Anti-cancer polypeptides, nucleic acid constructs and methods of using same

Abstract

Described are various embodiments of anti-cancer polypeptides, nucleic acid constructs. and methods of using same, particularly for the expression of protein comprising three thrombospondin-1 type 1 repeats (3TSR), as well as pharmaceutical compositions comprising any of the disclosed polypeptides or nucleic acid constructs for the treatment of solid tumor cancers such as ovarian cancer or pancreatic cancer.

Description

ANTI-CANCER POLYPEPTIDES, NUCLEIC ACID CONSTRUCTS AND METHODS OFUSING SAMECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to Canadian Patent Application No. 3,251,574 entitled “ANTI-CANCER POLYPEPTIDES, NUCLEIC ACID CONSTRUCTS AND METHODS OF USING SAME”, filed August 12, 2024, the entire disclosure of which is hereby incorporated herein by reference.BACKGROUND OF THE INVENTION

[0002] Solid tumors such as sarcomas, carcinomas, and lymphomas account for approximately 90 percent of cancer deaths (Dillekas H, Rogers MS, Straume O. Are 90% of deaths from cancer caused by metastases? Cancer Med. 2019 Sep;8(12):5574-5576. doi: 10.1002 / cam4.2474. Epub 2019 Aug 8. PMID: 31397113; PMCID: PMC6745820.)

[0003] It was estimated that in 2023 there will be 19,710 new cases of ovarian cancer (OC) in the United States and 13,270 deaths. Ovarian cancer has a 5-year survival rate of only about 50% according to the National Cancer Institute (https: / / seer.cancer.gov / statfacts / html / ovary.html accessed June 20, 2023).

[0004] Epithelial ovarian cancer (EOC) is an ovarian cancer that starts in the surface (epithelial) layer of the ovary. Epithelial ovarian cancer is the leading cause of death from gynecological malignancy and the fifth most common cause of cancer related death in women. Epithelial ovarian cancer is the most common type of ovarian cancer; about 90 out of 100 tumors of the ovary (90%) are epithelial.

[0005] It has been estimated that more than 50,000 deaths from pancreatic cancer (PC) will occur in the United States in 2023. Pancreatic cancer is the fourth leading cause of cancer death. The 5-year survival rate for pancreatic cancer in the United States is 12%. More than 90% of pancreatic cancers are exocrine adenocarcinoma that starts in the cells lining the pancreatic duct. Thus, there is an unmet need in the field for the development of novel therapies for effectively treating pancreatic cancer, to allow for more successful treatment outcomes.11420P-ACP-W001

[0006] Thrombospondin- 1 (TSP-1) is a negative regulator of tumor angiogenesis (Kerbel and Folkman. Nat. Rev. 107: 83-89, 2006). TSP-1 is an inhibitor of new blood vessel formation and tumor growth. TSP-1 inhibits endothelial cell growth, migration, and tube formation in vitro. Furthermore, it has been shown that TSP-1 is an extracellular matrix glycoprotein that can bind to a number of angiogenic factors, including FGF-2, VEGF, PDGF and TGF-pi (Lamszus etal., 1996, Margosio et al., 2003). The interaction of TSP-1 with CD36 is hypothesized to inhibit pro- angiogenic signaling by forming a complex with NRP-1 and VEGFR-2 that would prevent VEGF- induced VEGFR-2 activation. TSP-1 can further act on NOS, inhibit FGF-2-induced endothelial cell migration, and may also induce apoptosis of endothelial cells. It has been reported that the anti- angiogenic properties of TSP-1 implicated an inhibition of endothelial cell survival, proliferation, and migration (Jimenez B, Volpert OV, Crawford SE, Febbraio M, Silverstein RL, Bouck N. Nat Med. 2000 Jan; 6 (1): 41-8).

[0007] Australian Patent Application Publication Number AU2018200921A1 published March 1, 2018, discloses that TSP1 blocks the effects of nitric oxide (NO) in the vascular system, and prevents NO from dilating blood vessels and increasing blood flow to organs and tissues. This patent application discloses that TSP1 acts through the cell receptor CD47 to block the effects of NO on blood vessels. Relief of this inhibition in genetically altered (knockout) mice lacking either TSP1 or CD47 results in improved blood flow and increased tissue oxygenation. Further, by using monoclonal antibodies (mAbs), and peptides that block the TSP1-CD47 interaction, or agents (such as antisense oligonucleotides or morpholinos) that reduce the level of CD47 or TSP1, blood flow was increased to ischemic tissues.

[0008] Reduced expression of TSP- 1 facilitates the growth of several tumor types including bladder, breast, and ovarian cancer, fibrosarcoma, and glioblastoma (Hsu et al. Cancer Res. 56: 5684-5691, 1996; Campbell el ci\. Cancer Res. 58: 1298-1304, 1998; Alvarez etal. Gynecol. Oneal. 82: 273-278, 2001). TSP-1 inhibits angiogenesis by stimulating endothelial cell apoptosis and inhibiting endothelial cell migration (Jimenez et al. Nat. Med. 6: 4148, 2000) and by binding and sequestering proangiogenic growth factors such as vascular endothelial growth factor (VEGF) (Greenaway eta\. J. Cell Physiol. 210: 807-818, 2007). TSP-1 binds CD36, a receptor expressed on the surface of endothelial and steroidogenic cells in the ovary (Dawson et al. J. Cell Biol. 138: 707- 717, 1997; Petrik et al. Biol. Reprod. 67: 1522-1531, 2002) as well as numerous other cell surface receptors such as integrin-associated protein / CD47, the low-density lipoprotein receptor-related21420P-ACP-W001protein-1, integrins, and various heparan sulfate proteoglycans (Ren et al. Biochim Biophys Acta. 765: 178-188, 2006).

