WO2021151005A1

WO2021151005A1 - Inhaled das181 to treat cancer in the lung

Info

Publication number: WO2021151005A1
Application number: PCT/US2021/014772
Authority: WO
Inventors: Jennifer Ho; Nancy Chang; Jianying Dong
Original assignee: Ansun Biopharma Inc
Current assignee: Ansun Biopharma Inc
Priority date: 2020-01-22
Filing date: 2021-01-22
Publication date: 2021-07-29
Anticipated expiration: 2022-07-22
Also published as: WO2021151005A9

Abstract

Provided herein are methods of treating lung cancer by administering a therapeutically effective amount of a polypeptide having sialidase activity, e.g., DAS181, topically to the lungs, wherein the method of administration includes inhalation of a dry powder formulation containing, e,g,. DAS181 with a metered dose inhaler and/or inhalation of a liquid nebulized formulation containing, e.g., DAS181 with a nebulizer.

Description

INHALED DAS 181 TO TREAT CANCER IN THE LUNG

BACKGROUND

Lung cancer, also known as lung carcinoma, is a malignant lung tumor characterized by uncontrolled cell growth in tissues of the lung. This growth can spread beyond the lung by the process of metastasis into nearby tissue or other parts of the body. Most cancers that start in the lung, known as primary lung cancers, are carcinomas. The two main types are small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC). The three main subtypes of NSCLC are adenocarcinoma, squamous-cell carcinoma, and large-cell carcinoma. Rare subtypes include pulmonary enteric adenocarcinoma. In SCLC, the cells contain dense neurosecretory granules (vesicles containing neuroendocrine hormones), and most cases arise in the larger airways (primary and secondary bronchi). Sixty to seventy percent of patients have extensive disease (which cannot be targeted within a single radiation therapy field) at presentation.

Worldwide in 2012, lung cancer occurred in 1.8 million people and resulted in 1.6 million deaths. This makes it the most common cause of cancer-related death in men and second most common in women after breast cancer. Overall, 17.4% of people in the United States diagnosed with lung cancer survive five years after the diagnosis, which makes this a devastating disease and diagnosis.

Treatment and long-term outcomes depend on the type of cancer, the stage (degree of spread), and the person's overall health. Most cases are not curable. Common treatments include surgery, chemotherapy, and radiotherapy. NSCLC is sometimes treated with surgery, whereas SCLC usually responds better to chemotherapy and radiotherapy. More recent treatment options include monoclonal antibodies and immunotherapies, such as immune checkpoint inhibitors, all of which have their limitations. There are two types of immune checkpoint inhibitor therapy currently approved for lung cancer: CTLA-4 inhibitors and PD-1 inhibitors. CTLA-4 is a protein on the surface of T cells that helps keep the body’s immune responses in check. When CTLA-4 binds to another protein called B7 on an antigen presenting cell, it stops T cell activation and keeps the T cell from killing other cells including the cancer cell. CTLA-4 inhibitors bind to CTLA-4 and allow the T cells to kill cancer cells. Ipilimumab is a type of CTLA-4 inhibitor. PD-1 is a protein on the surface of T cells that helps keep the body’s immune responses in check. When PD-1 binds to another protein called PD-L1 on a cancer cell, it stops the T cell from killing the cancer cell. PD-1 inhibitors bind to PD-1 or PD-L1 and allow the T cells to kill cancer cells. Pembrolizumab and Nivolumab are anti-PD-1 types of PD-1 inhibitors approved for NSCLC treatment. In addition, an anti-PD-Ll drug (atezolizumab) has been approved as monotherapy for second-line treatment for NSCLC, and another anti-PD-Ll drug (Durvalumab) has been approved for unresectable stage III NSCLC. Although these checkpoint inhibitors have shown promising results in other cancers, their use in treating lung cancers has been marginal at best, and a cure or improvement in long term survival for lung cancer remains elusive.

In addition to NSCLC and SCLC, almost any type of cancer has the ability to spread to the lungs. The tumors that most commonly do so include kidney cancer, head and neck cancer, testicular cancer, osteo sarcoma, soft tissue sarcoma, melanoma, thyroid cancer, bladder cancer, colon or colorectal cancer, breast cancer, prostate cancer, sarcoma, Wilms tumor, and neuroblastoma. There are no adequate treatments for any of these cancers once they have spread to the lungs.

Thus, there is a need for novel treatments for lung cancer and other cancers that have metastasized to the lung.

