Compositions and methods for treating cancer

Abstract

The present disclosure is based, at least in part, on the unexpected discovery that novel compositions of leukotoxin (LtxA) polypeptides isolated from Aggregatibacter actinomycetemcomitans can maintain stability and biological activity for extended periods of time, even after lyophilization, storage, reconstitution and / or further storage, or under accelerated conditions, and that a specific range of dosages and administration schedules of LtxA polypeptides are highly effective and have low toxicity in treating cancer in patients in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 286,236, filed December 6, 2021, which is incorporated by reference in its entirety.

[0002] The present invention relates to pharmaceutical and biological compositions containing leukotoxins and methods of using them to treat cancer. [Background technology]

[0003] More than 60,500 people die each year from hematological malignancies (leukemia, lymphoma, myeloma), with more than 110,000 new cases diagnosed each year in the United States alone. Lymphomas are generally classified as Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL) and may be T-cell (NK natural killer cell) lymphoma or B-cell lymphoma, such as, but not limited to, mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, and others. Currently, treatment of these hematological cancers involves the use of synthetic compounds that target the cell division process not only in cancer cells, but in almost all cells of the body. As a result, devastating side effects are common. Furthermore, a significant proportion of patients eventually develop resistance to many medications, rendering treatment largely ineffective and leaving many patients with recurrent, resistant, and / or refractory disease. For example, MCL is a fatal and incurable disease, with an average overall survival of approximately 3-4 years, even with new treatment methods. In contrast to FL, indolent NHL is the most common, and the disease is considered incurable with no consensus treatment protocol. Approximately 30-40% of DLBCL patients still die from the cancer. Many of these deaths are due to resistance of cancer cells to treatment upon disease recurrence. Thus, there is a strong need for new drugs that target B-cell lymphoma. However, currently used drugs are toxic to cells and are not highly specific. New therapeutic agents for treating hematological malignancies and cancers generally include drugs that are primarily specific for cancer cell types. Examples of targeted therapeutic agents include rituximab, a monoclonal antibody against B lymphocytes, and Mylotarg, an antitumor antibiotic-conjugated antibody against myelomonocytic cells.

[0004] Recently, the US FDA issued an initiative draft guidance to facilitate the combined medical research and clinical development of experimental therapeutics and to improve the clinical outcomes, efficacy, and safety profiles of anti-cancer drug regimens. Conventional standard chemotherapy agents are not specific to cancer cells, have high cytotoxicity, and have severe side effects.

[0005] Therefore, there is a need to develop new anti-cancer drugs and therapies that target cancer cells, are less toxic, and are effective in treating cancer. Summary of the Invention

[0006] The present disclosure has been made to solve the above problems from multiple angles. In one aspect, the present disclosure provides a liquid composition for cancer treatment. The liquid composition contains about 0.1 mg / ml to about 0.5 mg / ml of leukotoxin (LtxA) polypeptide (or protein) isolated from Aggregatibacter (Actinobacillus) actinomycetemcomitans, about 5 mM to about 50 mM Tris, about 100 mM to about 300 mM NaCl, and about 0.05 mM to about 0.5 mM CaCl2, and is formulated to have a pH of about 7.0 to about 8.0.

[0007] In some embodiments, the liquid composition comprises about 0.3 mg / ml of LtxA polypeptide. In some embodiments, the liquid composition comprises about 20 mM Tris, about 250 mM NaCl, and about 0.2 mM CaCl2. In some embodiments, the liquid composition is formulated to have a pH of about 7.5.

[0008] In some embodiments, the liquid composition is formulated to be stable at 4° C. for at least 24 hours.

[0009] In some embodiments, the LtxA polypeptide is isolated from Aggregatibacter actinomycetemcomitans strain NJ4500. In some embodiments, the LtxA polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:1, or comprises the amino acid sequence of SEQ ID NO:1.

[0010] In another aspect, the present disclosure provides a lyophilized composition prepared from a liquid composition according to any one of the preceding claims, the lyophilized composition comprising from about 0.2 mg to about 2 mg of LtxA polypeptide, from about 2 mg to about 8 mg of Tris, from about 10 mg to about 50 mg of NaCl, and from about 0.01 mg to about 0.5 mg of CaCl2, formulated to have a pH of about 7.0 to 8.0 upon reconstitution.

[0011] In some embodiments, the lyophilized composition comprises about 0.6 mg of LtxA polypeptide, about 4.85 mg of Tris, about 29.2 mg of NaCl, and about 0.04 mg of CaCl 2 .

[0012] In some embodiments, the lyophilized composition is formulated to have a pH of about 7.5 after reconstitution. In some embodiments, the lyophilized composition is reconstituted in sterile water or buffered saline. In some embodiments, the lyophilized composition is reconstituted as a liquid composition comprising about 0.3 mg / ml of LtxA polypeptide.

[0013] In some embodiments, the lyophilized composition is formulated to remain stable after storage at −20±5° C. for up to 24 months. In some embodiments, the lyophilized composition is formulated to remain stable after storage at temperatures below −20° C., reconstitution, and storage at temperatures below −20° C. for up to 7 days. In some embodiments, the lyophilized composition is formulated to remain stable after storage at temperatures below −20° C., reconstitution, and storage at about 4° C. for up to 24 hours.

[0014] In another aspect, the present disclosure provides a kit comprising a liquid composition or a lyophilized composition described herein.

[0015] In yet another aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a liquid composition described herein, the therapeutically effective amount of the liquid composition being from about 1 μg / kg to about 1200 μg / kg based on the subject's body weight, or the ratio of the mass (e.g., μg) of the liquid composition to the body surface area (e.g., meters squared = M 2 ) (e.g., μg / M 2 ) is used for the calculation.

[0016] In some embodiments, the therapeutically effective amount of the liquid composition is about 1.4 μg / kg based on the body weight of the subject. In some embodiments, the therapeutically effective amount of the liquid composition is about 1020 μg / kg based on the body weight of the subject, or the ratio of the mass of the liquid composition (e.g., μg) to the body surface area (e.g., square meters = M 2 ) (e.g., μg / M 2 ) is used for the calculation.

[0017] In some embodiments, the liquid composition administered to a subject is formulated as a dosage form selected from a modified release dosage form, a sustained release (depot) dosage form, a controlled release dosage form, a sustained release dosage form, a delayed release dosage form, an extended release dosage form, and / or an extended release dosage form.

[0018] In some embodiments, the liquid composition is administered parenterally by intravenous injection over a period of 1 to 10 hours. In some embodiments, the liquid composition is administered intravenously over a period of 3 to 4 hours. In some embodiments, the liquid composition is administered intravenously over a period of 1 to 2 hours.

[0019] In some embodiments, the liquid composition is administered parenterally to a subject in a modified release, sustained release (depot), controlled release, timed release, delayed release, prolonged release, and / or extended release dosage form.

[0020] In some embodiments, the cancer may be any LFA-1-expressing tumor. In some embodiments, the cancer is selected from adrenal tumor, bile duct cancer, bladder cancer, brain cancer, breast cancer, epithelial carcinoma, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer, hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, Li-Fraumeni tumor, liver cancer, lung cancer, leukemia, lymphoma, melanoma, meningioma, multiple neuroendocrine tumors type I and II, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell carcinoma, parathyroid cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, kidney cancer, respiratory tract cancer, non-epithelial carcinoma, skin cancer, gastric cancer, testicular cancer, thyroid cancer, tracheal cancer, genitourinary tract cancer, and uterine cancer.

[0021] In some embodiments, the cancer is leukemia and any subtype thereof, hi some embodiments, the cancer is lymphoma or any subtype thereof.

[0022] In some embodiments, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including anaplastic large cell lymphoma, angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma (small noncleaved cell lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, enteropathy-type T-cell lymphoma, follicular lymphoma, hepatosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-cell lymphoma, childhood lymphoma, peripheral T-cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, therapy-related T-cell lymphoma, and Waldenstrom's macroglobulinemia.

[0023] In some embodiments, the method further comprises administering to the subject a second agent or therapy. In some embodiments, the second agent comprises an anti-tumor or anti-cancer agent. In some embodiments, the second agent or therapy is administered sequentially before or after the composition. In some embodiments, the second agent or therapy is administered simultaneously with the composition.

[0024] The above summary is not intended to define all aspects of the disclosure, and additional aspects are described in other sections, such as the detailed description below. It should be understood that the entire document is intended to be related as an integrated disclosure, and that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of this document. Other features and advantages of the present invention will become apparent from the detailed description below. However, the detailed description and specific examples, while showing specific embodiments of the disclosure, are merely illustrative, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0025] [Figure 1] Qualitative Western blot results for GMP lot 599-0818-003 are shown. [Diagram 2] Qualitative Western blot results for GMP lot 599-0718-002 are shown. [Diagram 3] Western blot results of low dose concentration samples are shown. [Figure 4] The results of evaluating the biological activity of low-dose concentration samples are shown. [Diagram 5] Western blot results of high dose concentration samples are shown. [Figure 6] The results of evaluating the biological activity of high-dose concentration samples are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present disclosure is based, at least in part, on the unexpected discovery that novel compositions of leukotoxin (LtxA) polypeptides isolated from Aggregatibacter actinomycetemcomitans can maintain stability and biological activity for extended periods of time, even after lyophilization, storage, reconstitution and / or further storage, or under accelerated conditions, and that a specific range of dosages and administration schedules of LtxA polypeptides are highly effective and have low toxicity in treating cancer in patients in need thereof.