[0009] The interaction of the type 1 repeats (TSRs) of TSP-1 with CD36 plays a role in the regulation of angiogenesis in vitro and in vivo (Jimenez et al. Nat Med. 6: 41-48, 2000; Dawson et al. Mot. Pharmacol. 55: 332-338, 1999; Dawson et al. J. Cell Biol. 138: 707-717, 1997). In vitro, CD36 mediates the inhibition of endothelial cell migration and tube formation by TSP-1 (Dawson et al. Mot. Pharmacol. 55: 332-338, 1999; Dawson et al. J. Cell Biol. 138: 707-717, 1997).

[0010] Native 450 kDa TSP-1 has potent anti-angiogenic and antitumorigenic effects, but its large size and complex multiple functions limit its usefulness as a therapeutic in humans. It is generally known that certain peptide fragments of TSP- 1 as well as certain synthetic peptides whose structure is analogous to that of these peptide fragments show an activity similar to native TSP-1. Smaller TSP-1 proteins (e.g., a polypeptide including a TSP-1 domain or portion thereof, e.g., a Fc3TSR fusion polypeptide where the 3TSR peptide is fused to the C-terminus of each of the two heavy chains of the Fc region of human IgGl), have been studied.

[0011] Russel et al. (FASEB J. 2015 Feb; 29(2): 576-588. Published online 2014 Nov 13. doi: 10.1096 / fj .14-261636: 10.1096 / fj.14-261636) described that the treatment of EOC cells with a recombinant version of the thrombospondin- 1 type I repeats (3TSR) induced more apoptotic cell death in vitro compared to untreated controls. In vivo, tumors were induced in an orthotopic, syngeneic mouse model of advanced stage EOC and treated with 3TSR (4 mg / kg per day) alone or in combination with chemotherapy drugs delivered with maximum tolerated dose or metronomic scheduling. Pretreatment with 3TSR induced tumor regression, normalized tumor vasculature, and improved uptake of chemotherapy drugs. Combination 3TSR and metronomic chemotherapy induced the greatest tumor regression (6.2-fold reduction in size compared to PBS treated controls) and highest survival when treatment was initiated at advanced stage. 3TSR binding to its receptor, CD36 (cluster of differentiation 36), increased binding of CD36 and SHP-1, which significantly inhibited phosphorylation of the VEGF receptor.

[0012] Matuszewska et al. (Gynecologic Oncology 164 (2022) 154-169) demonstrated that Fc3TSR significantly decreased the viability and proliferative potential of EOC cells and endothelial cells in vitro compared to native 3TSR. High-resolution imaging followed by image correlation spectroscopy demonstrated enhanced clustering of the CD36 receptor in cells treated with Fc3TSR.31420P-ACP-W001This was associated with enhanced downstream signaling and greater in vitro and in vivo cellular responses. Fc3TSR induced greater vascular normalization and disease regression compared to native 3TSR in an orthotopic, syngeneic mouse model of advanced stage ovarian cancer.

[0013] International Patent Application Number PCT / FR2015 / 052133 published February 4, 2016, discloses a particular oligopeptide whose sequence is deduced from a part of the Thrombospondin type 1 repeat (TSR) motifs present in SCO-spondin, a glycoprotein specific to the central nervous system and present in all vertebrates. SCO-spondine is a large molecule composed of over 4500 amino acids including 26 TSR motifs. It is known that certain peptides derived from SCO-spondin TSR motifs demonstrate biological activity on nerve cells as described in the International Patent Application Publication Number. WO 1999 / 03890 published January 28, 1999, and M. Bambad et al., Cell & Tissue Research, Vol. 315, 15-25 (2004).

[0014] The TSR motifs are protein domains of about 55 residues, based on the alignment of conserved amino acids cysteine, tryptophan, and arginine. These motifs were first isolated in TSP- Iwhich is a molecule involved in coagulation. They have been described in many other molecules with diverse biological functions such as cell attachment, mobility, proliferation, cell aggregation, protease modulation, or inhibition of angiogenesis.

[0015] US Patent Serial Numbers: US6, 239,110 issued May 29, 2001; US5,840,692 issued November 24, 1998; US5, 849, 701 issued December 15, 1998; US5, 491, 130 issued February 13,1996; US6, 051, 549 issued April 18, 2000; US5, 190, 918 issued March 2, 1993; US5,200,397 issued April 6, 1993; and US6,384,189 issued May 7, 2002, describe the structure and the effects of peptide fragments derived from TSP-1 and its synthetic analogues.

[0016] Peptide derivatives mimicking the effects of TSP-1, in particular ABT-510 and ABT- 526, were studied clinically with disappointing results (Haviv F, et al. , Thrombospondin- 1 mimetic peptide inhibitors of angiogenesis and tumor growth: design, synthesis, and optimization of pharmacokinetics and biological activities. J. Med. Chem. 48(8): 2838-2846, 200). ABT-510 was studied in sarcomas and renal cell carcinomas, however, the efficacy of ABT-510 was insufficient (Baker LH, et al., Randomized, phase II study of the thrombospondin- 1 -mimetic angiogenesis inhibitor ABT-510 in patients with advanced soft tissue sarcoma. J Clin Oncol. 26(34): 5583-5588, 2008; Ebbinghaus S, et al., Phase 2 study of ABT-510 in patients with previously untreated advanced renal cell carcinoma. Clin Cane Res. 3(22 Pt l):6689-95, 2007).41420P-ACP-W001