SUMMARY

Provided herein are methods of treating lung cancer by administering a therapeutically effective amount of a polypeptide having sialidate activity to the lungs of a patient in need thereof. Within the tumor microenvironment the sialidase can remove terminal sialic acid residues on cancer cells, immune cells and other types of cells, thereby reducing the barrier for entry of immunotherapy reagents and promote cellular immunity against cancer cells in the location of the lungs. In one embodiment, the sialidase is a recombinant sialidase. In yet another embodiment, the sialidase is a bacterial derived recombinant sialidase. In yet another embodiment, the bacterial derived recombinant sialidase is DAS181.

Described herein is a method of treating a subject suffering from lung cancer, the method comprising administering a therapeutically effective amount of a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or 2 to the lungs of the subject. In various embodiments: the method of administration includes inhalation of a dry powder formulation comprising the polypeptide with a metered dose inhaler; the method of administration includes inhalation of a liquid nebulized formulation comprising the polypeptide; the method futher comprises administering a chemotherapeutic agent; the polypeptide comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1 or 2; the polypeptide comprises an amino acid sequence that has no more than 5 single amino acid substitutions or deletions compared to SEQ ID NO: 1 or 2; the polypeptide comprises an amino acid sequence that has no more than 5 single amino acid substitutions compared to SEQ ID NO: 1 or 2 (e.g., the amino acid substitutions are conservative substitutions); the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 2; the polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or 2.

Also described is a method for increasing the effectiveness of an antibody used the treat lung cancer, the method comprising administering a therapeutically effective amount of a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or 2 to the lungs of the subject and administering an immunotherapy used to treat lung cancer. In various embodiments: In various embodiments: the method of administration includes inhalation of a dry powder formulation comprising the polypeptide with a metered dose inhaler; the method of administration includes inhalation of a liquid nebulized formulation comprising the polypeptide; the method futher comprises administering a chemotherapeutic agent; the polypeptide comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1 or 2; the polypeptide comprises an amino acid sequence that has no more than 5 single amino acid substitutions or deletions compared to SEQ ID NO: 1 or 2; the polypeptide comprises an amino acid sequence that has no more than 5 single amino acid substitutions compared to SEQ ID NO: 1 or 2 (e.g., the amino acid substitutions are conservative substitutions); the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 2; the polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or 2; the antibody is evacizumab or ramucirumab.

Also provided herein are methods of treating lung cancer by administering a therapeutically effective amount of DAS181 topically to the lungs and administering a therapeutically effective amount of a chemotherapeutic agent, wherein the method of administration of DAS181 includes inhalation of a dry powder formulation containing DAS 181 with a metered dose inhaler and/or inhalation of a liquid nebulized formulation containing DAS 181 with a nebulizer.

The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.

A "sialidase catalytic domain protein" is a protein that comprises the catalytic domain of a sialidase, or an amino acid sequence that is substantially homologous to the catalytic domain of a sialidase, but does not comprise the entire amino acid sequence of the sialidase the catalytic domain is derived from, wherein the sialidase catalytic domain protein retains substantially the same activity as the intact sialidase the catalytic domain is derived from. A sialidase catalytic domain protein can comprise amino acid sequences that are not derived from a sialidase, but this is not required. A sialidase catalytic domain protein can comprise amino acid sequences that are derived from or substantially homologous to amino acid sequences of one or more other known proteins, or can comprise one or more amino acids that are not derived from or substantially homologous to amino acid sequences of other known proteins.

FIGURES

FIGURE 1: SNA-detected glycans remaining after DAS 181 exposure. CFG glycan microarray v3.2 was exposed to 0, 0.5, 5, or 50 nM DAS181 (A, B, C and D, respectively) and then remaining glycans were detected with SNA lectin. Information for the top 20 glycans detected by SNA in each graph are listed on the right; glycan number, shorthand glycan name/structure, and relative fluorescence units (RFU) are shown. Glycans with an a2,3-linked sialic acid terminus are in bold, glycans with an a2,6-linked sialic acid terminus are in italics.

FIGURE 2: MAL2-detected glycans remaining after DAS181 exposure. CFG glycan microarray v3.2 was exposed to 0, 0.5, 5, or 50 nM DAS181 (A, B, C and D, respectively) and then remaining glycans were detected with MAL2 lectin. Information for the top 20 glycans detected by MAL2 in each graph are listed on the right; glycan number, shorthand glycan name/structure, and relative fluorescence units (RFU) are shown. Glycans with an a2,3-linked sialic acid terminus are in bold, glycans with an a2,6-linked sialic acid terminus are in italics.

