provided.

Description

COMPOSITIONS AND METHODS FOR TREATING ATII CELL-DEPENDENT LUNG

DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] The present application claims priority to U.S. Provisional Application No. 62/715,035, filed August 6, 2018, the entirety of which is incorporated herein by reference.

SEQUENCE LISTING

[2] The instant application contains a Sequence Listing which has been submitted

electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 6, 2019, is named l8-500l3-WO_SL.txt and is 14,196 bytes in size.

BACKGROUND

[3] Pulmonary function is dependent on the alveolar homeostasis of the distal lung maintained by alveolar type II (ATII) cells secreting and recycling surfactant. Consequently, mutation or injury to the ATII cells has been implicated in the pathogenesis of multiple acute and chronic lung diseases. The dysfunction or injury of alveolar type II (ATII) cells have been implicated in the pathogenesis of multiple acute and chronic lung diseases. In some diseases, ATII cells are severely injured or completely missing ( e.g ., acute respiratory distress syndrome (ARDS)). In others, they are genetically defective, causing lung fibrosis (e.g., surfactant proteins A2, B and C deficiency ( SFTPA2 , SFTPB, and SFTPC ), ABCA3 deficiency and Hermansky- Pudlak syndrome (HPS)). In others, there is a perturbation of the epithelial-mesenchymal homeostasis (e.g. idiopathic, interstitial pneumonia, chronic obstructive pulmonary disease (COPD), and Birt-Hogg Dube syndrome).

[4] Despite empiric medical therapy, unfortunately, lung transplantation— the only definitive treatment for patients with end stage lung disease— remains hampered by a severe shortage of donor organs with an overall survival at 5 years post-transplant, which remains unchanged at around 50%.

[5] There is a need for treatment alternatives which can avoid the prolonged hospitalization and long-term complications of lung disease and the need for transplantation. As such, there is a need for improved therapeutic strategies that reduce dependence on transplants for end-stage lung disease.

SUMMARY

[6] The present disclosure is directed to compositions and methods for treating ATII cell- dependent lung diseases.

[7] ATII cells play a pivotal role in the alveolar homeostasis of the distal lung: (i) they produce and recycle pulmonary surfactant, (ii) perform xenobiotic metabolism, and (Hi) participate in lung repair serving as progenitors following lung injury to replace damaged ATI and ATII cells. ATII cellular dysfunction or injury has been implicated in the pathogenesis of multiple acute and chronic lung diseases. Targeted deletion of only injured or defective epithelial cells, such as, ATII cells, while preserving all the surrounding healthy compartments (epithelium, vasculature and ECM) remains a challenge in regenerative medicine.

[8] The present disclosure provides for a fusion protein which includes at least a portion portion of the catalytic domain of Diphtheria toxin which can be coupled with at least a portion of Surfactant Protein A (SPA). Such fusion proteins can be used in methods for treating patients with lung diseases to selectively ablate ATII cells. The fusion proteins can be formulated for inhalation or for intra-tracheal injection. Fusion proteins of the present disclosure can be formulated in pharmaceutical compositions with pharmaceutically acceptable excipients. The fusion proteins of the present disclosure can be formulated in pharmaceutical compositions suitable for inhalational delivery or for administration by intra-tracheal injection. Selective ablation of ATII cells can serve a therapeutic effect by eliminating injured and/or dysfunctional cells and can also provide a void for engraftment of healthy ATII cells to restore lung function and treat disease.

[9] This technology utilizes targeted therapy to treat defective alveolar pulmonary diseases. By designing a fusion protein, it can selectively ablate defective ATII cells to allow future repopulation of healthy ATII cells with cell transplants. In some embodiments, this fusion protein links Surfactant Protein A (SPA) to a shortened version of Diphtheria toxin (DT388), with SPA triggering the uptake of DT388 to induce apoptosis of ATII cells. Importantly, fusion proteins of the present disclosure can be delivered via inhalation or intra-tracheal injection, reducing potential adverse side effects from systemic administration. After treatment, the patient’s lungs may then be repopulated by healthy exogenous ATII cells for long-lasting repair and tissue regeneration.

[10] In some embodiments, a fusion protein is provided which includes a cytotoxic domain that comprises the first 388 amino acids of Diphtheria toxin of Corynebacterium diphtheriae (DT388) and Surfactant Protein A (SPA). In certain aspects, the fusion protein is provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is inhalable. In some embodiments, the pharmaceutical composition is suitable for administration by intra-tracheal injection.

[11] In some embodiments, a method for treating an ATII cell-dependent lung diseases includes the step of administering a fusion protein which includes a cytotoxic domain comprises the first 388 amino acids of Diphtheria toxin of Corynebacterium diphtheriae (DT388) and Surfactant Protein A (SPA) to a patient in need thereof. In some embodiments, the fusion protein is administered at a dose sufficient to kill at least a portion of ATII cells in the patient. In certain aspects, the fusion protein is administered at a dose sufficient to kill a substantial proportion of ATII cells in the patient. In some embodiments, the fusion protein is administered at a dose sufficient to kill all ATII cells in said patient.

BRIEF DESCRIPTION OF THE DRAWINGS

[12] FIGURE 1 A depicts a schematic diagram depicting the Diphtheria toxin of

Corynebacterium diphtheriae (DT), the catalytic domain of the Diphtheria toxin (DT388), Surfactant Protein A (SPA) and the fusion protein (DT388-SPA).

[13] FIGURE 1B depicts Coomassie stains and Western blots of His-tagged DT388-SPA expressed in BL21 E. coli cells.

[14] FIGURE 2 depicts an exemplary experimental strategy for removal of ATII cells from lung tissue and repopulation with therapeutic ATII cells.

[15] FIGURE 3 depicts luciferase activity for rabbit reticulocyte lysate incubated with luciferase mRNA and vehicle or 10 mM of DTA388-SPA, DT388, SPA or cycloheximide.