[0027] Composition of LtxA Polypeptides In one aspect, the present disclosure provides a liquid composition for the treatment of various cancers, the liquid composition comprising about 0.1 mg / ml to about 0.5 mg / ml of a leukotoxin (LtxA) polypeptide (or protein) isolated from Aggregatibacter actinomycetemcomitans or a variant / fragment thereof, about 5 mM to about 50 mM Tris, about 100 mM to about 300 mM NaCl, and about 0.05 mM to about 0.5 mM CaCl2, and is formulated to have a pH of about 7.0 to about 8.0.

[0028] In some embodiments, the liquid composition comprises about 0.3 mg / ml of LtxA polypeptide. In some embodiments, the liquid composition comprises about 20 mM Tris, about 250 mM NaCl, and about 0.2 mM CaCl2. In some embodiments, the liquid composition is formulated to have a pH of about 7.5. In some embodiments, the liquid composition is formulated to be stable at 4° C. for at least 24 hours.

[0029] In some embodiments, the LtxA polypeptide is isolated from Aggregatibacter actinomycetemcomitans strain NJ4500. In some embodiments, the LtxA polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:1, or comprises the amino acid sequence of SEQ ID NO:1.

[0030] In another aspect, the present disclosure provides a lyophilized composition prepared from a liquid composition according to any one of the preceding claims, the lyophilized composition comprising from about 0.2 mg to about 2 mg of LtxA polypeptide, from about 2 mg to about 8 mg of Tris, from about 10 mg to about 50 mg of NaCl, and from about 0.01 mg to about 0.5 mg of CaCl2, formulated to have a pH of about 7.0 to 8.0 upon reconstitution.

[0031] In some embodiments, the lyophilized composition comprises about 0.6 mg of LtxA polypeptide, about 4.85 mg of Tris, about 29.2 mg of NaCl, and about 0.04 mg of CaCl2. In some embodiments, the lyophilized composition is formulated to have a pH of about 7.5 after reconstitution.

[0032] In some embodiments, the lyophilized composition is reconstituted in sterile water or buffered saline, hi some embodiments, the lyophilized composition is reconstituted as a liquid composition comprising about 0.3 mg / ml of LtxA polypeptide.

[0033] In some embodiments, the lyophilized composition is formulated to remain stable after storage at −20±5° C. for up to 24 months. In some embodiments, the lyophilized composition is formulated to remain stable after storage at temperatures below −20° C., reconstitution, and storage at temperatures below −20° C. for up to 7 days. In some embodiments, the lyophilized composition is formulated to remain stable after storage at temperatures below −20° C., reconstitution, and storage at about 4° C. for up to 24 hours.

[0034] Table 1 Representative sequences [Table 1] TIFF2024546686000003.tif255166TIFF2024546686000004.tif19167Aggregatibacter actinomycetemcomitans is a Gram-negative pathogen present in the human oral cavity. It is the causative agent of limited aggressive periodontitis (LAP), a rapidly progressive destructive disease of the gingiva and periodontal ligament. Among its many virulence factors, Aggregatibacter actinomycetemcomitans produces RTX (repeats in toXin) leukotoxin, an approximately 115 kDa protein that specifically kills human and Old World primate leukocytes. Leukotoxin (LtxA) is a member of the RTX family that includes Escherichia coli α-hemolysin (HlyA) and Bordetella pertussis adenylate cyclase (CyaA). Leukotoxin plays an important role in the pathogenesis of Aggregatibacter actinomycetemcomitans by helping the bacterium destroy crevicular polymorphonuclear leukocytes (PMNs) and monocytes, thereby suppressing local immune defenses.

[0035] LtxA binds leukocyte function antigen-1 (LFA-1) to white blood cells (WBCs) and induces cell death by apoptosis or necrosis. We found that LtxA selectively targets WBCs with high levels of activated LFA-1, a hallmark of hematological malignancies such as leukemia and lymphoma. In many cases, LtxA represents a natural immunotoxin because it is toxic and highly specific within the same molecule. The advantages of natural LtxA over engineered molecules are its high stability, high specificity, low toxicity, and minimal side effects.

[0036] LtxA is able to identify, target, and kill white blood cells arising from various types of hematological malignancies, such as lymphoma, making it an ideal agent for detecting and treating these diseases. For example, blood from a patient can be analyzed using LtxA-FITC staining. The finding of a significant percentage of activated WBCs indicates that the patient should receive LtxA treatment. The effectiveness of leukotoxin treatment can be monitored by using LtxA-FITC reagents to initially diagnose the disease. If the patient responds positively to treatment, a decrease in the number of WBCs with upregulated activated surface LFA-1 should be seen. Furthermore, LtxA is expected to have few side effects due to its high specificity and targeting. LtxA can kill many leukemia and lymphoma cell lines, and preclinical studies have shown that LtxA is an effective targeted therapy for the treatment of hematological malignancies. In non-human primates, it was found that a single LtxA treatment dramatically reduced white blood cell counts after 12 hours (rapid onset of activity). Importantly, it was found to be non-toxic when administered to mice at high doses.

[0037] Many LtxA preparations can be used, but preferably high purity LtxA is used. Examples include LtxA polypeptide (SEQ ID NO: 1) purified from Aggregatibacter actinomycetemcomitans and other variants with substantially the same biological activity as that having the sequence of SEQ ID NO: 1. It was discovered that Aggregatibacter actinomycetemcomitans secretes active LtxA into the culture supernatant (Kachlany, SC et al., 2000, Infect Immun 68: 6094-100), and an efficient method for its purification was described in Kachlany, SC et al., 2002, Protein Expr Purif 25: 465-71. Thus, this method can be used to prepare isolated or purified LtxA polypeptide.

[0038] In one embodiment, the toxin purification procedure includes the steps of (a) inoculating a single colony of Aggregatibacter actinomycetemcomitans into fresh culture medium and growing the culture; (b) adding the grown culture to fresh culture medium and adding glass beads and culturing; (c) centrifuging the cultivated culture to generate a precipitate and a supernatant; (d) filtering the supernatant through a membrane to obtain a filtered supernatant; (e) mixing (NH4)2SO4 and the filtered supernatant together to obtain a mixture; (f) centrifuging the mixture to generate a mixed precipitate; (g) resuspending the mixed precipitate in a buffer to generate a protein resuspension; (h) passing the protein resuspension through a column; and (i) collecting the proteins eluted from the column. See also PCT / US2006 / 45258 (International Publication No. 2007 / 062150) and U.S. Patent Application Publication No. 20090075883 (U.S. Application No. 12 / 154,843), both of which are incorporated by reference herein in their entireties.

[0039] In the present disclosure, various bacterial LtxA or variants thereof can be used. For example, forms of LtxA include the JP2 form (isolated from the JP2 strain of Aggregatibacter actinomycetemcomitans) and the NJ4500 form (isolated from the NJ4500 strain of Aggregatibacter actinomycetemcomitans). The NJ4500 strain of Aggregatibacter actinomycetemcomitans was deposited at the American Type Culture Collection (ATCC), Boulevard College, Manassas, Va. 20110-2209, USA, under the accession number PTA-11721, on March 2, 2011.

[0040] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also include amino acid polymers modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, PEGylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or unnatural amino acids or synthetic amino acids, amino acid analogs and peptidomimetics, including glycine and D or L optical isomers.

[0041] An "isolated polypeptide" refers to a polypeptide that is separated from other proteins, lipids, and nucleic acids with which it is naturally associated. The polypeptide constitutes at least 10% by dry weight of a purified preparation (i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 99%). Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. An isolated polypeptide of the invention can be purified from a natural source, produced by recombinant DNA technology or chemical methods. A functional equivalent of LtxA refers to a polypeptide derivative of an LtxA polypeptide, e.g., a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or combinations thereof. It substantially retains the activity of the LtxA polypeptide, i.e., the ability to target and kill leukocytes expressing an activated form of LFA-1 on their surface, while having little or no toxic effect on other cells or organs in the body. The isolated polypeptide can comprise SEQ ID NO:1 or a functional fragment of SEQ ID NO:1. Generally, functional equivalents have at least 75% identity with SEQ ID NO:1 (e.g., any value between 75% and 100%, e.g., 70%, 80%, 85%, 90%, 95% and 99%).

[0042] Naturally occurring LtxA, genetically engineered LtxA, and chemically synthesized LtxA can all be used to practice the invention disclosed herein. LtxA obtained by recombinant DNA technology may have the same amino acid sequence as naturally occurring LtxA (SEQ ID NO: 1) or its functional equivalent. The term "LtxA" also includes chemically modified LtxA. Examples of chemically modified LtxA include LtxA whose sugar chains have undergone structural changes, additions or deletions, and LtxA to which compounds such as polyethylene glycol have been attached. Once purified and detected by standard methods well known in the art, LtxA can be added to pharmaceutical and / or biological compositions.

[0043] As described herein, the LtxA polypeptide can be obtained as a naturally occurring polypeptide or a recombinant polypeptide. To prepare a recombinant polypeptide, the nucleic acid encoding it (e.g., SEQ ID NO:2) can be combined with a fusion partner, such as glutathione-s-transferase (GST), a 6xHis epitope tag, or other nucleic acid encoding an M13 Gene3 protein. The resulting fusion nucleic acid expresses a fusion protein in a suitable host cell that can be isolated by methods well known in the art. The isolated fusion protein can be further processed, for example, by enzymatic digestion, to remove the fusion partner and obtain the recombinant polypeptide of the present disclosure.

[0044] As mentioned above, mutants of LtxA proteins or polypeptides are also included within the scope of the present disclosure. As used herein, the term "mutant" refers to a first molecule in relation to a second molecule (also referred to as a "parent" molecule). A mutant molecule may be derived from, isolated from, based on, or homologous to a parent molecule. As used herein, a "functional mutant" of a protein refers to a mutant of a protein that maintains at least some activity of the protein. Functional mutants may include mutants, including polymorphisms, which may be insertion, deletion, or substitution mutants. Functional mutants also include fusion products of such proteins with another, usually unrelated, nucleic acid, protein, polypeptide, peptide. Functional mutants may be of natural origin or man-made.