DETAILED DESCRIPTION

DAS181 is a particularly potent sialidase that can reduce the level of sialic acid residues on the surface of tumor cells. The high level of sialic acid on tumor cells can serve to interfere with the killing of tumor cells by cells of the immune system such as NK cells. By eliminating sialic acid residues from the surface of cancer cells, the tumor micro environment becomes less hostile to immune cells, such as T cells, NK cells and macrophages, allowing the better infiltration of the tumor micro-environment by cancer killing immune cells and the reduction in inactivation of immune cells by tumor cells.

In some embodiments, DAS 181 or another sialidase may be administered in combination with a second therapeutic modality. In one embodiment, the second therapeutic modality may be a chemotherapeutic agent. Examples of chemotherapeutic agents that may be administered in combination with DAS 181 or another sialidase include without limitation the following: platinum; platinum analogs (e.g., platinum coordination complexes) such as cisplatin, carboplatin, oxaliplatin, DWA2114R, NK121, IS 3 295, and 254-S; anthracenediones; vinblastine; alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; substituted ureas; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide; procarbazine; anti-cancer polysaccharides; polysaccharide-K; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2,2-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytosine arabinoside; cyclophosphamide; thiotepa; taxoids, such as paclitaxel, docetaxel and albumin-bound paclitaxel (nab-paclitaxel, abraxane); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; etoposide (VP- 16); pemetrexed, ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; XELODA; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins; capecitabine; methylhydrazine derivatives; Erlotinib (TARCEVA); sunitinib malate (SUTENT); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Polypeptides with Sialidase Activity

DAS181 is a recombinant sialidase that is capable of cleaving sialic acid from tumor cells.

Other polypeptides that include all or a catalytic portion of a sialidase are summarized below. Table 1. Nomenclature of NeuSAc sialyltraiisferases

HGNC: Hugo Gene Community Nomenclature (www.genenames.org)

Domains within Polypeptides having Sialidase Activity

The polypeptide, in addition to the sialidase or catalytic portion thereof can, optionally, include peptide or protein sequences that contribute to the therapeutic activity of the protein. For example, the protein can include an anchoring domain that promotes interaction between the protein and a cell surface. The anchoring domain and sialidase domain can be arranged in any appropriate way that allows the protein to bind at or near a target cell membrane such that the therapeutic sialidase can exhibit an extracellular activity that removes sialic acid residues. The protein can have more than one anchoring domains. In cases in which the polypeptide has more than one anchoring domain, the anchoring domains can be the same or different. The protein can have more than one sialidase domain. In cases in which a compound has more than one sialidase domain, the sialidase domains can be the same or different. Where the protein comprises multiple anchoring domains, the anchoring domains can be arranged in tandem (with or without linkers) or on alternate sides of other domains, such as sialidase domains. Where a compound comprises multiple sialidase domains, the sialidase domains can be arranged in tandem (with or without linkers) or on alternate sides of other domains.

Sialidase Domain

The sialidase domain can be specific for Neu5 Ac linked via alpha 2,3 linkage, specific for Neu5Ac linked via an alpha 2,6 or can cleave Neu5Ac linked via an alpha 2,3 linkage or an alpha 2,6 linkage. A variety of sialidases are described in Tables 2-5.

A sialidase that can cleave more than one type of linkage between a sialic acid residue and the remainder of a substrate molecule, in particular, a sialidase that can cleave both alpha(2, 6)-Gal and alpha(2, 3)-Gal linkages can be used in the compounds of the disclosure. Sialidases included are the large bacterial sialidases that can degrade the receptor sialic acids Neu5Ac alpha(2,6)-Gal and Neu5Ac alpha(2,3)-Gal. For example, the bacterial sialidase enzymes from Clostridium perfringens (Genbank Accession Number X87369), Actinomyces viscosus (GenBankX62276), Arthrobacter ureafaciens GenBank (AY934539), or Micromonospora viridifaciens (Genbank Accession Number DO 1045) can be used. Sialidase domains of compounds of the present disclosure can comprise all or a portion of the amino acid sequence of a large bacterial sialidase or can comprise amino acid sequences that are substantially homologous to all or a portion of the amino acid sequence of a large bacterial sialidase. In one preferred embodiment, a sialidase domain comprises a sialidase encoded by Actinomyces viscosus, such as that of SEQ ID NO: 1 or 2, or such as sialidase sequence substantially homologous to SEQ ID NO: 12. In yet another preferred embodiment, a sialidase domain comprises the catalytic domain of the Actinomyces viscosus sialidase extending from amino acids 274-666 of SEQ ID NO: 12, or a substantially homologous sequence.