[16] FIGURE 4 depicts laser scanning micrographs of rat RLE-6NT cells incubated under standard culture conditions (“Control”) or with 50 pM of Alexa 488 labled DT388-SPA, DT388 or SPA for up to 12 hours. [17] FIGURE 5 depicts cell viability versus protein concentration for rat RLE-6NT cells incubated with vehicle, DT388-SPA (“DT-SPA”), DT388 or SPA for 72 hours.

DETAILED DESCRIPTION

[18] The present disclosure provides compositions and methods for treating ATII cell- dependent lung diseases. Specifically, the present disclosure provides a toxic fusion protein,

DT388-SPA, which includes the catalytic domain of the Diphtheria toxin from Corynebacterium diphtheriae (DT388) and Surfactant Protein A (SPA). The fusion protein can be used to treat ATII cell-dependent lung diseases by administration to a patient in need thereof.

Definitions

[19] As used herein, the singular forms“a”,“an” and“the” include plural referents unless the context clearly dictates otherwise.

[20] The use of the term“or” in the claims and the present disclosure is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[21] Use of the term“about”, when used with a numerical value, is intended to include +/- 10%. For example, if a number of amino acids is identified as about 200, this would include 180 to 220 (plus or minus 10%).

[22] Use of the terms“homology” or“homologous” herein refers to the identity between two or more nucleic acid or amino acid sequences. Homology can be determined by aligning the sequences and counting the number of identical nucleotides or amino acids over the aligned region and dividing by the number of positions in the aligned region and multiplying by 100 to obtain a percentage value of homology. Where the alignment of two sequences results in staggered ends or the aligned region is less than the full length of at least one of the sequences, the homology is calculated only for the aligned region. Homology analysis can be performed manually or by using available software algorithms such as BLAST or BLAST 2.0.

[23] The terms“patient,”“individual,” and“subject” are used interchange- ably herein, and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the present disclosure find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates. [24] As used herein, the term“therapeutically effective amount” is meant an amount effective of a composition to yield the desired therapeutic response. By way of example, but not limitation, a therapeutically effective amount of therapeutic ATII cells can be an amount sufficient to engraft to the lung tissue of a patient to repopulate the lung tissue with ATII cells after abalation with a fusion protein of the present disclosure. By way of further example, but not limitation, a therapeutically effective amount of a fusion protein of the present disclosure can be an amount sufficient to kill at least a desired portion of ATII cells in patient. The specific therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the composition administered or its derivatives.

[25] “Treatment” is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly,“treatment” can refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.

[26] SPA is a lung surfactant protein that is produced and recycled by ATII cells. In combination with other surfactant proteins, such as Surfactant Proteins B, C and D, SPA and the lipid components of surfactant helps to avoid alveolar collapse during expiration.

[27] ATII cells are primary producers and recyclers of lung surfactant and mediate lung repair. The dysfunction or injury of ATII cells has been implicated in the pathogenesis of multiple acute and chronic lung diseases.

[28] The present disclosure provides an innovative and novel approach for treating defective and/or injured ATII cell-dependent lung diseases by specifically targeting and removing only ATII cells. This approach can selectively ablate the ATII cells while leaving the rest of the lung epithelium intact.

[29] In accordance with the present disclosure, fusion proteins are provided which comprise (1) the catalytic domain of the Diphtheria toxin of Corynebacterium diphtheriae , which can include the first 388 amino acids of the full-length toxin and (2) at least a portion of SPA. FIGURE 1 A schematically depicts the structure of a fusion protein of the present disclosure which includes DT388 fused to the N-terminus of SPA. DT388 alone is not functional unless transported inside a cell by a carrier, however, by coupling DT388 to SPA, selective and specific targeting of ATII cells can be achieved. Without being bound to theory, it is expected that upon uptake of DT388-SPA by ATII cells, DT388 will be released from the fusion protein, which will inhibit protein synthesis and induce targeted apoptosis. In some embodiments, the fusion protein can further include a linker between the DT388 and SPA. The linker can comprise amino acids and effectively couple DT388 to SPA.

[30] This targeted apoptosis of ATII cells only can leave a void that can then be repopulated by the engrafting of healthy exogenous ATII cells. This strategy is depicted in FIGURE 2 which shows injured ATII cells (left), removal of targeted injured ATII cells (middle) and the engraftment of therapeutic ATII cells (right).

[31] In some embodiments, the catalytic domain of the Diphtheria toxin of Corynebacterium diphtheriae portion of the fusion protein comprises the amino acid sequence of SEQ ID NO: 1 (GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTD NKYD AAGY S VDNENPL SGK AGGVVK VTYPGLTKVL ALK VDNAETIKKELGL SLTEPLM EQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQ DAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSES PNKT V SEEK AKQ YLEEFHQT ALEHPEL SELKT VT GTNP VF AGAN Y A AW A VN V AQ VID S ETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELV DIGF AAYNF VESIINLF Q VVHN S YNRP AY SPGHKTRP) or a portion thereof.

[32] In some embodiments, the SPA portion of the fusion protein comprises the amino acid sequence of SEQ ID NO: 2

(NVTDVCAGSPGIPGAPGNHGLPGRDGRDGVKGDPGPPGPMGPPGGMPGLPGRDGLPG APGAPGERGDKGEPGERGLPGFPAYLDEELQTELYEIKHQILQTMGVLSLQGSMLSVGD KVFSTNGQSVNFDTIKEMCTRAGGNIAVPRTPEENEAIASIAKKYNNYVYLGMIEDQTP GDFHYLDGASVNYTNWYPGEPRGQGKEKCVEMYTDGTWNDRGCLQYRLAVCEF) or a portion thereof.

[33] In some embodiments, the fusion protein comprises SEQ ID NO: 1 and SEQ ID NO: 2.