[0045] As used herein, a peptide or polypeptide "fragment" refers to a peptide, polypeptide, oligopeptide, or protein that is less than full length. For example, a peptide, oligopeptide, or polypeptide fragment may be at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40 amino acids in length, or may be a single unit length. For example, a fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more amino acids in length. There is no upper limit to the size of a peptide fragment. However, in some embodiments, a peptide fragment may be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less than about 250 amino acids in length.

[0046] The amino acid composition of the LtxA polypeptides described herein may be altered without destroying the ability of the polypeptides to target and kill WBCs. For example, one or more conservative amino acid substitutions may be included. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, predicted non-essential amino acid residues in SEQ ID NO: 1 are preferably replaced with other amino acid residues from the same side chain family.Also, mutations can be introduced randomly in all or part of SEQ ID NO: 1, such as by saturation mutagenesis, and the resulting mutants can be screened for their ability to identify mutants that ameliorate skin conditions and maintain activity as described in the Examples below.

[0047] As used herein, the percentage of homology between two amino acid sequences corresponds to the percentage of identity between the two sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps to be introduced for optimal alignment of the two sequences and the length of each gap. The alignment of two sequences and the determination of the percentage of identity can be achieved using a mathematical algorithm, as described in the non-limiting examples below.

[0048] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4,11-17 (1988)) as incorporated into the ALIGN program, using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) as incorporated into the GAP program of the GCP software package (available at www.gcg.com), using either a Blossum62 matrix or a PAM250 matrix, using gap weights of 16, 14, 12, 10, 8, 6, or 4, or length weights of 1, 2, 3, 4, 5, or 6.

[0049] The term "homolog" or "homology" when used in reference to polypeptides refers to high sequence identity between two polypeptides, or high similarity between three-dimensional structures, or high similarity between active sites and mechanisms of action. In some embodiments, a homolog has 60% or more sequence identity with a reference sequence, more preferably 75% or more sequence identity, and even more preferably 90% or more sequence identity. When applied to polypeptides, the term "substantial identity" means that two peptide sequences share at least 75% sequence identity when optimally aligned with the programs GAP or BESTFIT using default gap weights.

[0050] Variants, mutants and homologs having significant identity to the disclosed LtxA polypeptides are also included within the scope of the present disclosure. For example, such variants and homologs have at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the sequences of the LtxA polypeptides described herein.

[0051] In some embodiments, the detectable tag can be conjugated or attached to the N-terminus and / or C-terminus of the LtxA polypeptide or its variant. The detectable tag and the affinity tag may be separated by one or more amino acids. In some embodiments, the detectable tag can be conjugated or attached to the variant via a cleavable moiety. In the context of the present invention, the term "cleavable moiety" refers to a peptide sequence that is susceptible to cleavage by chemical agents or enzymatic means such as proteases. Proteases may be sequence specific (e.g. thrombin) or may have limited sequence specificity (e.g. trypsin). Cleavable moieties I and II may also be included in the amino acid sequence of the detection tag or polypeptide, in particular the amino acid sequence of the detection tag or polypeptide where the last amino acid is K or R.

[0052] As used herein, the term "joining" or "conjugation" or "bonding" refers to the joining of two or more entities to form one entity. Conjugates include both peptide-small molecule conjugates and peptide-protein / peptide conjugates.

[0053] The term "fusion polypeptide" or "fusion protein" or "fusion oligopeptide" refers to a protein produced by joining two or more polypeptide sequences. Fusion polypeptides encompassed by the present invention include the translation product of a chimeric gene construct in which a nucleic acid sequence encoding a first polypeptide is joined to a nucleic acid sequence encoding a second polypeptide to form a single open reading frame. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins linked by a peptide bond or via several peptides. A fusion protein may also include a peptide linker between the two domains.

[0054] The term "linker" refers to any means, entity or moiety used to connect two or more entities. The linker may be a covalent or non-covalent linker. Examples of covalent linkers include a covalent bond or a linker moiety covalently attached to one or more of the proteins or domains to be connected. The linker may also be a non-covalent linker, for example an organometallic bond via a metal center such as a platinum atom. For covalent attachment, various functional groups may be used, such as amide groups including carbonic acid derivatives, ethers, esters including organic esters and inorganic esters, amino, urethane, urea, etc. To provide the bond, the domains may be modified by oxidation, hydroxylation, substitution, reduction, etc. to provide sites for coupling. Methods of conjugation are well known to those skilled in the art and are included in the use of the present invention. Linker moieties include, but are not limited to, chemical linker moieties or peptide linker moieties (linker sequences).

[0055] In some embodiments, the linker may be a peptide linker or a non-peptide linker. Examples of peptide linkers include, but are not limited to, [S(G)n]m or [S(G)n]mS (n is an integer from 1 to 20 and m is an integer from 1 to 10).

[0056] As used herein, the term "stability" refers to the stability and / or biological activity of a protein (e.g., the ability of a polypeptide to target and kill WBCs). In some embodiments, as used herein, the term "stability" refers to the ability of a molecule to remain folded under certain conditions (e.g., storage conditions) such that the molecule maintains at least one of its normal functional activities, such as binding to a target molecule, targeting or killing WBCs. In some embodiments, a polypeptide may become less stable if denaturation, aggregation, oligomerization occurs upon storage, lyophilization, or freezing / refrozen. Measurements of protein stability and protein instability can be viewed as the same or different aspects of protein integrity. Proteins are sensitive or "unstable" to denaturation caused by heat, ultraviolet or ionizing radiation, changes in the surrounding osmolarity and pH when in solution, filtration through small pore sizes, ultraviolet radiation, ionizing radiation such as by gamma irradiation, chemical or thermal dehydration, or any other action or force that may cause the destruction of the protein structure. Molecular stability can be determined using standard methods. For example, the stability of a molecule can be determined by measuring the thermal melting ("TM") temperature, which is the temperature in degrees Celsius (°C) at which half of the molecule is unfolded, using standard methods. Typically, the higher the TM, the more stable the molecule. In addition to heat, the chemical environment also alters the ability of a protein to maintain a specific three-dimensional structure. In some embodiments, protein stability is characterized by Western blot, mass spectrometry (MS), high performance liquid chromatography (HPLC), immunoassays (e.g., ELISA), or ATP-based cell viability assays.

[0057] Any suitable reagent may be used to adjust the pH of the composition. Typical reagents used to adjust the pH may be one or more of NaOH, NH4OH, hydrochloric acid, acetic acid, sulfuric acid, EDTA, Tris buffer, and the like. In one embodiment, the pH of the sample is adjusted using a base such as sodium hydroxide or an acid such as hydrochloric acid. The pharmaceutical compositions of the present invention may be formulated with suitable carriers, excipients, and other reagents that provide suitable migration, delivery, tolerance, and the like. Many suitable formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. formulary, known to all pharmacists. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN), DNA complexes, anhydrous absorption pastes, oil-in-water emulsions, water-in-oil emulsions, emulsions of carbowax (polyethylene glycol of similar molecular weight), semi-solid gels, semi-solid mixtures containing carbowax. See also Powell et al., PDA, (1998), J Pharm Sci Technol, 52:238-311.

[0058] As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one component useful in the present disclosure with other components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. A pharmaceutical composition facilitates administration of one or more components of the present invention to a living organism.

[0059] A pharmaceutical composition generally comprises substantially purified LtxA and a pharma- ceutically acceptable carrier in a form suitable for administration to a subject. The pharma- ceutically acceptable carrier is determined in part by the particular composition being administered, and in part by the particular method by which the composition is administered. Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0060] The terms "pharmaceutical acceptable" and "physiologically acceptable" are used interchangeably when referring to compositions, carriers, diluents, and reagents, and indicate that the substance can be administered to a subject without producing undesirable physiological effects that would prohibit administration of the composition. For example, a "pharmaceutical acceptable excipient" includes excipients that are generally safe, non-toxic, and desirable and useful in the preparation of pharmaceutical compositions, including excipients that are acceptable for veterinary as well as human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutical active substances is well known in the art. Any conventional media or compounds are contemplated for use in the composition, provided they are incompatible with LtxA. Additional active compounds may also be incorporated into the composition.

[0061] A pharmaceutical composition is formulated to suit its intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents, antibacterial compounds such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating compounds such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetic acid, citric acid, or phosphate, and compounds for adjusting isotonicity such as sodium chloride or dextrose. The pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0062] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions and dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injectability with a syringe exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, to maintain the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by using various antibacterial and antifungal compounds, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic compounds in the composition, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of the injectable compositions can be achieved by including in the composition compounds which delay absorption, such as aluminum monostearate and gelatin.

[0063] Sterile injectable solutions can be prepared by incorporating the required amount of LtxA in a suitable solvent together with one or a combination of the above listed ingredients as needed. Generally, dispersions are prepared by incorporating LtxA in a sterile vehicle containing a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which produces a powder containing the active ingredient and any additional desired ingredients from the previously sterile-filtered solution. LtxA can be administered in the form of a depot injection or implant preparation, and can be formulated to allow sustained or pulsatile release of the active ingredient.

[0064] The present disclosure also includes within its scope a kit comprising the liquid or lyophilized composition described herein, for example, for treating or inhibiting tumor growth in a patient. In some embodiments, the kit also comprises a container containing the composition, and optionally, informational material. The informational material may be explanatory, instructional, marketing, or other material related to the methods described herein and / or the use of the agent for therapeutic utility. In one embodiment, the kit also comprises another therapeutic agent, as described herein. For example, the kit comprises a first container containing the composition and a second container containing the other therapeutic agent.