[0017] As recombinant proteins have come to play a larger and larger role in research as well as diagnostics and therapeutics, numerous issues have developed around the efficient production and purification of full recombinant proteins. A significant issue is the inadvertent production of truncated proteins. According to Jennings et al., undesirable truncated recombinant protein products pose a special expression and purification challenge because such products often share similar chromatographic properties as the desired full-length protein. (Protein Expr Purif. 2016 May; 121 : 17-21. doi: 10.1016 / j.pep.2015.12.001). In some systems that rely on the random insertion of the genetic code for the target protein into a genome (often using a viral vector, for example), such problems can be significant and can impact efficacy. In fact, such issues may lead to the erroneous conclusion that a recombinant protein is not effective when in fact the issue is the presence of truncated versions of the protein. This issue is present in the production of Fc3TSR according to the methods previously used and such issues may be solved, or at least ameliorated, by use of the constructs and methods according to this invention.

[0018] The instant disclosure provides in some embodiments, an effective and well-tolerated specific peptide, which overcomes certain disadvantages of existing anti-cancer therapies, or at least provides a useful alternative thereto.

[0019] This background information is provided to reveal information believed by the applicant to be of possible relevance. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art or forms part of the general common knowledge in the relevant art.SUMMARY OF THE INVENTION

[0020] The following presents a simplified summary of the general inventive concept(s) described herein to provide a basic understanding of some aspects of the disclosure. This summary is not an extensive overview of the disclosure. It is not intended to restrict key or critical elements of embodiments of the disclosure or to delineate their scope beyond that which is explicitly or implicitly described by the following description and claims.

[0021] The present invention comprises nucleic acid and amino acid constructs and methods for producing purified novel compositions comprising fusion polypeptides of the Fc3TSR type. The Fc3TSR compositions according to the instant disclosure comprise one or more nucleic acid51420P-ACP-W001constructs comprising a DNA sequence coding for 3TSR (three type-l-repeat regions of thrombospondin- 1) and hereinafter termed “MOL608” in the instant disclosure. Furthermore, methods of killing cancer cells utilizing the MOL608 compositions as herein described are also provided. As used hereinafter the terms “MOL608 composition” or, as applicable, “MOL608 compositions”, shall be understood to refer to nucleic acid constructs of the instant disclosure and one or more proteins resultant from such nucleic acid constructs shall be termed “MOL608 compositionresultant protein”, or as applicable, “MOL608 composition-resultant proteins”.

[0022] In one aspect, there is provided a deoxyribonucleic acid (DNA) construct comprising SEQ ID NO: 2 or SEQ ID NO: 4, encoding for a protein comprising three thrombospondin- 1 type 1 repeats (3TSR), or a portion of such protein comprising 3TSR.

[0023] In another aspect, there is provided a mammalian cell comprising a DNA construct comprising SEQ ID NO: 2 or SEQ ID NO: 4, encoding for a protein comprising three thrombospondin- 1 type 1 repeats (3TSR), or a portion thereof. In some embodiments, the DNA sequence of the instant disclosure (encoding for MOL608 composition) is stably inserted into the genome of the mammalian cell at least 5 times. In some embodiments, the mammalian cell comprises a rodent cell, a primate cell, a canine cell, a feline cell, or a human cell. In some embodiments, a full- length protein (MOL608 composition-resultant protein) from the DNA sequence is produced in the mammalian cell. In some embodiments, a majority of the protein resultant from the DNA sequence is full-length protein (MOL608 composition-resultant protein of the instant disclosure). In some embodiments, substantially all of the protein resultant from the DNA sequence is full-length protein (MOL608 composition-resultant protein).

[0024] In another aspect, there is provided a protein comprising three thrombospondin- 1 type 1 repeats (3TSR), produced using a DNA construct comprising SEQ ID NO: 2 or SEQ ID NO: 4 (encoding for the MOL608 composition-resultant protein or a portion thereof). In some embodiments, there is provided a protein comprising three thrombospondin- 1 type 1 repeats (3TSR) (MOL608 composition-resultant protein) produced as disclosed herein using a mammalian cell, where the mammalian cell comprises a DNA construct comprising SEQ ID NO: 2 or SEQ ID NO: 4.

[0025] In another aspect, there is provided a deoxyribonucleic acid (DNA) construct comprising SEQ ID NO: 3.61420P-ACP-W001

[0026] In another aspect, there is provided a method of treating cancer comprising administering a composition including a MOL608 composition-resultant protein as disclosed herein. In some embodiments of the method, the cancer is ovarian cancer. In some embodiments of the method, the cancer is pancreatic cancer. In some embodiments of the method, the cancer is a solid tumor cancer.

[0027] In another aspect, there is provided an anti-cancer agent comprising a MOL608 composition-resultant protein made using cells comprising numerous full length functional copies of the 3TSR gene resultant from the MOL608 composition being inserted into the genome of the production cells using the construct of SEQ ID NO: 3.

[0028] In another aspect, there is a provided a use of a composition comprising a protein resultant from a deoxyribonucleic acid (DNA) construct comprising at least one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, encoding for a protein comprising three thrombospondin- 1 type 1 repeats (3TSR), or a portion thereof, as an anti-cancer agent.

[0029] In another aspect, there is provided a use of a protein comprising three thrombospondin- 1 type 1 repeats (3TSR), resultant from a deoxyribonucleic acid (DNA) construct comprising at least one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, for the manufacture of a medicament for the treatment of cancer.

[0030] In some embodiments of the aforementioned uses, the cancer is ovarian cancer, pancreatic cancer, and / or a solid tumor cancer.