[34] In the foregoing embodiments, a portion of SEQ ID NO: 1 and/or SEQ ID NO: 2 can include a portion of the sequence that retains the activity of the SEQ ID NO: 1 and/or SEQ ID NO: 2, respectively. For example, a portion of SEQ ID NO: 1 can retain at least a portion of the cytotoxic activity of SEQ ID NO: 1. By way of further example, a portion of SEQ ID NO: 2 can retain the function triggering uptake of the fusion protein by ATII cells. By way of example but not limitation, the portion of SEQ ID NO: 1 can include at least 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386 or 387 amino acids of SEQ ID NO: 1. By way of example but not limitation, the portion of SEQ ID NO: 2 can include at least 200, 210, 215, 220, 221, 222, 223, 224, 225, 226 or 227 amino acids of SEQ ID NO: 2.

In some embodiments, the fusion protein can include sequences having homology to SEQ ID NO: 1 or SEQ ID NO: 2 and portions of each thereof, respectively. Said homology can, by way of example but not limitation, be at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% for each of SEQ ID NO: 1 and SEQ ID NO: 2 or the portions thereof. In certain aspects, the fusion protein can comprise at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of each of SEQ ID NO: 1 and SEQ ID NO: 2. One of skill in the art can assess whether a portion or homologue of the sequences demonstrates the desired cytotoxicity and cellular uptake potential by methods known in the art and disclosed herein.

[35] In some embodiments, a portion of SEQ ID NO: 1 or a homologue includes cysteine residues at positions corresponding to amino acid positions 186 and 201 of SEQ ID NO: 1. In some embodiments, a portion of SEQ ID NO: 1 or homologue includes the furin cleavage site at a position corresponding to positions 190-193 of SEQ ID NO: 1. In some embodiments, a portion of SEQ ID NO: 1 or homologue can include a sequence corresponding to amino acid positions 1-193 of SEQ ID NO: 1 which represents the catalytic domain of the sequence.

[36] In some embodiments, SEQ ID NO: 1 or the portion thereof is positioned N-terminally to SEQ ID NO: 2 or the portion thereof in the fusion protein. In some embodiments, the fusion protein comprises SEQ ID NO: 1 positioned N-terminally to SEQ ID NO: 2.

[37] In some embodiments, the fusion protein can further comprise a linker sequence positioned between the catalytic domain of the Diphtheria toxin of Corynebacterium diphtheriae or portion thereof and SPA or the portion thereof. By way of example, but not limitation, the linker can comprise an amino acid sequence HM or a (Glycine4Serine) repeat such as

(Glycine4Serine)3 (SEQ ID NO: 5). Linkers are well known to those of skill in the art. [38] In some embodiments the fusion protein comprises the amino acid sequence of SEQ ID NO: 3

(HMHHHHHHGADD VVDS SKSF VMENF SS YHGTKPGYVD SIQKGIQKPKSGTQGNYDDD WKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL GL SLTEPLMEQ V GTEEFIKRF GDGASRVVL SLPF AEGS S S VEYINNWEQ AK AL S VELEIN FETRGKRGQD AMYE YM AQ AC AGNRVRRS VGS SL S CINLD WD VIRDKTKTKIE SLKEHG PIKNKMSESPNKT V SEEKAKQ YLEEFHQT ALEHPEL SELKT VT GTNP VF AG ANY AAW A VNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQ AIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTRPHMNVTDVCAGSPGI PGAPGNHGLPGRDGRDGVKGDPGPPGPMGPPGGMPGLPGRDGLPGAPGAPGERGDKG EPGERGLPGFPAYLDEELQTELYEIKHQILQTMGVLSLQGSMLSVGDKVFSTNGQSVNF DTIKEMCTRAGGNIAVPRTPEENEAIASIAKKYNNYVYLGMIEDQTPGDFHYLDGASVN YTNWYPGEPRGQGKEKCVEMYTDGTWNDRGCLQYRLAVCEF). In some embodiments, the fusion protein can include a portion of SEQ ID NO: 3. In some embodiments, the portion of SEQ ID NO: 3 can include at least 500, 525, 550, 575, 600, 605, 610, 615, 616, 617, 618, 619, 620, 621, 622, 623 or 624 amino acids of SEQ ID NO: 3. By way of example, but not limitation, the fusion protein can include amino acids 9-626 of SEQ ID NO: 3 which omits the N-terminal sequence which include a His tag. In some embodiments, the fusion protein can include the amino acid sequence of SEQ ID NO: 3 or a portion thereof with homology to SEQ ID NO: 3.

By way of example but not limitation, said homology can be at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[39] In some embodiments, a nucleic acid molecule can include a first nucleotide sequence encoding SEQ ID NO: 1 or a portion thereof and a second nucleotide sequence encoding SEQ ID NO: 2 or a portion thereof. In some embodiments, the first nucleotide sequence can encode an amino acid sequence with at least 70% homology to the amino acid sequence of SEQ ID NO: 1. By way of example, but not limitation, the first nucleotide sequence can encode an amino acid sequence with at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the second nucleotide sequence can encode an amino acid sequence with at least 70% homology to the amino acid sequence of SEQ ID NO: 2. By way of example, but not limitation, the second nucleotide sequence can encode an amino acid sequence with at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the amino acid sequence of SEQ ID NO: 2. In some embodiments the nucleic acid molecule can further include a third nucleotide sequence encoding a linker. By way of example, but not limitation, the linker can be the amino acid sequence HM or (Glycine4Serine)3 (SEQ ID NO: 5). In some embodiments, the third nucleotide sequence is positioned between the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the nucleic acid molecule is DNA. In some embodiments, the nucleic acid molecule is RNA. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are operably linked such that they can express a fusion protein of the amino acids sequences encoded by the first nucleotide sequence and the second nucleotide sequence. In some embodiments, the first nucleotide sequence is positioned relative to the second nucleotide sequence such that a fusion protein that can be expressed from the nucleic acid molecule will include the amino acid sequence encoded by the first nucleotide sequence positioned N-terminally to the amino acid sequence encoded by the second nucleotide sequence.