[0065] The informational material of the kit is not limited in its form. In some embodiments, the informational material may include information regarding the preparation of the composition, concentration, expiration date, batch or place of manufacture information, and the like. In one embodiment, the informational material relates to methods of administering the composition, e.g., administering the composition in an appropriate dose, dosage form, or administration form (e.g., a dose, dosage form, or administration form described herein) to treat a subject in need thereof. In one embodiment, the instructional material provides a dosing regimen, dosage amount, and / or route of administration of the composition or other therapeutic agent. The information can be provided in a variety of formats, including printed text, computer readable materials, video or audio recordings, or information that includes a link or address to the substantive material.

[0066] The kit may include one or more containers for the composition. In some embodiments, the kit includes separate containers, dividers, or compartments for the composition and the informational material. For example, the composition can be in a bottle or vial and the informational material can be in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are in a single, undivided container. For example, the composition is in a bottle or vial to which the informational material is attached in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each container containing one or more unit dosage forms of the agent (e.g., a dosage form described herein).

[0067] The kit may optionally include a device suitable for administration of the composition, or other suitable delivery device. The device may be pre-loaded with one or both agents, or may be empty if suitable for loading. Such a kit may optionally include a syringe capable of injecting an antibody contained in the kit into an animal, such as a human.

[0068] Cancer Treatment Methods In yet another aspect, the disclosure includes methods of treating, slowing or inhibiting tumor growth. In some embodiments, the disclosure includes methods of promoting tumor growth inhibition, cancer regression, and / or inducing cancer apoptosis. In some embodiments, the disclosure includes methods of reducing tumor cell burden or reducing tumor burden. In some embodiments, the disclosure includes methods of preventing tumor recurrence, resistance, relapse, and / or refractory disease.

[0069] In one aspect, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a liquid composition described herein, the therapeutically effective amount of the liquid composition being about 1 μg / kg to about 1200 μg / kg (e.g., 1.4, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200 μg / kg) based on the subject's body weight.

[0070] In some embodiments, the therapeutically effective amount of the liquid composition is about 1.4 μg / kg based on the subject's body weight. In some embodiments, the therapeutically effective amount of the liquid composition is about 1020 μg / kg based on the subject's body weight.

[0071] The dosage of the pharmaceutical composition of the present invention is determined according to the age, weight, skin surface area, general health, sex, diet, administration time, administration method, clearance rate, and / or the level of disease for which the patient is currently being treated, or is determined taking into account other factors. The daily dose of the compound of the present invention varies depending on the condition and weight of the patient, the type of compound, the route of administration, etc., and is administered parenterally, for example, 0.001 to 100 mg / patient / day by subcutaneous administration, intravenous administration, intramuscular administration, transdermal administration, ocular administration, pulmonary bronchial administration, or nasal administration. Given the variety of compounds available and the differences in efficacy of various routes of administration, it is expected that the required dosage will vary. As is well understood in the art, variations in these dosage levels can be adjusted using standard empirical routines for optimization. Delivery efficiency can be improved by encapsulating the compound in an appropriate delivery vehicle (e.g., polymeric microparticles or implantable devices).

[0072] Oral dosage forms may include capsules, tablets, emulsions, aqueous suspensions, dispersions and solutions. For tablets, commonly used carriers include, but are not limited to, lactose and cornstarch. Lubricants such as magnesium stearate are also commonly added, but are not limited to this. When administered orally in capsule form, useful diluents include, but are not limited to, lactose and dry cornstarch. When administered orally as an aqueous suspension or emulsion, the active ingredient can be suspended or dissolved in an oily phase in combination with an emulsifier or suspending agent. If necessary, certain sweeteners, flavorings, colorants, and the like can be added. In some embodiments, the oral dose range administered orally in a single or divided dose is about 1.0 to about 100 mg / kg body weight, including about 1.0 to about 50 mg / kg body weight, about 1.0 to about 25 mg / kg body weight, and about 1.0 to about 10 mg / kg body weight (assuming an average body weight of approximately 70 kg, and adjusting values ​​accordingly for individuals who are larger or smaller than the average body weight). For oral administration, the composition is provided in the form of a tablet containing, for example, about 50 to about 1000 mg of the active ingredient, in particular about 75 mg, about 100 mg, about 200 mg, about 400 mg, about 500 mg, about 600 mg, about 750 mg, or about 1000 mg of the active ingredient, for symptomatic adjustment of the dosage to the subject being treated.

[0073] In some embodiments, the pharmaceutical compositions described herein are administered to subjects in a variety of ways, which may include continuous or intermittent administration, depending on the nature of the cancer. The pharmaceutical compositions may be administered by a route independently selected from the group consisting of oral administration, intravenous administration, intraarterial administration, intramuscular administration, intracolonic administration, intracranial administration, intrathecal administration, intraventricular administration, intraurethral administration, intravaginal administration, subcutaneous administration, intraocular administration, intranasal administration, and any combination thereof. Accordingly, the pharmacologic effective composition may include a pharmacologic acceptable additive, carrier, or excipient. Such pharmaceutical compositions may include the active ingredient formulated together with one or more non-toxic pharmacologic acceptable carriers specifically formulated for oral administration in solid or liquid, for injection, or for rectal administration according to standard methods well known in the art.

[0074] The term "parenteral" administration refers to methods of administration including intravenous, intramuscular, intraperitoneal, intracapsular, intratarsal, subcutaneous, intraarticular injection and intravenous drip. Injectable mixtures are well known in the art and include pharma- ceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), vegetable oils (e.g., olive oil), injectable organic esters (e.g., ethyl oleate), and suitable mixtures thereof.

[0075] In some embodiments, the liquid composition is administered to the subject intravenously, subcutaneously, or intraperitoneally.

[0076] In some embodiments, the liquid composition is administered by intravenous injection. In some embodiments, the liquid composition is administered by intravenous injection over a period of 1-10 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours). In some embodiments, the liquid composition is administered by intravenous injection over a period of 3-4 hours (+ / - 15 minutes). In some embodiments, the liquid composition is administered by intravenous injection over a period of 1-2 hours (+ / - 15 minutes).

[0077] In some embodiments, the liquid composition is administered to a subject in a sustained, controlled, or delayed release dosage form.

[0078] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (see, e.g., Wu et al., 1987, J.Biol.Chem.262:4429-4432). Methods of administration include, but are not limited to, intravesical, intradermal, intramuscular, intratumoral, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any convenient route, for example, by intravenous drip or bolus injection, absorption through epithelial or mucocutaneous (e.g., oral, rectal, intestinal, etc.), and may be administered together with other biologically active agents.

[0079] As used herein, the term "subject" may be used interchangeably with the term "patient." The phrase "subject in need thereof" refers to a human or non-human mammal that exhibits one or more symptoms or signs of cancer and / or has been diagnosed with cancer. In some embodiments, the human subject has been diagnosed with a primary or metastatic tumor and / or exhibits one or more symptoms or signs, including, but not limited to, enlarged lymph nodes, abdominal distension, chest pain / tightness, unexplained weight loss, fever, night sweats, persistent fatigue, loss of appetite, enlarged spleen, itching, and the like. The phrase includes patients who have undergone one or more chemotherapy treatments that have toxic side effects. In some embodiments, the phrase "subject in need thereof" includes cancer patients who have been treated but have subsequently relapsed or metastasized. For example, patients who have undergone treatment with one or more anti-cancer agents that led to tumor regression but have subsequently relapsed with cancer that is resistant to the one or more anti-cancer agents (e.g., chemotherapy-resistant cancer) are treated with the methods of the present disclosure.

[0080] As used herein, the terms "treatment", "treating" and the like mean alleviating or reducing the severity of at least one symptom or sign, eliminating the cause of a symptom, either temporarily or permanently, slowing or inhibiting tumor growth, reducing tumor cell mass or tumor burden, promoting tumor regression, causing tumor shrinkage, necrosis and / or disappearance, preventing tumor recurrence, preventing or inhibiting metastasis, inhibiting metastatic tumor growth, eliminating the need for radiation or surgery, and / or prolonging the survival of a subject.

[0081] The term "effective amount", "effective dose" or "effective dosage" is defined as an amount sufficient to obtain or at least partially obtain a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a pharmaceutical or therapeutic agent is any amount of the pharmaceutical agent that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of functional impairment or disability due to disease affliction. A "prophylactically effective amount" or "prophylactically effective dosage" of a pharmaceutical agent is an amount of the pharmaceutical agent that inhibits the onset or recurrence of a disease when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or developing a disease recurrence. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit the onset or recurrence of a disease can be evaluated using a variety of methods known to those skilled in the art, such evaluations being performed in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0082] In many embodiments, the terms "tumor," "lesion," "tumor lesion," "cancer," and "malignancy" are used interchangeably to refer to one or more cancerous growths. In some embodiments, the cancer may be any LFA-1-expressing tumor. In some embodiments, the cancer is selected from adrenal gland tumor, bile duct cancer, bladder cancer, brain tumor, breast cancer, epithelial carcinoma, central or peripheral nervous system tissue cancer, cervical cancer, colon cancer, endocrine or neuroendocrine cancer, hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, Li-Fraumeni tumor, liver cancer, lung cancer, leukemia, lymphoma, melanoma, meningioma, multiple neuroendocrine tumors type I and type II, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, islet cell carcinoma, parathyroid cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, kidney cancer, respiratory tract cancer, non-epithelial carcinoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tracheal cancer, genitourinary tract cancer, and uterine cancer.