[0031] In another aspect, there is provided a pharmaceutical composition comprising a MOL608 composition-resultant protein secreted or extracted from production cells transfected with numerous full length functional copies of a deoxyribonucleic acid (DNA) construct comprising at least one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, and a pharmaceutically acceptable excipient.

[0032] In another aspect, there this provided pharmaceutical composition comprising a MOL608 composition deoxyribonucleic acid (DNA) construct vector comprising at least one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, and a pharmaceutically acceptable excipient.71420P-ACP-W001

[0033] In another aspect, there is provided a method of treating solid tumor cancer, comprising administering to a patient an effective amount of a composition including a protein comprising three thrombospondin- 1 type 1 repeats (3TSR), where such protein is resultant from a deoxyribonucleic acid (DNA) construct comprising at least one of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0034] In some embodiments, the solid tumor cancer is ovarian cancer, or pancreatic cancer is a solid tumor cancer.

[0035] In one aspect, there is provided a deoxyribonucleic acid (DNA) construct comprising at least one of SEQ ID NO: 2 or SEQ ID NO: 4, or portion thereof.

[0036] In another aspect, there is provided mammalian cell comprising a DNA construct having a DNA sequence comprising at least one of SEQ ID NO: 2 or SEQ ID NO: 4.

[0037] In some embodiments of the mammalian cell, the DNA sequence is stably inserted into the genome of said mammalian cell at least 5 times. In some embodiments of the mammalian cell, the is a rodent cell, a primate cell, or a human cell.

[0038] In some embodiments of the mammalian cell, a full-length protein resultant from is produced in the mammalian cell.

[0039] In some embodiments of the mammalian cell, a majority of protein resultant from the DNA sequence is full-length protein.

[0040] In some embodiments of the mammalian cell, substantially all of the protein resultant from the DNA sequence is full-length protein.

[0041] In yet another aspect, there is provided a protein produced using a DNA construct of at least one of SEQ ID NO: 2 or SEQ ID NO: 4.

[0042] In another aspect, there is provided a protein produced using the mammalian cell comprising a DNA construct of at least one of SEQ ID NO: 2 or SEQ ID NO: 4

[0043] In another aspect, there is provided a deoxyribonucleic acid (DNA) construct comprising SEQ ID NO: 3.81420P-ACP-W001

[0044] In yet another aspect, there is provided a use of a composition comprising a protein resultant from a deoxyribonucleic acid (DNA) construct comprising at least one of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a portion thereof, as an anti-cancer agent. In some embodiments, the cancer is at least one of ovarian cancer, pancreatic cancer, or a solid tumor cancer.

[0045] In yet another aspect, there is provided a use of a protein resultant from a deoxyribonucleic acid (DNA) construct comprising at least one of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, for the manufacture of a medicament for the treatment of cancer. In some embodiments, the cancer is at least one of ovarian cancer, pancreatic cancer, or a solid tumor cancer.

[0046] In yet another aspect, there is provided a pharmaceutical composition comprising a MOL608 composition-resultant protein secreted from production cells transfected with numerous functional copies of a deoxyribonucleic acid (DNA) construct comprising at least one of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 being inserted into the genome of the production cells; and a pharmaceutically acceptable excipient.

[0047] In yet another aspect, there is provided a pharmaceutical composition comprising a MOL608 composition deoxyribonucleic acid (DNA) construct vector comprising at least one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; and a pharmaceutically acceptable excipient.

[0048] In another aspect, there is provided method of treating solid tumor cancer, comprising administering to a patient an effective amount of a composition comprising a protein resultant from a deoxyribonucleic acid (DNA) construct comprising at least one of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments of the method, the solid tumor cancer is ovarian cancer or pancreatic cancer.

[0049] Other aspects, features and / or advantages will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying figures.91420P-ACP-W001BRIEF DESCRIPTION OF THE FIGURES

[0050] Several embodiments of the present disclosure will be provided, by way of examples only, with reference to the appended figures, wherein:

[0051] Figure l is a depiction of an exemplary deoxyribonucleic acid (DNA) construct used to express a MOL608 composition-resultant protein (based on SEQ ID NO: 1) of the present disclosure, in a CHO-K1 cell line, in accordance with one embodiment;

[0052] Figure 2 is a depiction of an exemplary expression and secretion plasmid map (designated pGFTR) comprising SEQ ID NO: 3 used to transfect CHO-K1 cells with the DNA construct shown in Figure lof the present disclosure, in accordance with one embodiment;

[0053] Figure 3 is a photograph of an exemplary western blot of the expression of a MOL608 composition-resultant protein of the present disclosure in CHO-K1 cells and using the pGFTR plasmid, in accordance with one embodiment;

[0054] Figure 4 is a fluorescent microscope photograph of an exemplary fluorescent in situ hybridisation (FISH) experiment of the CHO-K1 cells used for Figure 3, demonstrating the stably integrated and amplified DNA sequence encoding for the MOL608 composition-resultant protein disclosed herein, in accordance with one embodiment;

[0055] Figure 5 is a photograph of an exemplary SDS-PAGE gel electrophoresis of a purified MOL608 composition-resultant protein purified from the culture medium of GFTR cells shown in Figure 3, in accordance with one embodiment;

[0056] Figure 6 is a photograph of an exemplary western blot of a purified MOL608 composition-resultant protein of the instant disclosure from GFTR cells, wherein purified fractions were confirmed with Western blotting using an anti-human-HRP / ECL system, in accordance with one embodiment;

[0057] Figure 7 is a depiction of an exemplary deoxyribonucleic acid (DNA) construct used to express a MOL608 composition-resultant protein (based on SEQ ID NO: 6) of the instant disclosure for use in an IFTR cell line (optionally in a pIFTR plasmid vector), in accordance with one embodiment;101420P-ACP-W001