Additional sialidases include the human sialidases such as those encoded by the genes NEU2 (SEQ ID NO:8; Genbank Accession Number Y16535; Monti, E, Preti, Rossi, E., Ballabio, A and Borsani G. (1999) Genomics 57:137-143) andNEU4 (SEQ ID NO: 9; Genbank Accession Number NM080741 ; Monti et al. (2002) Neurochem Res 27:646-663). Sialidase domains of compounds of the present diclosure can comprise all or a portion of the amino acid sequences of a sialidase or can comprise amino acid sequences that are substantially homologous to all or a portion of the amino acid sequences of a sialidase. Preferably, where a sialidase domain comprises a portion of the amino acid sequences of a naturally occurring sialidase, or sequences substantially homologous to a portion of the amino acid sequences of a naturally occurring sialidase, the portion comprises essentially the same activity as the intact sialidase. The present disclosure also includes sialidase catalytic domain proteins. As used herein a "sialidase catalytic domain protein" comprises a catalytic domain of a sialidase but does not comprise the entire amino acid sequence of the sialidase from which the catalytic domain is derived. A sialidase catalytic domain protein has sialidase activity. Preferably, a sialidase catalytic domain protein comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 50%, at least 70% of the activity of the sialidase from which the catalytic domain sequence is derived. More preferably, a sialidase catalytic domain protein comprises at least 90% of the activity of the sialidase from which the catalytic domain sequence is derived.

A sialidase catalytic domain protein can include other amino acid sequences, such as but not limited to additional sialidase sequences, sequences derived from other proteins, or sequences that are not derived from sequences of naturally occurring proteins. Additional amino acid sequences can perform any of a number of functions, including contributing other activities to the catalytic domain protein, enhancing the expression, processing, folding, or stability of the sialidase catalytic domain protein, or even providing a desirable size or spacing of the protein.

A preferred sialidase catalytic domain protein is a protein that comprises the catalytic domain of the A. viscosus sialidase. Preferably, an A. viscosus sialidase catalytic domain protein comprises amino acids 270-666 of the A. viscosus sialidase sequence (SEQ ID NO: 12). Preferably, an A. Viscosus sialidase catalytic domain protein comprises an amino acid sequence that begins at any of the amino acids from amino acid 270 to amino acid 290 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and ends at any of the amino acids from amino acid 665 to amino acid 901 of said A. viscosus sialidase sequence (SEQ ID NO: 12), and lacks any A. viscosus sialidase protein sequence extending from amino acid 1 to amino acid 269. In some preferred embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 274-681 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and lacks other A. viscosus sialidase sequence. In some preferred embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 274-666 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and lacks any other A. viscosus sialidase sequence. In some preferred embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 290-666 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and lacks any other A. viscosus sialidase sequence. In yet other preferred embodiments, an A. viscosus sialidase catalytic domain protein comprises amino acids 290-681 of the A. viscosus sialidase sequence (SEQ ID NO: 12) and lacks any other A. viscosus sialidase sequence. Useful sialidases for use in in the present invention include polypeptides comprising a sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to the sequences set forth in Table 2 below and the sequences set forth in SEQ IDs listed herein, particularly DAS 181 as represented in SEQ ID. No. 2.

Table 2: Engineered Sialidases

Table 3: Human Sialidases

Table 4: Sialidases in organisms that are largely commensal with humans

Table 5: Additional sialidases

Anchoring Domain

As used herein, an "extracellular anchoring domain" or "anchoring domain" is any moiety that interacts with an entity that is at or on the exterior surface of a target cell or is in close proximity to the exterior surface of a target cell. An anchoring domain serves to retain a compound of the present disclosure at or near the external surface of a target cell. An extracellular anchoring domain preferably binds 1) a molecule expressed on the surface of a cancer cell, or a moiety, domain, or epitope of a molecule expressed on the surface of a cancer cell, 2) a chemical entity attached to a molecule expressed on the surface of a cancer cell, or 3) a molecule of the extracellular matrix surrounding a cancer cell.