[0083] In some embodiments, the cancer is leukemia and its subtypes. For example, leukemia can be acute or chronic leukemia of lymphocytic or myeloid origin, including but not limited to acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), juvenile myelomonocytic leukemia (JMML), hairy cell leukemia (HCL), acute promyelocytic leukemia (subtype of AML), large granular lymphocytic leukemia, or adult T-cell chronic leukemia. In one embodiment, the patient suffers from acute myeloid leukemia, such as anaplastic AML (M0), myeloblastic leukemia (M1: with / without minimal cellular maturation), myeloblastic leukemia (M2: with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6) or megakaryoblastic leukemia (M7).

[0084] Lymphomas contain lymphoma cells that express activated LFA-1, and leukotoxins can treat lymphomas by binding to activated LFA-1 on lymphoma cells and destroying the lymphoma cells by apoptosis or necrosis. In some embodiments, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including anaplastic large cell lymphoma, angioimmunoblastic lymphoma, blastic NK-cell lymphoma, Burkitt's lymphoma, Burkitt-like lymphoma (small noncleaved cell lymphoma), chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous T-cell lymphoma, diffuse large B-cell lymphoma, enteropathy-type T-cell lymphoma, follicular lymphoma, hepatosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, nasal T-cell lymphoma, childhood lymphoma, peripheral T-cell lymphoma, primary central nervous system lymphoma, transformed lymphoma, therapy-related T-cell lymphoma, and Waldenstrom's macroglobulinemia.

[0085] In some embodiments, the method of the present disclosure further comprises administering to the subject a second agent or therapy. Antitumor therapy includes, but is not limited to, conventional antitumor therapy such as chemotherapy, radiation, surgery, or other therapies described herein. The second agent or therapy may be administered to improve antitumor efficacy, reduce toxicity of one or more therapies, and / or reduce the dosage of one or more therapies.

[0086] In some embodiments, the second agent may include a chemotherapy drug. Non-limiting examples of chemotherapy drugs include idarubicin, cytarabine, etoposide, daunorubicin, mitoxantrone, and melphalan. Other common chemotherapy drugs for treating leukemia and lymphoma include chlorambucil, fludarabine phosphate, cytarabine, and daunorubicin hydrochloride. These drugs have the common property of being highly toxic to humans and affecting many different tissues and organ systems in the body. Bone marrow suppression, serious neurological side effects, infertility, pulmonary disease, and gastrointestinal effects are some of the side effects that occur with these drugs. Many of these drugs work by inhibiting DNA synthesis, a process that all dividing cells undergo. These non-specific drugs target most cells in the body. Any suitable drug may be used in conjunction with LtxA, and the combination of the drug and leukotoxin aims to reduce the dose of the drug required to achieve an effective effect in the patient.

[0087] In some embodiments, the second agent or treatment is radiation, surgery, a cancer vaccine, imiquimod, an antiviral drug (e.g., cidofovir), photodynamic therapy, any immune checkpoint molecule, CTLA4, PD-1 / PD-L1 pathway inhibitor (e.g., anti-PD-1 antibody, anti-PD-L1 antibody), lymphocyte activation gene 3 (LAG3) inhibitor (e.g., anti-LAG3 antibody), glucocorticoid-induced tumor necrosis factor receptor (GITR) agonist (e.g., anti-GITR antibody), T cell immunoglobulin and mucin-containing 3 (TIM3) inhibitor, B and T lymphocyte attenuator (BTLA) inhibitor, T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitor, CD38 inhibitor, CD47 inhibitor, indoleamine-2,3-dioxygenase (IDO) inhibitors, CD28 activators, vascular endothelial growth factor (VEGF) antagonists (e.g., "VEGF-Trap" such as aflibercept, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab or ranibizumab), or small molecule kinase inhibitors of the VEGF receptor (e.g., sunitinib, sorafenib tocilizumab, or pazopanib)), angiopoietin 2 (Ang2) inhibitors, transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors, antibodies against tumor-specific antigens (e.g., CA9, CA125, melanoma associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor M2-PK, prostate specific antigen (PSA), mucin 1, MART-1, and CA19-9), Bacillus Calmette-Guerin (BCG) therapy, vaccines (e.g., The therapeutic agent may include one or more of the following: bacillus casei (BCG), granulocyte macrophage colony stimulating factor (GM-CSF), a second oncolytic virus, a cytotoxin, a chemotherapeutic agent (e.g., pemetrexed, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine hydrochloride, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, topotecan, irinotecan, vinorelbine tartrate, and vincristine), cytokines such as IL-6R inhibitors, IL-4R inhibitors, IL-10 inhibitors, IL-2, IL-7, IL-12, and IL-21, antibody drug conjugates, anti-inflammatory agents such as corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), cryotherapy, anti-HPV therapy, laser therapy, electrosurgical removal of HPV cells, and combinations thereof.

[0088] In some embodiments, the method further comprises administering a second agent, such as an anti-cancer agent. As used herein, an anti-cancer agent refers to any agent useful in the treatment of cancer, including, but not limited to, cytotoxins, agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotics, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), biologics (e.g., antibodies, interferons, etc.), and radiopharmaceuticals. As used herein, "cytotoxins or cytotoxic agents" also refers to chemotherapeutic agents, and refers to any agent that is detrimental to cells. Examples include taxol (paclitaxel), temozolomide, cytochalasin B, gramicidin D, ethidium bromide, emetine, cisplatin, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0089] In some embodiments, the second agent or therapy is administered before or after the composition, hi some embodiments, the second agent or therapy is administered simultaneously with the composition.

[0090] As used herein, the term "in combination with" includes sequential or simultaneous administration of an LtxA polypeptide and a second agent (e.g., therapeutic agent) or therapy. For example, when administered "before" a second agent or therapy, one or more doses of an LtxA polypeptide may be administered about 12 weeks, about 11 weeks, about 10 weeks, about 9 weeks, about 8 weeks, about 7 weeks, about 6 weeks, about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, about 150 hours, about 150 hours, about 100 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes or more before administering one or more doses of an LtxA polypeptide.

[0091] When administered "after" a second agent or therapy, the LtxA polypeptide may be administered about 12 weeks, about 11 weeks, about 10 weeks, about 9 weeks, about 8 weeks, about 7 weeks, about 6 weeks, about 5 weeks, about 4 weeks, about 3 weeks, about 2 weeks, about 150 hours, about 150 hours, about 100 hours, about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes or about 10 minutes after administration of the IL-15 polypeptide.

[0092] "Co-administered" with a second agent or therapy means that the LtxA polypeptide is administered to the subject in a separate dosage form within 10 minutes of (before, after, or simultaneously with) administration of the second agent or therapy, or is administered to the subject as a single combined dosage formulation containing both the LtxA polypeptide and the second agent or therapy.

[0093] In some embodiments, the treatment produces one or more therapeutic effects selected from delayed tumor growth, reduction in tumor cell count, tumor regression, prevention or delayed tumor recurrence, increased survival, partial response, and complete regression. In some embodiments, tumor growth in the patient is delayed by at least 10 days compared to tumor growth in an untreated patient. In some embodiments, tumor growth is inhibited by at least 20% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%) compared to an untreated patient.

[0094] Pharmaceutical compositions comprising LtxA polypeptides can be delivered, for example, intratumorally, intravesically, subcutaneously, intraperitoneally or intravenously by standard needles and syringes. For subcutaneous delivery, pen delivery devices are also readily adapted to deliver pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and easily replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded.

[0095] In some embodiments, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla.). In yet another embodiment, the controlled release system can be placed in the vicinity of the target of the composition, thereby requiring only a fraction of the systemic dose. (See, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0096] The injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip infusion, and the like. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared, for example, by dissolving, suspending, or emulsifying the antibody or its salt in a sterile aqueous or oily medium that is conventionally used for injection. Examples of aqueous media for injection include physiological saline, glucose-containing isotonic solutions, and other adjuvants, which may be used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 moles) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil, soybean oil, and the like, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injections thus prepared are preferably filled into suitable ampoules.

[0097] Advantageously, the above-mentioned oral or parenteral pharmaceutical compositions are prepared in a dosage unit form suitable for a dosage of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0098] Additional definitions To aid in the understanding of the detailed description of the compositions and methods according to the present disclosure, several terminology definitions that clearly disclose various aspects of the present disclosure are provided. 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.

[0099] As used herein, the term "recombinant" refers to an LtxA polypeptide of the disclosure that is produced, expressed, isolated or obtained by techniques or methods known as recombinant DNA technology, including DNA splicing, transgenic expression, etc. The term refers to an antibody that is expressed in an expression system of a non-human mammal (including a genetically modified non-human mammal, e.g., a genetically modified mouse) or cell (e.g., CHO cell), or that is isolated from a recombinant fully human antibody library.

[0100] "Nucleic acid" or "polynucleotide" refers to a DNA molecule (e.g., but not limited to, cDNA or genomic DNA) or an RNA molecule (e.g., but not limited to, mRNA), including DNA or RNA analogs. DNA or RNA analogs can be synthesized from nucleotide analogs. DNA or RNA molecules can contain non-naturally occurring portions, such as modified bases, modified backbones, and deoxyribonucleotides in RNA. Nucleic acid molecules can be single-stranded or double-stranded.

[0101] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, there is nucleotide sequence identity in at least 90%, more preferably at least 95%, 96%, 97%, 98% or 99% of the nucleotide bases as measured by any well-known algorithm, such as FASTA, BLAST or GAP, as described below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in some cases, encode a polypeptide having an amino acid sequence identical or substantially similar to the polypeptide encoded by the reference nucleic acid molecule.

[0102] As used herein, the term "disease" is intended to be generally synonymous and is used interchangeably with the terms "disorder" and "disease" (physical illness), both of which reflect an abnormal condition of the human or animal body or parts thereof that interferes with normal functioning (e.g., inflammatory disorders), typically presents with characteristic signs or symptoms, and results in a reduced survival or quality of life in humans or animals.