[0058] Figure 8 is a depiction of an exemplary expression and secretion plasmid map (designated pIFTR) used to transfect CH0-K1 cells with the DNA construct shown in Figure 7, in accordance with one embodiment;

[0059] Figure 9 is a photograph of an exemplary western blot of the expression of a MOL608 composition-resultant protein of the instant disclosure in CHO-K1 cell lines and using the pIFTR plasmid, in accordance with one embodiment;

[0060] Figure 10 are fluorescent microscope photographs of an exemplary FISH experiment of the CHO-K1 cells expressing a MOL608 composition-resultant protein used in the exemplary western blot of Figure 9, demonstrating a stably integrated and amplified DNA sequence encoding for the MOL608 composition-resultant protein of the instant disclosure, in accordance with one embodiment;

[0061] Figure 11 is a photograph of an exemplary SDS-PAGE gel electrophoresis of a purified MOL608 composition-resultant protein of the instant disclosure purified from the culture medium of CHO-K1 cells and using the pIFTR plasmid of Figure 8, as shown in Figure 10, in accordance with one embodiment;

[0062] Figure 12 is a photograph of an exemplary western blot of purified MOL608 composition-resultant protein CHO-K1 cells and using the pIFTR plasmid of Figure 8, as shown Figure 9, wherein purified fractions (F1-F4) were confirmed with Western blotting using an anti- human-HRP / ECL system;

[0063] Figure 13 is an exemplary graph of the percent cytotoxicity in murine ovarian cancer cells (specifically, Mouse Ovarian Surface Epithelial, “ID8 cells”) following in vitro treatment with MOL608 composition-resultant protein of the instant disclosure, repeated in triplicate, showing dose dependent cytotoxicity, in accordance with one embodiment;

[0064] Figure 14 is a series of exemplary images of the murine ovarian cancer cells (specifically, ID8 cells) associated with the graph of Figure 13, after exposure to crystal violet dye, visually illustrating dose dependent cytotoxicity at different concentrations of MOL608 composition-resultant protein, in accordance with one embodiment;

[0065] Figure 15 is an exemplary graph of the percent cytotoxicity in murine pancreatic cancer cells (specifically, KPC mouse cells, “KPC cells”) following in vitro treatment with MOL608 111420P-ACP-W001composition-resultant protein of the instant disclosure, repeated in triplicate, showing dose dependent cytotoxicity, in accordance with one embodiment;

[0066] Figure 16 is a series of exemplary images of the pancreatic cancer cells (specifically, KPC cells) associated with the graph of Figure 15, after exposure to crystal violet dye, visually illustrating dose dependent cytotoxicity at different concentrations of MOL608 compositionresultant protein, in accordance with one embodiment;

[0067] Figure 17 is an exemplary graph of the percent cytotoxicity in primary lung adenocarcinoma cancer cells (specifically, HDC canine mouse cells, “HDC cells”) following in vitro treatment with MOL608 composition-resultant protein of the instant disclosure, repeated in triplicate, showing dose dependent cytotoxicity, in accordance with one embodiment;

[0068] Figure 18 is an exemplary graph of the percent cytotoxicity in primary lung adenocarcinoma cancer cells (specifically, LuBi canine mouse cells, “LuBi cells”) following in vitro treatment with MOL608 composition-resultant protein of the instant disclosure, repeated in triplicate, showing dose dependent cytotoxicity, in accordance with one embodiment;

[0069] Figure 19 is an exemplary graph of the percent cytotoxicity in canine melanoma cancer cells (specifically, CML1 cells, “CML1 cells”) following in vitro treatment with MOL608 composition-resultant protein of the instant disclosure, repeated in triplicate, showing dose dependent cytotoxicity, in accordance with one embodiment;

[0070] Figure 20 is an exemplary graph of the percent cytotoxicity in canine melanoma cancer cells (specifically, CML6M cells, “CML6M cells”) following in vitro treatment with MOL608 composition-resultant protein of the instant disclosure, repeated in triplicate, showing dose dependent cytotoxicity, in accordance with one embodiment; and

[0071] Figure 21 is an exemplary graph of the percent cytotoxicity in canine osteosarcoma cancer cells (specifically, DHARMA cells, “DHARMA cells”) following in vitro treatment with MOL608 composition-resultant protein of the instant disclosure, repeated in triplicate, showing dose dependent cytotoxicity, in accordance with one embodiment.121420P-ACP-W001REFERENCE TO SEQUENCE LISTINGS SUBMITTED ELECTRONICALLY

[0072] The contents of the sequence listing submitted electronically, with reference made in the specification of the instant application to the SEQ ID NOs. listed therein is as follows:

[0073] SEQ ID NO: 1 is an amino acid sequence resultant from the inclusion of the nucleotide sequence of SEQ ID NO: 2 in the DNA construct represented in Figure 1, in an exemplary embodiment as disclosed herein;

[0074] SEQ ID NO: 2 is a DNA nucleotide sequence (MOL608 composition) included in the construct represented in Figure 1, in an exemplary embodiment as disclosed herein;

[0075] SEQ ID NO: 3 is a DNA nucleotide sequence (MOL608 composition) included in the construct represented in Figure 2, in an exemplary embodiment as disclosed herein;

[0076] SEQ ID NO: 4 is a DNA nucleotide sequence (MOL608 composition) included in the construct represented in Figure 7, in an exemplary embodiment as disclosed herein;

[0077] SEQ ID NO: 5 is a DNA nucleotide sequence (MOL608 composition) included in the construct represented in Figure 8, in an exemplary embodiment as disclosed herein;