[40] In some embodiments, a nucleic acid molecule can encode the amino acid sequence of SEQ ID NO: 3 or a portion thereof. In some embodiments, the nucleic acid molecule can include a nucleotide sequence that encodes an amino acid sequence with at least 70% homology to the amino acid sequence of SEQ ID NO: 3. By way of example, but not limitation, the nucleic acid molecule can encode an amino acid sequence with at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to SEQ ID NO:

3 or a portion thereof.

[41] In some embodiments, a nucleic acid molecule can include the nucleotide sequence of SEQ ID NO: 4 (CATATGCATCACCACCACCACCACGGTGCGGACGATGTGG

TTGACAGCAGCAAAAGCTTCGTTATGGAAAACTTTAGCAGCTACC ACGGCACCAAACCGGGTTATGTGGATAGCATCCAGAAGGGTATTC AAAAGCCGAAAAGCGGT ACCC AGGGC AACT ACGACGAT GACTGGA AAGGCTTCTATAGCACCGATAACAAGTACGACGCGGCGGGTTATA GCGTTGACAACGAGAACCCGCTGAGCGGTAAAGCGGGTGGCGTGG TT A AGGT G AC C T AC C C GGGT C T G AC C A A AGT GC T GGC GC T G A AGG TT GAT AACGCGGAAACC ATC AAGA AAGAGCTGGGTCTGAGCCTGA CCGAACCGCTGATGGAGCAGGTTGGCACCGAGGAATTCATTAAGC

GTTTTGGTGATGGTGCGAGCCGTGTGGTTCTGAGCCTGCCGTTCG

CGGA AGGT AGC AGC AGC GTTGAGT AC ATC A AC A AC T GGGA AC A AG

CGAAAGCGCTGAGCGTGGAGCTGGAAATTAACTTTGAGACCCGTG

GC AAGCGT GGCC AGGACGCGAT GT ACGAAT AT AT GGCGC A AGCGT

GCGCGGGT AACCGT GT GCGTCGT AGCGTTGGC AGC AGCCTGAGCT

GCATCAACCTGGATTGGGACGTTATTCGTGATAAGACCAAAACCA

AGATCGAAAGCCTGAAAGAGCACGGTCCGATTAAAAACAAGATGA

GCGAAAGCCCGAACAAGACCGTGAGCGAGGAAAAAGCGAAGCAGT

ACCTGGAGGAATTCCACCAAACCGCGCTGGAGCACCCGGAACTGA

GCGAGCTGAAAACCGTGACCGGTACCAACCCGGTTTTTGCGGGTG

CGAACTATGCGGCGTGGGCGGTGAACGTTGCGCAGGTGATCGATA

GCGAGACCGCGGACAACCTGGAAAAAACCACCGCGGCGCTGAGCA

TCCTGCCGGGTATTGGCAGCGTGATGGGCATTGCGGACGGTGCGG

TTC AC C AC A AC AC CGAGGA A AT C GT GGC GC AG AGC ATT GCGC T GA

GCAGCCTGATGGTTGCGCAAGCGATCCCGCTGGTTGGTGAGCTGG

TTGATATTGGTTTCGCGGCGTACAACTTTGTGGAAAGCATCATTA

ACCTGTTCCAAGTGGTTCACAACAGCTACAACCGTCCGGCGTATA

GCCCGGGTCACAAAACCCGTCCGCACATGAACGTGACCGATGTTT

GCGCGGGTAGCCCGGGCATCCCGGGTGCGCCGGGCAACCATGGTC

TGCCGGGTCGTGATGGTCGTGATGGTGTTAAGGGTGATCCGGGTC

CGCCGGGTCCGATGGGCCCGCCGGGTGGCATGCCGGGTCTGCCGG

GCCGTGACGGTCTGCCGGGTGCGCCGGGTGCGCCGGGTGAACGTG

GTGATAAGGGTGAACCGGGCGAGCGTGGCCTGCCGGGTTTTCCGG

CGTACCTGGACGAGGAACTGCAGACCGAACTGTATGAGATCAAGC

ACCAGATTCTGCAAACCATGGGCGTGCTGAGCCTGCAGGGTAGCA

T GCTGAGCGT GGGCGAC AAAGTTTT C AGC ACC AACGGT C AAAGCG

TTAACTTTGATACCATCAAGGAGATGTGCACCCGTGCGGGTGGCA

ACATTGCGGTGCCGCGTACCCCGGAGGAAAACGAAGCGATCGCGA

GCATTGCGAAGAAATACAACAACTACGTTTATCTGGGTATGATCG

AGGATCAAACCCCGGGTGACTTCCACTATCTGGATGGCGCGAGCG TTAACTACACCAACTGGTATCCGGGCGAACCGCGTGGTCAGGGCA

A AG A A A AGT GC GT GG AG AT GT AC AC C G AC GGT AC C T GG A AC GAT C GTGGCTGCCTGCAATATCGTCTGGCGGTGTGCGAGTTTTAAGGATCC).

[42] In some embodiments, a nucleic acid molecule can include a nucleotide sequence which comprises nucleotides 25-1188 of SEQ ID NO: 4 and 1195-1878 of SEQ ID NO; 4. In some embodiments, the nucleic acid molecule can include a nucleotide sequence which comprises nucleotides 25-1878 of SEQ ID NO: 4. In some embodiments, the nucleic acid molecule can include a nucleotide sequence with at least 70% homology to the nucleotide sequence of SEQ ID NO: 4. By way of example, but not limitation, the nucleic acid molecule can include a nucleotide sequence with at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to the nucleotide sequence of SEQ ID NO: 4. SEQ ID NO: 4 includes nucleotide sequences encoding DT388 and SPA connected by a linker with an N-terminal sequence that includes HM and a His tag. In some embodiments, the linker can be removed or replaced by another linker or nucleotide sequence encoding another amino acid sequence. In some embodiments, the HM and His tag (including the HM prior to the His tag) sequences at the N-terminal end can be removed.