[0103] As used herein, the terms "reduce", "reduced", "reduction", "reduction", and "suppression" generally refer to a statistically significant amount of reduction. However, without any doubt, "reduce", "reduced" or "reduction" or "suppression" refers to a reduction of about at least 10% compared to a reference level, such as a reduction of about at least 20%, about at least 30%, about at least 40%, about at least 50%, about at least 60%, about at least 70%, about at least 80%, about at least 90%, or even up to 100% (e.g., disappearance compared to a reference sample), or any value between 10% and 100% compared to a reference level.

[0104] As used herein, the term "agent" refers to a compound, a mixture of compounds, a biological macromolecule (e.g., a nucleic acid, an antibody, a protein, or portions thereof, such as a peptide), or an extract made from biological material such as a bacterial, plant, fungal, or animal (especially mammalian) cell or tissue. The activity of such agents may qualify them as therapeutic agents, which are biologically, physiologically, or pharmacologically active substances (or agents) that act locally or systemically in a subject.

[0105] As used herein, the terms "therapeutic agent", "therapeutic agent" or "therapeutic agent" are used interchangeably and refer to a molecule or compound that provides some beneficial effect when administered to a subject. Beneficial effects include possible diagnostic determination, amelioration of a disease, symptom, disorder or condition, reduction or prevention of the onset of a disease, symptom, disorder or condition, and generally neutralization of a disease, symptom, disorder or condition.

[0106] The term "therapeutic effect" is art-recognized and refers to a localized or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance.

[0107] Doses are often expressed in relation to body weight. Thus, a dose expressed as [g, mg or other unit] / kg (or g, mg, etc.) usually refers to [g, mg or other unit] per kg (or g, mg, etc.) body weight, even if the term "body weight" is not explicitly stated.

[0108] As used herein, the term "pharmacologically acceptable" refers to a relatively non-toxic material, such as a carrier or diluent, that does not interfere with the biological activity or properties of a composition, and that may be administered to an individual without producing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0109] As used herein, the term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, that are involved in carrying or transporting the compounds of the present disclosure in a subject so that they perform their intended function. Typically, such compounds are carried or transported from one organ or part of the body to another. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials which can function as pharma- ceutically acceptable carriers include sugars such as lactose, glucose and saccharose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate, ethyl laurate; agar; buffers such as magnesium hydroxide, aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Examples of suitable carriers include Ringer's solution, ethyl alcohol, phosphate buffer, diluents, granulating agents, lubricants, binders, disintegrants, wetting agents, emulsifiers, colorants, release agents, coating agents, sweeteners, flavoring agents, flavoring agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, stiffening agents, humectants, carriers, stabilizers, and other non-toxic compatible substances used in pharmaceutical formulations, and combinations thereof. As used herein, "pharmaceutical acceptable carrier" also includes any coating agent, antibacterial agent, antifungal agent, and absorption retardant, etc., that is compatible with one or more components of the present disclosure and physiologically tolerated by the subject. Additional active compounds may also be incorporated into the composition.

[0110] As used herein, unless otherwise clear from the context, "combination" therapy is meant to include the coordinate administration of two or more therapeutic agents, including but not limited to simultaneous administration. Specifically, combination therapy includes both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and sequential or sequential administration, provided that the administration of one therapeutic agent is in some way regulated on the administration of the other therapeutic agent. For example, one therapeutic agent may be administered after a different therapeutic agent has been administered and allowed to act for a period of time. See Kohrt et al. (2011) Blood 117:2423.

[0111] As used herein, "administration" refers to the introduction of a composition containing a therapeutic agent into the body of a subject using a variety of methods and delivery systems known to those skilled in the art. Examples of routes of administration of the antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, intratumoral, intravesical, spinal, or other parenteral routes of administration by injection or infusion. As used herein, the phrase "parenteral administration" refers to forms of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, substernal injection or infusion, and in vivo electroporation. The antibodies described herein can also be administered via parenteral routes, such as topical, epidermal, mucosal, etc., for example, intranasally, orally, vaginally, rectally, sublingually, or topically. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0112] As used herein, the term "co-administration" or "co-administered" refers to the administration of at least two agents or therapies to a subject. In some embodiments, the co-administration of two or more agents / therapies is simultaneous. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those skilled in the art will appreciate that the formulation and / or route of administration of the various agents / therapies used may vary.

[0113] As used herein, the term "in vitro" refers to events that take place not within a multicellular organism, but in an artificial environment, such as a test tube or reactor, cell culture, etc.

[0114] As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as a non-human animal.

[0115] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0116] As used herein, the terms "including," "comprising," "containing," "having," and variations thereof, unless expressly stated otherwise, are meant to encompass the items listed below and equivalents thereof as well as additional items.

[0117] As used herein, the phrases "one embodiment," "various embodiments," "some embodiments," etc. are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but may, unless the context clearly dictates otherwise.

[0118] As used herein, the term "and / or" or " / " means any item, any combination of items, or all items associated with this item.

[0119] As used herein, the word "substantially" does not exclude "completely", e.g., a composition that is "substantially free" of Y may be completely free of Y. Where appropriate, the word "substantially" may be omitted from the definitions of the present disclosure.

[0120] As used herein, the term "each," when used in reference to a collection of items, is intended to identify each individual item in the set, but does not necessarily refer to every item in the set. Exceptions may occur unless express disclosure or context clearly indicates otherwise.

[0121] As used herein, the term "approximately" or "about" refers to a value similar to the reference value set forth above when used with respect to one or more values ​​of interest. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (more or less) of the stated reference value, unless otherwise stated or otherwise clear from the context (except when such number exceeds 100% of the maximum possible value). Unless otherwise indicated, the term "about" is intended herein to include values, e.g., weight percent, close to the stated range that are equivalent with respect to the function of each component, composition, or embodiment.

[0122] As disclosed herein, several ranges of values ​​are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, is also specifically disclosed between the upper and lower limits of that range, unless the context clearly dictates otherwise. Each smaller range between a stated value or intervening value in a stated range and another stated or intervening value in that stated range is included in the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and either or both of the lower and upper limits included in the smaller range are included within the disclosure, subject to the specifically excluded limit in the stated range. The stated ranges include one or both of the limits, and ranges excluding either or both of these limits are also included in the disclosure.

[0123] Any examples provided herein, or the use of exemplary language (e.g., "etc.") are merely intended to further clarify the invention and do not impose limitations on the scope of the disclosure unless otherwise recited in the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0124] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. For any of the methods provided, the steps of the method may occur simultaneously or sequentially. When method steps occur sequentially, the steps may occur in either order unless otherwise stated. When a method includes a combination of steps, any and all combinations or subcombinations of steps are included within the scope of the present disclosure unless otherwise stated herein.

[0125] Each publication, patent application, patent, and other references described herein are incorporated by reference in their entirety to the extent that they are not inconsistent with this disclosure. Publications disclosed herein are provided solely for their disclosure prior to the filing date of this disclosure. This disclosure should not be construed as an admission that such publications are not entitled to antedate such publications by prior disclosure. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0126] The examples and embodiments described in this specification are merely illustrative, and a person skilled in the art may make various modifications or changes based on the examples and embodiments, and it should be understood that these modifications or changes are within the spirit and scope of the present disclosure and the scope of the claims.

[0127] Working Example [Example 1] composition The composition of this formulation, e.g., Leukothera, includes (a) a leukotoxin protein isolated from bacteria as the active pharmaceutical ingredient, (b) tromethamine (Tris) as a buffer, and (c) sodium chloride (NaCl) and calcium chloride (CaCl2) stabilizers for activation.

[0128] The composition is weighted according to an internally developed methodology to provide an end concentration of 0.30 mg / mL of leukotoxin when reconstituted in sterile water for clinical use / IV infusion. Leukothera for Injection is a white, sterile, lyophilized powder. It is filled into clear glass vials and reconstituted with 2 mL of sterile water for IV infusion. After reconstitution, the vial is gently swirled until all of the powder is dissolved. The appropriate volume for each dose is then added to an IV bag and slowly infused / injected. The concentration of leukotoxin in Leukothera for Injection is 0.30 mg / mL (300 μg / mL).

[0129] Leukothera for Injection is a lyophilized powder of the pharmaceutical ingredient leukotoxin. Leukothera is prepared in bulk solution at pH 7.5 in a buffer consisting of 20 mM tromethamine (Tris), 250 mM sodium chloride (NaCl) and 0.2 mM calcium chloride (CaCl2). Note that the preparation occurs as part of the manufacture of the pharmaceutical ingredient and no additional preparation steps occur during the manufacture of the drug product. Leukothera for Injection is manufactured to provide a final concentration of 0.30 mg / mL leukotoxin when reconstituted in sterile water at the time of use. The intended fill is 0.6 mg leukotoxin per vial. The composition of Leukothera for Injection is shown in Table 2.

[0130] Table 2. Composition of Leukothera for injection [Table 2] Table 3. Specifications for injectable Leukothera preparations [Table 3]

[0131] Protein identity can be assessed by a qualitative Western blot. Protein samples are electrophoretically separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. After SDS-PAGE electrophoresis, proteins are transferred to a nitrocellulose membrane. The nitrocellulose membrane is probed with a primary monoclonal antibody raised against the LtxA protein. A conjugated secondary antibody probe binds to the primary monoclonal antibody to detect the protein, which is measured using a ChemDoc MP system and Image Lab software. A qualitative Western blot is qualified to show specificity by its ability to determine whether nonspecific interactions occur on the Western blot on a nonspecific protein (bovine serum albumin [BSA]) with the molecular weight of the specific protein. The acceptance criteria established require the observation of a major sample band at 120 kilodaltons (kDa) that matches the migration pattern of a reference standard, ensuring protein identity. Data from Western blot analysis of GMP lot 599-0818-003 and engineering lot 599-0718-002 are shown in Figures 1 and 2, respectively.