[0078] SEQ ID NO: 6 is an amino acid sequence of the protein (MOL608 compositionresultant protein) produced by an exemplary MOL608 composition nucleic acid construct (MOL608 composition) in one exemplary embodiment as disclosed herein; and

[0079] SEQ ID NO: 7 is an amino acid sequence of the protein (MOL608 compositionresultant protein) produced by an exemplary MOL608 composition nucleic acid construct (MOL608 composition) in another exemplary embodiment as disclosed herein.DETAILED DESCRIPTION OF THE INVENTION

[0080] Various implementations and aspects of the specification will be described with reference to details discussed below. The following description, figures and sequence listings are illustrative of the specification and are not to be construed as limiting the specification. Numerous specific details are described to provide a thorough understanding of various implementations of the present specification. However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of implementations of the present specification.131420P-ACP-W001

[0081] Various methods and processes will be described below to provide examples of implementations of the disclosure disclosed herein, or otherwise examples of experimental data. No implementation or experimental data described below limits any claimed implementation and any claimed implementations may cover processes or methods that differ from those described below. The claimed implementations are not limited to methods or processes having all of the features of any one method or process described below or to features common to multiple or all of the methods or processes described below. It is possible that a method or process described below is not an implementation of any claimed subject matter.

[0082] Furthermore, numerous specific details are set forth in order to provide a thorough understanding of the implementations or examples described herein. However, it will be understood by those skilled in the relevant arts that the implementations described herein may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the implementations described herein.

[0083] It is understood that for the purpose of this specification, language of “at least one of X, Y, and Z” and “one or more of X, Y and Z” may be construed as X only, Y only, Z only, or any combination of two or more items X, Y, and Z (e.g., XYZ, XY, YZ, ZZ, and the like). Similar logic may be applied for two or more items in any occurrence of “at least one ...” and “one or more...” language.

[0084] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0085] Embodiments of the present invention are based in part on novel nucleic acid constructs, cell lines and methods of making and using these to make MOL608 composition-resultant fusion proteins, such as those of SEQ ID NO: 6 and SEQ ID NO: 7 that are surprisingly efficacious in treating solid tumor cancers such as epithelial ovarian cancer and pancreatic cancer, without limitation.

[0086] Embodiments feature polynucleotides encoding one or more (e.g., 1, 2, 3, or 4 or more) of the polypeptides (e.g., MOL608 composition-resultant fusion proteins) of the instant disclosure. Figure 1 depicts a particularly useful polynucleotide construct according to one embodiment of the instant disclosure, as shown for example at 10. Polynucleotide sequences141420P-ACP-W001encoding one or more polypeptides of the instant disclosure may be obtained using standard recombinant techniques.

[0087] The instant disclosure features vectors including one or more of the polynucleotides as disclosed herein. For example, a polynucleotide (SEQ ID NO: 2) of the instant disclosure as shown in Figure 1 at 10 is inserted into the pGFTR plasmid of Figure 2 as shown, for example at 10, (comprising SEQ ID NO: 3), in one embodiment.

[0088] In one embodiment, the novel construct according to the exemplary DNA construct depicted in Figure 1 (comprising SEQ ID NO: 2) and the exemplary DNA plasmid vector depicted Figure 2 (comprising SEQ ID NO: 3), used the human growth hormone (GH) secretion signal peptide to secrete a MOL608 composition-resultant protein of the instant disclosure into the culture medium. The pGFTR plasmid was then transfected into CHO-K1 cells using a transfection protocol known to those of skilled in the art and, for example, described in Example 1. Individual cell lines were constructed carrying and expressing the MOL608 composition gene construct resultant from transfection with the MOL608 composition of the instant disclosure. The pGFTR plasmid was transfected into the hamster genome. The proteins produced by this plasmid (see Figure 1) are surprisingly efficacious as demonstrated in Example 6 and Figures 13 to 16.

[0089] The GFTR cell line showed a high level of expression as demonstrated by exemplary Western Blotting experiments (Figure 3). The GFTR cell line, with reference to Figure 3 shows, in the lane labelled as GFTR, strong expression of the band at ~53 kDa which represents a MOL608 composition-resultant protein of the instant disclosure while the weaker band at 42 kDa represents beta-actin as a loading and internal control. The lane labeled as CHO-K1 represents the parental cell line and it does not show any MOL608 composition-resultant protein, but beta-actin is present as an internal control for total protein loading.

[0090] With regard to Figure 4, the green staining indicates the MOL608 composition (pGFTR plasmid) and the blue staining shows 4',6-diamidino-2-phenylindole (DAPI) counterstaining of hamster chromosomes in a fluorescence in situ hybridization (FISH) experiment. The FISH experiment of Example 2 and Figure 4 shows the presence of pGFTR plasmid which ensures long-term and stable protein production of a MOL608 composition-resultant protein of the instant disclosure into the culture medium.151420P-ACP-W001

[0091] In another embodiment, depicted in Figures 7 and 8, the secretion signal peptide is that of the hamster IgK (“IgK signal peptide”).

[0092] The instant disclosure also features, in some embodiments, vectors including one or more of the polynucleotides as disclosed herein. For example, a polynucleotide of the embodiment of Figure 7 comprises a MOL608 composition, as shown for example at 12, (based on SEQ ID NO: 6) being inserted into the pIFTR plasmid of Figure 8 (comprising SEQ ID NO: 5), as shown, for example at 12.

[0093] The novel construct according to the instant disclosure as depicted in Figure 8 which includes SEQ ID NO: 5, in this embodiment, uses the IgK (I) secretion signal peptide to secrete a MOL608 composition-resultant protein of the instant disclosure into the culture medium.