Useful anchoring domains bind to heparin/sulfate, a type of GAG that is ubiquitously present on cell membranes. Many proteins specifically bind to heparin/heparan sulfate, and the GAG-binding sequences in these proteins have been identified (Meyer, F A, King, M and Gelman, R A. (1975) Biochimica et BiophysicaActa 392: 223-232; Schauer, S. ed., pp 233. Sialic Acids Chemistry, Metabolism and Function. Springer-Verlag, 1982). For example, the GAG-binding sequences of human platelet factor 4 (PF4) (SEQ ID NO:2), human interleukin 8 (IL8) (SEQ ID NO:3), humanantithrombin III (AT III) (SEQ ID NO:4), human apoprotein E (ApoE) (SEQ ID NO:5), human angio-associated migratory cell protein (AAMP) (SEQ ID NO:6), or human amphiregulin (SEQ ID NO:7) have been shown to have very high affinity to heparin. Linkers

A protein that includes a sialidase or a catalytic domain thereof can optionally include one or more polypeptide linkers that can join domains of the compound. Linkers can be used to provide optimal spacing or folding of the domains of a protein. The domains of a protein joined by linkers can be sialidase domains, anchoring domains, or any other domains or moieties of the compound that provide additional functions such as enhancing protein stability, facilitating purification, etc. Some preferred linkers include the amino acid glycine. For example, linkers having the sequence: (GGGGS (SEQ ID NO:10))n, where n is 1 20

It should be noted that not all sialidases are created equal. Some are much more potent than others, and the potency of the sialidase will affect its efficacy. Some are also more broadly active, while others are very specific to the substrate on which they are active. Applicants have unexpectedly discovered that with respect to the desialylation of tumor cells, DAS 181 has a higher potency than virtually all other sialidases, including naturally occurring ones, and it is broadly active against all sialic acids no matter the structure of the underlying oligosaccharide chains. DAS 181 has the ability to remove sialic acid residues from the surface of cancer cells much more efficiently than other sialidases. This is a discovery that was not expected. For example, DAS181 when expressed in cells, either in a secreted form or anchored on the cell surface, showed unexpected potent activity at removal of tumor cell surface sialic acids in comparison to a human sialidase Neu2 constructed in the same format. The Neu2 showed much lower activity in sialic acid removal from tumor cells.

The pharmaceutical compositions of the present invention allow treatment of lung cancers and non-lung cancers that have metastasized to the lungs.

When the pharmaceutical compositions of the present invention are used, they may usually be mixed appropriately with pharmaceutical aids used in formulation, such as excipients, carriers, and diluents. These formulations may be administered orally, nasally, or parenterally in a form such as a tablet, a capsule, a powder, a syrup, a granule, a pill, a suspension, an emulsion, a solution, a powdered preparation, a suppository, eye drops, nasal drops, liquids for nebulized delivery, eardrops, a patch, an ointment, or an injection according to a conventional method. Moreover, an administration method, a dose, and the frequency of administration may be selected appropriately according to the age, body weight, cancer type and stage of cancer. It may usually be administered orally, nasally, or parenterally (e.g., injection, intravenous drips, administration to a rectal site, nebulized delivery of a liquid, inhalation of dry powder through a metered dose inhaler) to an adult in one to several divided dose(s) at dose(s) of 0.01 to 1000 mg/kg per day.

In one embodiment, DAS 181 is administered in a liquid formulation through a nebulizer such that it is inhaled or breathed into the lungs. In another embodiment, DAS 181 is administered in a dry powder formulation through a metered dose inhaler or other type of dry powder inhaler that is inhaled or breathed into the lungs.

The therapeutic formulation contains between about 1 mg and about 50 mg per dose of DAS 181, or more particularly between about 2 mg and about 10 mg of DAS 181 per dose, or still more particularly between about 4 mg and about 10 mg of DAS181 per dose, or still more particularly between about 8 mg and 10 mg of DAS 181 per dose, or more particularly between about 9 mg and about 10 mg of DAS 181 per dose or most particularly about 9 mg of DAS181 per dose. The therapeutic formulation can also contain between about 20 mg and about 50 mg of DAS 181 per dose, or between about 30 mg and about 50 mg of DAS181 per dose, or between about 40 mg and about 50 mg of DAS181 per dose. DAS 181 can be administered, e.g., once per day (per the dosages set forth above) for up to ten consecutive days, or up to seven consecutive days, or up to five consecutive days for a treatment course, followed by a break in treatment of about 20 days, followed by another treatment course of up to 10 consecutive days, or seven consecutive days or five consecutive days, or twice per week (per the dosages set forth above) for up to 5 consecutive weeks, followed by a break in treatment of about 20 days. . This cycle can be repeated as needed to reduce tumors in the lung. DAS 181 can be administered as a monotherapy or in combination with approved immune checkpoint inhibitors or standard chemotherapy.