[43] Where the nucleic acid molecule is RNA, it should be understood that thymine (T) and uracil (U) can be considered equivalent for purposes of determining homology.

[44] In some embodiments, a polynucleotide vector can include a nucleic acid molecule of the present disclosure. By way of example, but not limitation, the polynucleotide vector can be a plasmid. By way of example, but not limitation, the polynucleotide vector can be pET-l la. In some aspects, where the vector is pET-l la, the cloning site for the nucleic acid molecule can be the Ndel-BamHI site. In some embodiments, a cell is provided that includes a nucleic acid molecule of the present disclosure. In some embodiments, a cell is provided that includes a polynucleotide vector of the present disclosure. By way of example, but not limitation, the cell is an Escherichia coli cell or a eukaryotic cell. Methods for transfecting cells to contain the polynucleotide vector are well known to those of skill in the art.

[45] In some embodiments, the fusion protein can be formulated in inhalable form. In some embodiments, the fusion protein can be formulated in a form suitable for intra-tracheal injection. In certain aspects the fusion protein can be included in a pharmaceutical composition. The pharmaceutical composition can further comprise pharmaceutically acceptable excipients. Suitable pharmaceutically acceptable excipients can include, by way of example but not limitation, sodium chloride, glycerol and surfactants. For example, the pharmaceutical composition can be formulated in buffered aqueous sodium chloride. In some embodiments, the pharmaceutical composition can be in liquid form. In some embodiments, the pharmaceutical composition can be in lyophilized form. In some embodiments, the pharmaceutical composition can be in dry powder form. The pharmaceutical composition can, by way of example but not limitation, be formulated in inhalable form or a form suitable for intra-tracheal injection. Such inhalable or intra-tracheal formulations can aid in the delivery of the fusion protein directly to the lungs where it can exert its cytotoxic effect specifically on ATII cells and avoid systemic diffusion.

[46] The fusion protein of the present disclosure can be produced by methods well known to those of skill in the art. By way of example, but not limitation, the fusion protein of the present disclosure can be made in a bacterial cell such as E. coli from an expression vector which encodes the fusion protein. Similarly, by way of example, but not limitation, the fusion protein of the present disclosure can be made in eukaryotic cells. Methods for expression and purification of such expressed fusion protein can be performed by standard methods well known in the art. By way of example, but not limitation, the fusion proteins of the present disclosure can be produced by transfecting a cell with a nucleic acid molecule encoding the fusion protein and incubating the cell under conditions sufficient to express the fusion protein, followed by isolating the fusion protein.

[47] In some embodiments, a method for treating an ATII cell-dependent lung disease is provided which includes a step of administering a fusion protein of the present disclosure to a patient in need thereof. In some embodiments, the fusion protein is administered at a dose sufficient to kill at least a portion of ATII cells in the patient. In certain aspects, the fusion protein is administered at a dose sufficient to kill a substantial proportion of ATII cells in the patient. In some embodiments, the fusion protein is administered at a dose sufficient to kill all ATII cells in said patient. By way of example, but not limitation, the fusion protein can be administered at a dose sufficient to kill at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more of ATII cells in the patient. Such methods can permit the selective ablation of ATII cells while leaving lung epithelium intact. In some embodiments, the fusion protein or composition thereof is administered in a therapeutically effective amount. One of skill in the art can assess the appropriate dose to achieve the desired therapeutic effect including the extent of ATII cell ablation. In certain aspects, the administration of the fusion protein or pharmaceutical composition can be repeated to achieve the desired effect.

[48] ATII cell-dependent lung diseases are lung diseases which result from injury and/or dysfunction of ATII cells. Such ATII cell-dependent lung diseases include, by way of example but not limitation, acute respiratory distress syndrome (ARDS), fibrosis ( e.g ., surfactant proteins A2, B and C deficiency ( SFTPA2 , SFTPB, and SFTPC ), ABCA3 deficiency, Hermansky-Pudlak syndrome (HPS)), idiopathic interstitial pneumonia, chronic obstructive pulmonary disease (COPD), childhood interstitial lung disease (ChILD), idiopathic pulmonary fibrosis (IPF), familial IPF, interstitial lung disease, infantile desquamative interstitial pneumonitis, nonspecific interstitial pneumonia (NSPIP), congenital pulmonary alveolar proteinosis (PAP), usual interstitial pneumonia (UIP), chronic pneumonitis of infancy, and Birt-Hogg-Dube syndrome.

[49] In some embodiments, the fusion protein is administered as part of a pharmaceutical composition. In certain aspects, the fusion protein or pharmaceutical composition is

administered to the patient via inhalation. Other formulations and methods of administration can be used including, but not limited to, liquid formulations and intravenous or nasal administration. In some embodiments, the fusion protein or pharmaceutical composition is administered via a mesh nebulizer or jet nebulizer. In some embodiments, the fusion protein or pharmaceutical composition can be administered by intra-tracheal injection. In some embodiments, the pharmaceutical composition can be formulated for intra-tracheal injection.

[50] In some embodiments, the foregoing methods can further include a step of administering therapeutic ATII cells to the lungs of said patient. The step of administering the therapeutic ATII cells to the patient can be performed under conditions sufficient to allow the therapeutic ATII cells to engraft in the lung tissue of the patient. The therapeutic ATII cells can be used to re-establish lung function with ATII cells that do not have the injury and/or dysfunction of the ablated ATII cells. The engrafting of healthy ATII cells can be performed after a period of time after the administration of the fusion proteins or compositions of the present disclosure. By way of example but not limitation, the period of time can be from about 2 to about 7 days and any range therebetween. By way of example, but not limitation, the therapeutic ATII cells can be administered about 2, 3, 4, 5, 6, 7 or more days after administration of the fusion protein or pharmaceutical composition comprising the fusion protein. By way of example, but not limitation, engrafting of ATII cells can be performed by administration of said ATII cells to the airway such as by cannulation and inhalation administration. Such administration can be bolus administration and can be repeated as necessary. The amount of therapeutic ATII cells can be a therapeutically effective amount which can be determined by one of skill in the art. Therapeutic ATII cells can be obtained by methods known in the art including, by way of example, but not limitation, flow cytometry, magnetic beads or using specific antibodies.