[0094] The IFTR cell line showed a high level of expression as demonstrated by exemplary Western Blotting experiments (Figure 9). The IFTR cell line on Figure 9 shows, in the lanes labeled IFTR, the strong expression of the band at ~53 kDa which represents a MOL608 compositionresultant protein of the instant disclosure while the weaker band at 42 kDa represents beta-actin as a loading and internal control. The lane labeled as CHO-K1 represents the parental cell line and it does not show MOL608 composition-resultant protein, but beta-actin is present as an internal control for total protein loading.

[0095] The exemplary FISH experiment of Example 4 and Figure 10 shows the presence of DNA sequence(s) stemming from the pIFTR plasmid (green signal) on a chromosome in the IFTR cell line. The green staining is of the DNA sequence(s) associated with the MOL608 composition of the instant disclosure, while the blue staining shows DAPI counterstaining of hamster chromosomes. This stable gene integration ensures long-term and stable protein production of a MOL608 composition-resultant protein of the instant disclosure into the culture medium, in this embodiment.

[0096] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, methods, and kits of the present invention, or indeed the nucleotide sequences herein described, without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.161420P-ACP-W001EXAMPLES

[0097] The present invention is illustrated by the following examples, which are in no way intended to be limiting of the invention.EXAMPLE 1. CHO-K1 CELL LINE PRODUCTION OF MOL6O8-COMPOSITION RESULTANT PROTEIN ENCODED BY A MOL608 COMPOSITION OF THE INSTANT DISCLOSURE WITH GFTR

[0098] A well-performing DNA sequence for expression of a MOL608 composition-resultant protein of the instant disclosure was determined, synthesized in a pUC plasmid, and transferred into an appropriate insertion vector as described herein for insertion into CHO-K1 cells. An exemplary plasmid, comprising SEQ ID NO: 3 is shown in Figure 2. This plasmid was then used to establish stable CHO-K1 cell lines transfected with an amplified MOL608 composition. CHO-K1 cells were seeded into a 60 mm Petri dish at approximately 200,000 cells, and on the following day, transfected with 6 pg of a MOL608 composition-containing plasmid utilizing a TurboFect™ reagent (see https: / / www.thermofisher.eom / order / catalog / product / R0532# / R0532, accessed May 31, 2021). The insertion plasmid was diluted with 600 pl of serum-free Dulbecco's Modified Eagle Medium (DMEM), and the reagent was mixed thoroughly by vortexing. 12 pL of the reagent was added to the insertion plasmid solution, which was then mixed by pipetting and incubated at room temperature for 20 minutes. This transfection mix was added evenly, drop by drop, to the cells in the 60 mm Petri dish, each containing 4 ml of serum-containing DMEM medium.

[0099] After 24 hours, antibiotic selection was initiated with the addition of 10 pg / ml puromycin. Three days later, the selection medium was exchanged for fresh medium, and after another three days, the process was repeated. Following an additional three days, the selection medium was replaced with fresh medium containing 50 pg / ml puromycin, and three days later, this was exchanged for medium containing 100 pg / ml puromycin.

[0100] The cells were grown in this selection medium until confluency, with regular medium exchanges. Once confluency was reached, the cells were split into five 60 mm Petri dishes by trypsinization. Four of these dishes were frozen in liquid nitrogen, each in a separate vial, while one dish was continued in culture. The growing cells were analyzed by lysing and purifying the total protein, and Western blotting (Figure 3) was used to detect production of the protein encoded by the171420P-ACP-W001MOL608 composition.

[0101] Cells were also examined using FISH experiments for the presence of nucleic acid derived from the insertion plasmid (Figure 4). Subsequently, cells were grown in serum-free medium, and the protein encoded by the MOL608 composition was purified from the medium. The concentration of this protein was then determined using a spectrophotometer, high-performance liquid chromatography (HPLC), or similar procedures.EXAMPLE 2. MOL608 COMPOSITION-RESULTANT PROTEIN PURIFICATION

[0102] The protein encoded by a MOL608 composition of the instant disclosure was purified from the supernatant of GFTR cells, which were grown in a 10 cm dish until the growing area of the 10 cm Petri dish was full, and the culture medium was removed. The cells were washed with 5 ml lx phosphate-buffered saline (PBS) (four times) and then put into 10 ml serum-free culture medium and cultured in this medium for three days. Serum-free culture medium was collected from the cells in a 15 ml centrifuge tube and centrifuged at 3800 rpm (2390 ref) for 10 minutes to remove cell debris. The supernatant was transferred into a fresh 15 ml tube, and a lx Protease inhibitor cocktail was added to the solution. The supernatant was stored in a 4-degree Celsius freezer. Of the supernatant, 7.2 ml was used for protein purification with Protein G SpinTrap™ columns (GE Healthcare™, 28903134). Since the MOL608 composition-resultant protein is secreted into the culture medium of GFTR cells, four fractions were eluted from the column (Fl, F2, F3, F4). As seen in Figure 5, the fractions are very clean and other proteins (and shorter truncated proteins) are barely visible (99% purity, the experiment is intentionally overexposed). Each fraction has a volume of 430 microliter, and we loaded 50-50 microliter of purified protein from the 430 microliter onto this Coomassie® Brilliant Blue stained SDS-PAGE protein gel, which is directly showing the proteins encoded by a MOL608 composition of the instant disclosure. Gel was run under reducing conditions (10% acrylamide gel). Purified fractions were checked using Western blotting experiments by using an anti-human-HRP / ECL system. 20 microliter purified protein (from total 430 microliter) was run on each lane. As depicted in Figure 6, the Western blotting confirmed that the proteins encoded by a MOL608 composition and with high purity because the Western blotting does not reflect any truncated proteins (GFTR: F1-F4 lanes).181420P-ACP-W001EXAMPLE 3. USE OF MOL608 COMPOSITION IN IFTR