Example 1 - Broad Activity and Potency of DAS181

The specific activity of DAS181 against a synthetic substrate is more than 100 times higher than the activity of the human neuraminidase Neu2. This difference in specific activity is surprising because DAS 181 is an engineered fusion protein yet retains high specific activity. Moreover, DAS181 efficiently cleaves sialylated glycans regardless of the structure of the more distant parts of the oligosaccharide chain (e.g. a2,3 vs. a2,6 linkage, chain length, or modification).

FIGURES 1A-D and 2A-D shown the results of DAS181 treatment on glycans in a glycan microarray. Tables 6-9, respectively, list the top 20 glycan detected by SNA lectin after the treatments in FIGURES 1A-D, respectively. Tables 10-13, respectively, list the top 20 glycan detected by MALI lectin after the treatments in FIGURES 2A-D, respectively. As can be seen, glycans with typical terminal sialic acid structures such as Neu5Ac (N- acetylneuraminic acid) are readily cleaved by DAS 181 with near complete removal at low DAS181 concentrations (e.g., 0.5 nM). Also, glycans with KDN terminal sialic acid structure (2-keto-3-deoxynononic acid) are still cleaved by DAS181, but require higher concentrations to achieve complete removal. Residues with internal sulfate and fucosyl groups are efficiently cleaved. This surprisingly broad substrate specificity means that DAS181 can remove a variety of sialic acid types from cells. DAS 181 can desialylate cell surfaces ofNeu5Ac and KDN terminal sialic acid structures. In addition, DAS181 can remove sialic acids from a variety of oligosaccharides irrespective of the underlying sugar structure. This broad specificity means that DAS 181 has the ability to remove sialic acid residues from the surface of cancer cells much more efficiently than other sialidases. This is a discovery that was not expected, because ability to cleave sialic acids from underlying sugar structures cannot be predicted and there is no basis to believe that all Neu5Ac and KDN terminal sialic acid structures would be cleaved by one sialidase.

TABLE 6

TABLE 7

TABLE 8

TABLE 9

TABLE 10

TABLE 11

TABLE 12

TABLE 13

While certain embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

What is claimed:

1. A method of treating a subject suffering from lung cancer, the method comprising administering a therapeutically effective amount of a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or 2 to the lungs of the subject.

2. The method of claim 1, wherein the method of administration includes inhalation of a dry powder formulation comprising the polypeptide with a metered dose inhaler.

3. The method of claim 1, wherein the method of administration includes inhalation of a liquid nebulized formulation comprising the polypeptide.

4. The method of claim 1, futher comprising administering a chemotherapeutic agent.

5. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1 or 2.

6. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that has no more than 5 single amino acid substitutions or deletions compared to SEQ ID NO: 1 or 2.

7. The method of claim 1, wherein the polypeptide comprises an amino acid sequence that has no more than 5 single amino acid substitutions compared to SEQ ID NO: 1 or 2.

8. The method of claim 7, wherein the amino acid substitutions are conservative substitutions.

9. The method of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 2.

10. The method of claim 1, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or 2.

32

11. A method for increasing the effectiveness of an antibody used the treat lung cancer, the method comprising administering a therapeutically effective amount of a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 1 or 2 to the lungs of the subject and administering an immunotherapy used to treat lung cancer.

12. The method of claim 11, wherein the method of administration includes inhalation of a dry powder formulation comprising the polypeptide with a metered dose inhaler.

13. The method of claim 11, wherein the method of administration includes inhalation of a liquid nebulized formulation comprising the polypeptide.

14. The method of claim 11, futher comprising administering a chemotherapeutic agent.

15. The method of claim 11, wherein the polypeptide comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1 or 2.

16. The method of claim 11, wherein the polypeptide comprises an amino acid sequence that has no more than 5 single amino acid substitutions or deletions compared to SEQ ID NO: 1 or 2.

17. The method of claim 11, wherein the polypeptide comprises an amino acid sequence that has no more than 5 single amino acid substitutions compared to SEQ ID NO: 1 or 2.

18. The method of claim 17, wherein the amino acid substitutions are conservative substitutions.

19. The method of claim 11, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 2.

20. The method of claim 11, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or 2.

33

21. The method of claim 11 , wherein the immunotherapy is an antibody.

22 The method of claim 21, wherein the antibody is evacizumab or ramucirumab.

34