[0132] formulation The pharmaceutical composition disclosed in this disclosure is a parenteral formulation. The formulation is a white sterile lyophilized powder for intravenous infusion solution formulated for injection, parenteral route of administration and filled into clear glass vials. Each vial contains 0.6 mg of lyophilized powder. The final dosage form is an intravenous infusion solution. To obtain the final dosage form, 0.6 mg of lyophilized powder is reconstituted with 2 mL of sterile water to a final concentration of 0.3 mg / mL. It is filled into a clear glass vial and reconstituted with 2 mL of sterile water at the time of clinical use for intravenous injection. After reconstitution, the vial is gently swirled until all the powder is dissolved. Then, the appropriate volume for each dose is added to an IV bag and slowly infused / injected. The intravenous infusion concentration of API in this formulation (Leukothera for Injection) is 0.30 mg / mL (300 μg / mL).

[0133] Storage and Stability Vials containing the lyophilized powder must be stored frozen at temperatures below -20°C (standard freezer). After reconstitution with sterile water, the formulation can be stored at 4°C (standard refrigeration) for up to 24 hours, after which it must be discarded. All investigational products at the study site must be stored under secure lock and key with limited access. Temperature must be controlled during shipping and storage at the study site.

[0134] Table 4 Stability research overview [Table 4] a Lot 093I0720 was formulated at 0.7 mg / vial because DS lot A599-LtxA-20-004 has a concentration of 353 μg / mL and will not be further diluted during drug product manufacturing.

[0135] Stability under long-term storage conditions Under long-term storage conditions of -20±5°C, data are available for up to 12 months for the primary lots (072I0918 and 081I1018). Stability data for supportive stability lots are available for up to 19 months. All lots met all specifications when stored at or below -20±5°C for 12 months, demonstrating that the formulation is stable under these storage conditions.

[0136] When stored at -20±5°C, data from lot 093I0720 do not show trends in the quality attributes related to formulation (pH, protein content), biological activity (potency) and purity (CE-SDS, RP-HPLC and cIEF) due to the use of Tier 1 specifications.

[0137] Stability during clinical use To mimic the extreme potential conditions of clinical use, the stability (activity assessed by an ATP-based cell viability assay) of injectable Leukothera was studied after reconstitution under the following clinically relevant conditions:

[0138] Refrigerated: Remove lyophilized samples at temperatures below -20°C, reconstitute and store at 4°C for up to 24 hours.

[0139] Conclusion: Samples frozen for less than 12 months can be stored at 4°C for a maximum of 24 hours.

[0140] Refreezing: Remove lyophilized samples at temperatures below -20°C, reconstitute and store at temperatures below -20°C for up to 7 days.

[0141] Conclusion: Freeze-dried samples frozen at temperatures below -20°C for up to 12 months can be reconstituted and refrozen (at temperatures below -20°C) for up to 7 days.

[0142] The results confirm that refrigeration or refrigeration does not significantly affect the product quality of injectable Leukothera in a clinical setting.

[0143] Storage period The primary stability batches supporting the determination of an 18-month shelf life for clinical material are based on appropriate extensions of available stability data from development studies of Leukothera for Injection, lot 072I0918 (engineering lot) and lot 081I1018 (GMP lot), which met all specifications for 12 months at -20 ± 5° C. Data from Leukothera for Injection, lot 003D0318, analyzed after 19 months of storage, supports an 18-month shelf life.

[0144] The recommended storage conditions for injectable Leukothera clinical specimens are at or below -20°C with a shelf life of 18 months.

[0145] Container Closure System The primary container closure for Leukothera for Injection is a 5 mL clear glass vial with a 20 mm Flurotec stopper. The vial is sealed with a flip-off TrueEdge seal.

[0146] Table 5 Primary container closures for Leukothera for injection [Table 5]

[0147] [Example 2] Manufacturing and Process Development This section describes the manufacturing process for injectable Leukothera as performed by the University of Iowa for lot 081I1018 for Good Manufacturing Practice (GMP). A manufacturing flow chart showing the process controls available is provided below.

[0148] Manufacturing process flow chart of Leukothera for injection TIFF2024546686000009.tif203160

[0149] Unzip Containers of Leukothera Bulk Drug Solution (BDS) are removed from freezer storage (-60°C to -90°C) and placed upright in refrigerated storage (2°C to 8°C) to thaw. Thawing time is allowed to be approximately 3-4 days. The thawed solution is removed from the refrigerator and transported to the compounding area.

[0150] Pool Each container of Leukothera bulk solution was gently rotated / swirled and then carefully poured into a 9 L sterile glass carboy using a stir bar. The solution was gently mixed with a magnetic stirrer until homogenous (minimum 5 minutes).

[0151] Disinfection Once the cleanroom is deemed suitable for use by standard operating procedures, the container, closure, and equipment (a Standard Hull HY-PRO freeze dryer) are transported into the cleanroom. Non-viable particulates within the room are monitored via particle sensors.

[0152] The pressure differential is also monitored relative to the cleanroom to ensure the water is at least 0.05 inches higher than the entry. The 9 L glass carboy containing the pooled Leukothera bulk solution is then transferred to the cleanroom for filtration. The live microflora in the cleanroom is monitored. The pooled Leukothera bulk solution is then sterilized into a sterile 9 L glass carboy using a sterile 0.22 micron Millipak 200 filter. The carboy remains covered with a sterile stopper until the start of the filling process. At the end of filtration, the filter integrity is inspected according to standard operating procedures.

[0153] The water bubble point should reach 50 PSI. Complete an initial wash of the filter with 70 / 30 IPA / WFI pre-wash followed by water, recording the number of flushes and the bubble point result. A sufficient bubble point should be obtained before starting the filling operation. If the filter bubble point fails, the filtration should be repeated with a new filter.

[0154] A sufficient bubble point must be achieved before the filling operation can begin.

[0155] Vial filling Using a sterile Flexicon FP50 filling machine, fill each sterile 5 mL vial with approximately 2.04 grams (g) of filtered Leukothera bulk solution. Stopper each vial to lyophilization depth according to standard operating procedures. Perform an in-process fill weight check on all vials. Vials not filled to the specified weight range of 2.04 g + / - 0.20 g are rejected.

[0156] Freeze drying Once the vials are filled, each tray of vials is placed into the freeze dryer. The freeze dryer is loaded at room temperature. A Hull HY-PRO freeze dryer is used to freeze dry the vials filled with Leukothera bulk solution according to a prescribed cycle. Table 6. Freeze-drying cycle for injectable Leukothera [Table 6] The vials containing the lyophilized product are kept at 25° C. for storage.

[0157] Stopper and seal After the lyophilization cycle is complete, purge the chamber and vials with nitrogen, NF atmosphere. Then cap the vials. Verify the vial height at the capping station (crimper). Remove the vials from the chamber and place a sterile seal on each vial. Crimp the seal per standard operating procedure (SOP). Store the sealed Leukothera for injection vials at 2-8°C.

[0158] Leukothera vials for injection undergo 100% manual visual inspection for acceptable quality levels (AQL) followed by spot testing for particulate matter and defects in the container closure system according to SOPs established by the University of Iowa.

[0159] Labelling, Packaging and Storage Label vials of Leukothera for injection with the approved vial label. Package labeled vials in bulk and store at -70°C until shipment.

[0160] Table 7. Description of investigational drugs [Table 7]

[0161] Biological characteristics The active pharmaceutical ingredient (API) is the pharmaceutical ingredient leukotoxin. Leukotoxin is post-translationally modified at lysine residues K561 and K686 with fatty acyl groups. These acylations are required for leukotoxin activity against LFA-1 expressing leukocytes. Leukotoxin does not contain cysteine ​​residues or glycosylation motifs.

[0162] [Example 3] Posology and Administration Target clinical dose The lyophilized formulation is reconstituted in a vial with 2 mL of sterile water to a concentration of 0.3 mg / mL. The reconstituted formulation solution is diluted with 0.9% saline in an IV bag and infused over up to 4 hours, with low doses of 1.4 μg / kg or 98 μg, concentration of 0.25 μg / mL, and volume of 400 mL for a 70 kg subject, and high doses of 1020 μg / kg or 71.4 mg, concentration of 0.18 mg / mL, and volume of 400 mL for a 70 kg subject.

[0163] The protein concentration results for all samples at the low dose concentration were below the lower limit of quantification (0.20 μg / mL) of the BCA assay for total protein content, reflecting the low dose concentration sample being at 0.25 μg / mL at that level. Western blot was used to demonstrate protein recovery, as shown in Figure 3. Purity measurements were not performed by scanning the Western blot, but there was no visible change in the number of bands retained in the IV bag over time compared to the T=0 and control samples. Bioactivity results at the low dose concentrations, as shown in Figure 4, demonstrate that active protein content affecting cell death was recovered at levels within the expected formulation release range (50-150%). Similar results were seen at the highest dose, as shown in Figures 5 and 6. There was also no change in the type of bands seen on the Western blot between the test samples, T=0 and control samples, indicating no decrease in purity over the 4 hour retention time.