[51] The foregoing methods can also be used in a patient that does not exhibit symptomology of lung disease but is at risk of developing an ATII cell-dependent lung disease. Thus, in certain aspects, the methods can be used to prophylactically treat a patient at risk for developing an ATII cell-dependent disease absent clinical or physical manifestations of the disease. By way of example, but not limitation, patients with a genetic defect that causes ATII cell dysfunction can be treated by such methods. By way of further example, but not limitation, the genetic defects can include surfactant protein A2, B and C deficiency, ABCA3 deficiency and Hermansky- Pudlak syndrome (HPS).

EXAMPLES

Example 1: Expression o/DT388-SPA in E. coli

[52] DT388-SPA was genetically made by fusing DT388 (first 388 amino acids of Diphtheria Toxin excluding the initial methionine) and Surfactant Protein A (SPA) through a linker HM. A Histidine tag was genetically added to the N-terminus of DT388-SPA, cloned in pETl la, and expressed in BL21 competent E. coli. Isolated colonies were selected and cultured to logarithmic phase. Inducer Isopropyl b-D-l-thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mmol/L, followed by incubation at 20°C for 24 h. The expression level of the target protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE). The recombinant protein was identified by Western blot with polyclonal anti-His-Tag antibody and by Coomassie staining as shown in FIGURE 1B. DT388-SPA was purified under native conditions. The bacterial clear lysate containing DT388-SPA of recombinant strain was incubated with Ni2+-charged affinity resin at 4°C for 1 h. After washing and elution, imidazole and other small molecules were removed by dialysis. The recombinant protein was cleared by endotoxins and quantified. [53] Nucleic acids encoding DT388, and SPA were also each cloned into pETl la and expressed in BL2l competent A. coli.

[54] Expressed protein for DT388 and SPA was also assessed by gel electrophoresis with Coomassie staining and Western blot as shown in FIGURE 1B. The upper panels depict the Western blot results while the lower panels depict the Coomassie stains for both transfected cells (+) and negative controls (0). These results show expression of SPA, DT388 and the fusion protein in transfected cells.

Example 2: Effect and Specificity of DT388-SP A in vitro

[55] Epithelial cells will be isolated from wild type and SPC (-/-) l29S2/SvPasOrlRj (l29/Sv) mice lungs. Briefly, after proteolytic digestion of lung tissue, fluorescence activated cell sorting (FACS) will be used to isolate the EPCAM+ and EPCAM- populations to distinguish between lung epithelial and non-epithelial cells, respectively. Both cell populations will be cultured in DMEM with 10% bovine serum and treated with increasing concentration of each targeting molecule at doses ranging from 10 ⁸ to 10 ¹⁰ M to evaluate the EC50 that inhibits cell

proliferation and induces apoptosis. The EPCAM- cells will be the negative control for targeting molecules uptake. We anticipate that the three fluorescent targeting molecules will act as follows: (i) SPA will be taken up by ATII cells without inducing apoptosis; DT388 will not be taken up by any cell since it lacks the cell binding domain; DT388-SPA will be taken up by ATII cells, internalized and induce apoptosis. The specificity, extent and efficiency of ATII cell death will be assessed at different time points (from 24 to 72 hours, as shown in vitro) from the last dose administered of the targeting molecule by: (i) Live cell imaging. Using time-lapse fluorescence microscopy, cells will be imaged at 10 min intervals to record the targeting molecules’ binding on cell surface, internalization, and the eventual induction of apoptosis (apoptotic bodies formation) (ii) Inhibition of proliferation by BrdU incorporation and counterstaining with lung epithelial and interstitial cell type makers: Aq5 (ATI), SPC (ATII),

SPB (ATII, club), Mucin 5 (goblet), p63 (basal), CC-10 (club), Acetyl-tubulin (ciliated), vimentin (fibroblast), NG2 (pericyte), SMA (myocite). (iii) Apoptosis. Cleaved caspase 3 staining will be counterstained with lung markers as in (ii). (iv) Quantitative RT-PCR for ATII cells markers (SFTPA, SFTPB, SFTPC, SFTPD, LAMP3) to show decreased amount of ATII cells after cytotoxic effects of DT388, compared to untreated cells. Example 3: Determining inhalational parameters using an ex vivo bioreactor

[56] Lungs will be harvested from 2-4 months old l29/Sv mice and mounted on an ex vivo bioreactor, where the trachea is cannulated and ventilated under physiologic conditions. A nebulizer connected to the tracheal cannula will be used to delivery each of three fluorescently labeled targeting molecule (DT388, DT388-SPA, and SPA) mixed with normal surfactant to the lungs by aerosol. Fluorescent molecules within the alveoli can be visualized externally using a real-time deep-tissue imaging system developed by our lab. Quantity, number and timing of each aerosolized dose will be titrated to reach 80-90% of the lung alveolar region. The delivery and deposition of the fluorescent targeting molecules onto the alveolar surface will be measured using our imaging system.

[57] Once we have the optimized dosage, we seek to determine if DT388-SPA could specifically target ATII cells in native lung setting. Lungs will be collected 1 and 3 hours post aerosol delivery and analyzed as follows: (i) Quantification of DT388-SPA in alveolar space.

DT388-SPA will be quantified by analysis of Alexa dye signal in pre-determined lung sections. Ratio of alveolar/non-alveolar signal will be quantified by ImageJ software (ii) Determination of DT388-SPA uptake by lung cells. Cells uptaking the DT388-SPA in lung sections will be identified by the Alexa dye and co-stained with specific cell markers.