[0164] Table 8. Administration parameters for infusion bag compatibility study [Table 8] Table 9. Analytical Test Results for Low-Dose Concentration Leukothera (0.25 μg / mL) During an IV Bag Compatibility Study [Table 9] LOQ - Lower limit of quantitation (20μg / mL) Table 10. Analytical Test Results for High-Dose Concentration Leukothera (0.18 mg / mL) During IV Bag Compatibility Study [Table 10]

[0165] The low human dose for the Phase 1 study was selected based on the 10% maximum effective concentration (EC10), calculated from an in vitro model using THP-1 cells, a human monocytic cell line derived from a patient with acute monocytic leukemia. Diseased WBCs have higher expression of LFA-1, the target of injectable Leukothera, compared to healthy WBCs. Thus, the EC10 estimate from the in vitro model using THP-1 cells derived from a patient with acute monocytic leukemia is most representative of the EC10 in patients being treated with injectable Leukothera. The EC10 calculated from the in vitro model was 20ng / mL. In a 70kg human with a blood volume of approximately 5L, a dose of 1.4μg / kg would predict a concentration of injectable Leukothera close to the EC10. Thus, 1.4μg / kg / week is the proposed human starting dose, initially administered as a single IV dose over 3-4 hours, with the intention of administering weekly, with the duration of the IV infusion freely modified as needed. Further support for the proposed starting dose of 1.4 μg / kg comes from ICH S9 and the standard approach for estimating starting doses in early phase clinical trials of anticancer drugs, which is 1 / 10 of the STD10 for rodents or 1 / 6 of the HNSTD for non-rodents on a body surface area (BSA) normalized basis.

[0166] Leukothera for injection is a biologically based drug that can be calculated to aid in the selection of an appropriate starting dose for humans. The STD10 for male rats during the 4-week study was 500-750 μg / kg, while the STD10 for female rats was 100 μg / kg. Using the most conservative dose of 500 μg / kg, which is the no observed adverse effect level, the starting dose for humans is estimated as follows: no observed adverse effect level for rats = 500 μg / kg ÷ 1000 μg = 0.5 mg / kg, BSA-normalized human equivalent dose = 0.5 mg / kg ÷ 6.2 = 0.08 mg / kg × 1000 = 81 μg / kg, human starting dose (BSA-normalized): 81 μg / kg ÷ 10 (safety factor) = 8.1 μg / kg. Based on the HNSTD of 300 μg / kg, a no observed adverse effect level during the 4-week dog study, the human starting dose is estimated as follows: no observed adverse effect level in dogs = 300 μg / kg ÷ 1000 μg = 0.3 mg / kg, BSA-normalized human equivalent dose = 0.3 mg / kg ÷ 1.8 = 0.167 mg / kg × 1000 = 167 μg / kg, human starting dose (BSA-normalized): 167 μg / kg ÷ 6 (safety factor) = 27.8 μg / kg. Therefore, the most conservative starting dose on the BSA-normalized basis is 8.1 μg / kg. The proposed starting dose of 1.4 μg / kg is more than 5-fold lower than the recommended starting dose on the BSA-normalized basis.

[0167] Rats are the most sensitive nonclinical species, and the proposed human starting dose is 57-fold lower than the body surface area (BSA)-normalized human dose at the lower end of the STD10 range (500 μg / kg) and 86-fold lower than the human equivalent dose (HED) associated with mortality in rats (750 mg / kg). The HNSTD in dogs is at the highest dose level tested (300 μg / kg), or 119-fold higher than the proposed starting dose on a BSA-normalized basis. Transient elevations of various cytokines indicate that patients should be appropriately monitored and treated as necessary due to the potential for lysis-associated cytokine release.

[0168] Also, a veterinary clinical study in dogs with lymphoma is underway in which eight dogs were given Leukothera for injection intravenously once a week at a dose level of 200 μg / kg, gradually increasing the dose over 30 minutes. None of the dogs showed any adverse reactions during or after the infusion of Leukothera for injection. On a BSA-normalized basis, this dose range is equivalent to 2.8-111 μg / kg in humans, 2-79 times higher than the proposed human starting dose of 1.4 μg / kg.

[0169] Preparation and Administration Procedure Each site will be provided with a dispensing manual containing detailed reconstitution and preparation instructions for the investigational drug. The investigational drug is reconstituted with 2 mL of sterile water to a dose concentration of 0.3 mg / mL. The solution is transferred to an infusion bag and pump and adjusted with saline to obtain the selected dose. The maximum holding time for the reconstituted investigational drug is 24 hours at 4°C (standard refrigeration), and if it exceeds 24 hours it must be discarded. The investigational drug is administered by slow intravenous injection. The first intravenous infusion of any dose of the investigational drug to a patient should be administered over 3-4 hours (+ / - 15 minutes). If the patient does not show an adverse reaction, subsequent infusions at the previously tolerated dose level will be administered over 1-2 hours (+ / - 15 minutes). The infusion time for any infusion should be at least 1 hour.

[0170] The present disclosure is not intended to be limited to the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

Claims

1. about 0.1 mg / ml to about 0.5 mg / ml of a leukotoxin (LtxA) polypeptide isolated from Aggregatibacter actinomycetemcomitans; about 5 mM to about 50 mM Tris; about 100 mM to about 300 mM NaCl; about 0.05 mM to about 0.5 mM CaCl ２ and, A liquid composition for treating cancer, formulated to have a pH of about 7.0 to about 8.

0.

2. 10. The liquid composition of claim 1, comprising about 0.3 mg / ml of LtxA polypeptide.

3. About 20 mM Tris, about 250 mM NaCl, and about 0.2 mM CaCl ２ The liquid composition of claim 1 , comprising:

4. 10. The liquid composition of claim 1, formulated to have a pH of about 7.

5.

5. 10. The liquid composition of claim 1, formulated to remain stable at 4°C for at least 24 hours.

6. 2. The liquid composition of claim 1, wherein the LtxA polypeptide is isolated from Aggregatibacter actinomycetemcomitans strain NJ4500.

7. The liquid composition of claim 1 , wherein the LtxA polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:1 or comprises the amino acid sequence of SEQ ID NO:

1.

8. about 0.2 mg to about 2 mg of an LtxA polypeptide; about 2 mg to about 8 mg of Tris; about 10 mg to about 50 mg of NaCl; about 0.01 mg to about 0.5 mg of CaCl ２ and, A lyophilized composition prepared from the liquid composition of claim 1, formulated to have a pH of about 7.0 to 8.0 upon reconstitution.

9. Approximately 0.6 mg of LtxA polypeptide; about 4.85 mg of Tris; Approximately 29.2 mg of NaCl; Approximately 0.04 mg of CaCl ２ The freeze-dried composition of claim 8, comprising:

10. 10. The lyophilized composition of claim 8 or 9, formulated to have a pH of about 7.5 after reconstitution.

11. The lyophilized composition of claim 8, which is reconstituted in sterile water or buffered saline.

12. The lyophilized composition of claim 8, reconstituted as a liquid composition containing about 0.3 mg / ml of LtxA polypeptide.

13. 9. The lyophilized composition of claim 8, formulated to remain stable after storage at -20±5°C for up to 24 months.

14. 9. The freeze-dried composition of claim 8, which is stored at a temperature below -20°C, reconstituted, and formulated to remain stable after storage at a temperature below -20°C for up to 7 days.

15. 9. The lyophilized composition of claim 8, which is formulated to remain stable after storage at temperatures below -20°C, reconstitution, and storage at about 4°C for up to 24 hours.

16. A kit comprising the liquid composition of claim 1 or the lyophilized composition of claim 8.

17. Use of a therapeutically effective amount of a liquid composition described in any one of claims 1 to 7 for treating cancer in a subject, wherein the therapeutically effective amount of the liquid composition is from about 1 μg / kg to about 1200 μg / kg based on the subject's body weight.

18. 18. The use of claim 17, wherein the therapeutically effective amount of the liquid composition is about 1.4 μg / kg based on the subject's body weight.

19. 18. The use of claim 17, wherein the therapeutically effective amount of the liquid composition is about 1020 μg / kg based on the subject's body weight.

20. 18. The use of claim 17, wherein the liquid composition is administered parenterally to a subject intravenously, subcutaneously, or intraperitoneally.

21. 18. The use of claim 17, wherein the liquid composition is administered parenterally to a subject by intravenous injection.

22. 18. The use of claim 17, wherein the liquid composition is administered to a subject by intravenous injection over a period of 1 to 10 hours.

23. 18. The use of claim 17, wherein the liquid composition is administered parenterally to a subject by intravenous injection over a period of 3 to 4 hours.

24. 18. The use according to claim 17, wherein the liquid composition is administered by intravenous injection over a period of 1 to 2 hours.

25. 18. The use of claim 17, wherein the liquid composition administered to a subject is formulated as a dosage form selected from a modified release dosage form, a sustained release (depot) dosage form, a controlled release dosage form, a sustained release dosage form, a delayed release dosage form, an extended release dosage form and / or an extended release dosage form.

26. 18. The use according to claim 17, wherein the cancer is selected from adrenal gland tumor, bile duct cancer, bladder cancer, brain tumor, breast cancer, epithelial carcinoma, cancer of central or peripheral nervous system tissue, cervical cancer, colon cancer, endocrine or neuroendocrine cancer, hematopoietic cancer, esophageal cancer, fibroma, gastrointestinal cancer, glioma, head and neck cancer, Li-Fraumeni tumor, liver cancer, lung cancer, leukemia, lymphoma, melanoma, meningioma, multiple neuroendocrine tumors type I and type II, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell carcinoma, parathyroid cancer, pheochromocytoma, pituitary tumor, prostate cancer, rectal cancer, kidney cancer, respiratory tract cancer, non-epithelial carcinoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, tracheal cancer, genitourinary tract cancer and uterine cancer.

27. 18. The use of claim 17, wherein the cancer is leukemia or any subtype thereof.

28. 18. The use of claim 17, wherein the cancer is lymphoma or any subtype thereof.

29. 18. The use of claim 17, further comprising administering to the subject a second agent or therapy.

30. 30. The use of claim 29, wherein the second agent comprises an anti-tumor or anti-cancer agent.

31. 30. The use of claim 29, wherein the second agent or therapy is administered before or after the composition.

32. 30. The use of claim 29, wherein the second agent or therapy is co-administered with the composition.

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