Example 4: In vivo evaluation o/DT388-SPA

[58] (i) First, DT388-SPA will be administered by non-invasive ventilation to wild type l29/Sv mice based on the results in Example 3. Animals will be sacrificed at 3 and 7 days from last administration of DT388-SPA, when potentially all ATII cells are deceased according to DT388 effect in vivo. ATII cell death will be assessed as in Example 2. (ii) SPC (-/-) l29/Sv mice will be used for healthy ATII cells isolation. In these mice, cells expressing SPC, will also express Tomato (red fluorescence), which allows SPC-expressing ATII cells to be isolated by FACS. We are planning to intratracheally inject 10, 15, and 20 millions of exogenous ATII cells to determine the number of cells required for efficient lung repopulation post ATII cells depletion (iii) When conditions are optimized for cell replacement in wild type mice, we are planning to treat mice as follows: 1) Treatment with saline solution in SPC (-/-) mice (negative control). 2) Treatment with DT388-SPA in SPC (-/-) mice (only cell death). 3) Treatment with DT388-SPA plus ATII cells replacement in SPC (-/-) mice (Animals will be treated with DT388- SPA at 6 months, when lung fibrosis is clearly evident. Prior to cell replacement, lung imaging (high resolution CT) will be performed to assess lung architecture (healthy versus fibrotic/emphysematous parenchyma). 99mTcAxV SPECT/CT imaging will be used to measure pulmonary cell apoptosis. For cell replacement, mice will be intratracheally cannulated and exogenous ATII cells will be injected into the airways. Animals will be harvested after 4 and 7 days. Exogenous ATII cells will be easily detected by fluorescence (Tomato). After harvest, lung tissue will be analyzed by: (i) Cell morphology and distribution by H&E, TEM, and

immunostaining for lung cell markers as in Example 2 to assess distribution and alveolar lining of the exogenous ATII cells (ii) Cell proliferation by using ki67 and cell apoptosis with cleaved caspase 3, counterstained with lung cell markers as in Example 2. (iii) Quantitative RT-PCR for ATII cells markers as in Example 2 on exogenous ATII cells to evaluate their expression in the recipient lung.

Example 5: In vitro evaluation of protein synthesis

[59] DT388 inhibits protein translation and consequently induces cell apoptosis. Rabbit reticulocyte lysate, containing all of the components necessary for protein translation, was used according to the manufacturer’s protocol (Promega L4960). Each mixture (in triplicate) was incubated with luciferase mRNA and with vehicle or 10 pm of DT388-SPA, DT388 (positive control), SPA (negative control) or cycloheximide (known protein synthesis inhibitor, positive control) for 90 minutes at 30°C. To measure protein synthesis, 2.5 pL of each reaction was added to 50 pL of Luciferase Assay Reagent and luciferase activity was read using a

luminometer per the manufacturer’s protocol.

[60] Results of the luciferase assay are shown in FIGEIRE 3. As shown in FIGURE 3,

DT388-SPA inhibits protein translation by 99% compared to vehicle, similar to both DT388 and cycloheximide. SPA (negative control) did not affect protein translation.

Example 6: Uptake o/DTA388-SPA by Rat RLE-6NT Cells

[61] To test if ATII cells can recognize and take up DT388-SPA, and potentially undergo apoptosis, rat RLE-6NT (rat lung epithelial T-antigen negative) cells derived from ATII cells of 56-day old Rattus norvegicus were used. 0.1 x 10⁶ RLE-6NT cells were plated in Ham’s F12 medium supplemented with 2 mM L-glutamine supplemented with 0.01 mg/mL bovine pituitary extract, 0.005 mg/mL insulin, 2.5 ng/mL insulin-like growth factor, 0.00125 mg/mL transferrin, and 2.5 ng/mL EGF, 90%; fetal bovine serum, 10%. After 24 hours of culture, cells were incubated with vehicle, 50 pM of Alexa 488 labeled DTA388-SPA, DT388 or SPA for up to 12 hours. Images of uptake were captured with a 2-photon confocal laser scanning microscope (Leica TCS SP8).

[62] The resulting images are shown in FIGURE 4. As shown in FIGURE 4, uptake and internalization was evident for SPA and DTA388-SPA, but not for DT388 alone.

Example 7: Cytotoxicity of DTA388-SPA in Rat RLE-6NT Cells

[63] 5 x 10³ RLE 6NT cells were plated in triplicates in 96 multi well plate in Ham's F12 medium supplemented with 2 mM L-glutamine supplemented with 0.01 mg/ml bovine pituitary extract, 0.005 mg/ml insulin, 2.5 ng/ml insulin-like growth factor, 0.00125 mg/ml transferrin, and 2.5 ng/ml EGF, 90%; fetal bovine serum, 10%. After 24 hours, Cells were treated with different concentrations (12.5, 6.25, 3.125, 1.625, 0 nM) of DT388-SPA, DT388, SPA or vehicle. As positive control of cytotoxicity, cells were treated with cycloheximide (100 pg/ml). At 72 hours, Dojindo’s highly water-soluble tetrazolium salt (WST-8) assay was performed to assess cell viability according to the manufacturer protocol (Cell Counting Kit-8, Dojindo Molecular Technologies, Inc.).

[64] Results are shown in FIGURE 5. As shown in FIGURE 5, at 10 ⁴M, DT388-SPA kills 95% of cells, whereas SPA and DT388 only 2-4%.

[65] Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as illustrated, in part, by the appended claims.

[66] The foregoing description of specific embodiments of the present disclosure has been presented for purpose of illustration and description. The exemplary embodiments were chosen and described in order to best explain the principles of the disclosure and its practical application, to thereby enable others skilled in the art to best utilize the subject matter and various embodiments with various modifications are suited to the particular use contemplated. Different features and disclosures of the various embodiments within the present disclosure may be combined within the scope of the present disclosure.

Claims

What is claimed is:

1. A fusion protein comprising the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.

2. The fusion protein of claim 1, wherein the amino acid sequence of SEQ ID NO: 1 is positioned N-terminally to the amino acid sequence of SEQ ID NO: 2.

3. The fusion protein of claim 1, further comprising a linker positioned between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.

4. The fusion protein of claim 2, further comprising a linker positioned between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.

5. The fusion protein of claim 3, wherein the linker comprises the amino acid sequence HM.

6. The fusion protein of claim 4, wherein the linker comprises the amino acid sequence HM.

7. The fusion protein of claim 3, wherein the linker comprises the amino acid sequence of

SEQ ID NO: 5.

8. The fusion protein of claim 4, wherein the linker comprises the amino acid sequence of SEQ ID NO: 5.

9. A fusion protein comprising the amino acid sequence of SEQ ID NO: 3.

10. A pharmaceutical composition comprising the fusion protein of any one of claims 1-9.

11. The pharmaceutical composition of claim 10, further comprising at least one

pharmaceutically acceptable excipient.

12. The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable excipient is selected from the group consisting of sodium chloride, glycerol and a surfactant.

13. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is formulated for inhalation.

14. The pharmaceutical composition of claim 10, wherein the pharmaceutical composition is in lyophilized form.

15. A nucleic acid molecule comprising the sequence of SEQ ID NO: 4.

16. A nucleic acid molecule comprising a first nucleotide sequence comprising nucleotides 25-1188 of SEQ ID NO: 4 and a second nucleotide sequence comprising nucleotides 1195-1878 of SEQ ID NO; 4.

17. The nucleic acid molecule of claim 16, further comprising a third nucleotide sequence positioned between the first nucleotide sequence and the second nucleotide sequence, the third nucleotide sequence encoding a linker.

18. The nucleic acid molecule of claim 17, wherein the linker has the amino acid sequence HM.

19. The nucleic acid molecule of claim 17, wherein the linker has the amino acid sequence of SEQ ID NO: 5.

20. The nucleic acid molecule of any one of claims 16-19, wherein the nucleic acid molecule is DNA, and wherein the first nucleotide sequence is positioned 3’ to the second nucleotide sequence.

21. The nucleic acid molecule of any one of claims 16-19, wherein the nucleic acid molecule is RNA, and wherein the first nucleotide sequence is positioned 5’ to the second nucleotide sequence.

22. A plasmid comprising the nucleic acid molecule of any one of claims 15-19.

23. A plasmid comprising the nucleic acid molecule of claim 20.

24. A cell comprising the nucleic acid molecule of any one of claims 15-19.

25. A cell comprising the nucleic acid molecule of claim 20.

26. A cell comprising the nucleic acid molecule of claim 21.

27. The cell of claim 24, wherein the cell is an Escherichia coli cell.

28. The cell of claim 25, wherein the cell is an Escherichia coli cell.

29. The cell of claim 26, wherein the cell is an Escherichia coli cell.

30. The cell of claim 24, wherein the cell is a eukaryotic cell.

31. The cell of claim 25, wherein the cell is a eukaryotic cell.

32. The cell of claim 26, wherein the cell is a eukaryotic cell.

33. A method for treating a alveolar type II (ATII) cell-dependent lung disease in a patient in need thereof, comprising: administering a pharmaceutical composition comprising a fusion protein comprising the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2 to said patient, wherein said fusion protein is administered at a dose sufficient to kill at least a portion of ATII cells in said patient.

34. The method of claim 33, wherein said patient is suffering from an ATII cell-dependent lung disease.

35. The method of claim 34, wherein the ATII cell-dependent lung disease is selected from the group consisting of acute respiratory distress syndrome (ARDS), fibrosis ( e.g ., surfactant proteins A2, B and C deficiency ( SFTPA2 , SFTPB, and SFTPC ), ABCA3 deficiency,

Hermansky-Pudlak syndrome (HPS)), idiopathic interstitial pneumonia, chronic obstructive pulmonary disease (COPD), childhood interstitial lung disease (ChILD), idiopathic pulmonary fibrosis (IPF), familial IPF, interstitial lung disease, infantile desquamative interstitial pneumonitis, nonspecific interstitial pneumonia (NSPIP), congenital pulmonary alveolar proteinosis (PAP), usual interstitial pneumonia (UTP), chronic pneumonitis of infancy, and Birt- Hogg-Dube syndrome.

36. The method of claim 33, wherein the pharmaceutical composition is suitable for inhalation, and wherein the pharmaceutical composition is administered by inhalation to said patient.

37. The method of claim 33, wherein the pharmaceutical composition is suitable for intra- tracheal injection, wherein the pharmaceutical composition is administered by intra-tracheal injection to said patient.

38. The method of claim 33, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

39. The method of claim 38, wherein the at least one pharmaceutically acceptable excipient is selected from the group consisting of sodium chloride, glycerol and a surfactant.

40. The method of claim 33, wherein the fusion protein is administered at a dose sufficient to kill all ATII cells in said patient.

41. The method of claim 33, wherein the fusion protein further comprises a linker positioned between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2

42. The method of claim 41, wherein the linker has the amino acid sequence HM.

43. The method of claim 41, wherein the linker has the amino acid sequence of SEQ ID NO: 5.

44. The method of claim 33, wherein the amino acid sequence of SEQ ID NO: 1 is positioned N-terminally to the amino acid of SEQ ID NO: 2 in the fusion protein.

45. The method of claim 33, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 3.

46. The method of any one of claims 33-45. further comprising, after administering the fusion protein to the patient: administering therapeutic ATII cells to the lungs of said patient after a period of time after administering the fusion protein to the patient, under conditions sufficient for the ATII cells to engraft to epithelium in the lungs of said patient.

47. The method of claim 46, wherein the period of time is from about 2 to about 4 days.