[0103] Using the same methods detailed in Examples 1 and 2, a MOL608 compositionresultant protein was produced and purified utilizing the pIFTR constructs shown in Figures 7 and 8. In the IFTR construct depicted in Figure 7, the hamster IgK secretion signal peptide was incorporated to facilitate secretion of the MOL608 composition-resultant protein (SEQ ID NO: 6) into the culture medium. The pIFTR plasmid was transfected into CHO-K1 cells, leading to the generation of individual cell lines carrying and expressing the MOL608 composition gene construct.

[0104] The protocol began by seeding approximately 20,000 CHO-K1 cells into a well of a 24-well plate, and transfecting them the next day with 1 pg of the MOL608 composition-containing plasmid using a TurboFect™ reagent (see https: / / www.thermofisher.eom / order / catalog / product / R0532# / R0532, accessed May 31, 2021). The plasmid was diluted in 100 pl of serum-free DMEM, and the reagent was thoroughly mixed via vortexing, after which 2 pl were added to the plasmid solution. This mixture was mixed by pipetting and incubated at room temperature for 20 minutes before being distributed evenly, dropwise, into the culture wells of the 24-well plate, each containing 1 ml serum-containing DMEM. After 24 hours, cells were harvested using TrypLE™ Select reagent, suspended in about 40 ml of DMEM before being redistributed into two 96-well plates. Accordingly, approximately 200 pl of DMEM having the cells suspended there was added to each of the 192 wells.

[0105] Antibiotic selection commenced 24 hours later with 10 pg / ml puromycin. After three days, the selection medium was replaced with fresh medium, and this process was repeated after another three days. Following another three-day incubation, the growing cell clones were collected from the 96-well plates using TrypLE™ Select and transferred into individual wells of a 24-well plate. After 24 hours, antibiotic selection was intensified to 50 pg / ml puromycin, and three days later, the selection medium was again exchanged, this time for medium containing 100 pg / ml puromycin.

[0106] The resulting cell lines were analyzed by lysing the cells, purifying the total protein, and using Western blotting (as in Figure 9) to verify production of the MOL608 protein. The highest producing cell lines were also subjected to FISH analysis for the presence of nucleic acids from the insertion plasmid, as shown in the embodiment of Figure 4. Further selection of optimal clones was achieved through increased antibiotic pressure, with cells growing at 100 pg / ml puromycin being subcultured into three wells of a 24-well plate. Surviving clones underwent re-examination by191420P-ACP-W001Western blotting for protein production and FISH for transgene presence. Top producing clones were subsequently expanded in serum-free media, with the encoded protein purified from the medium. The concentration of this protein was determined by spectrophotometry, HPLC, or similar analytical techniques, enabling the selection and purification of the highest-yielding cell lines and the corresponding MOL608 composition-resultant protein.

[0107] As seen in Figure 9, the IFTR cell line exhibited a high level of expression on Western blot analysis, with a strong band at approximately 53 kDa corresponding to the MOL608 protein and a weaker band at 42 kDa representing the beta-actin loading and internal control. Additional non-specific bands, attributable to protein overloading and secondary antibody recognition were also present. The protein was then purified as discussed in Example 5.EXAMPLE 4. pIFTR CHROMOSOMAL INSERTION

[0108] The IFTR cell line showed the presence of the transgenes (green signal) in one embodiment, as depicted in Figure 10 which demonstrates an exemplary FISH experiment. Nucleotides encoding proteins of the MOL608 composition of the instant disclosure are stained green and bright blue staining with DAPI is a counterstaining of hamster chromosomes. This stable gene integration ensures long-term and stable protein production of a MOL608 composition-resultant protein of the instant disclosure into the culture medium.EXAMPLE 5. IFTR MOL608 COMPOSITION-RESULTANT PROTEIN PURIFICATION

[0109] The MOL608 composition-resultant protein was purified from the supernatant of IFTR cells grown in a 10 cm Petri dish. Cells were grown until the dish was full and then the culture medium was removed and the cells were washed with 5 ml IxPBS four times. 10 ml of serum-free culture medium was added to the cells and the cells were cultured for three days. The serum-free culture medium from the cells was collected into a 15 ml centrifuge tube and centrifuged at 3800 rpm (2390 ref) for 10 minutes to remove cellular debris. The supernatant was transferred into a fresh 15 ml tube, and lx Protease inhibitor cocktail was added to the solution. The supernatant was stored at 4-degrees Celcius. 10 ml of supernatant from the cells was collected and 7.2 ml was used for protein purification. Protein G SpinTrap™ columns (GE Healthcare®, 28903134) were used according to the protocol that was included with this product. Since a MOL608 compositionresultant protein is secreted into the culture medium of IFTR cells, four fractions were eluted from the column (Fl, F2, F3, F4) as shown in Figure 11. The fractions are very clean and other proteins 201420P-ACP-W001are barely visible (99% purity was achieved - the experiment is intentionally overexposed). Each fraction had a volume of 430 microliter and 50 microliter of purified protein from the 430 microliter was loaded onto a Coomassie® Brilliant Blue stained SDS-PAGE protein gel as depicted. The gel was run under reducing conditions (10% acrylamide gel).

[0110] Results were confirmed with Western blotting using an anti-human-HRP / ECL system. 20 microliter purified protein (from 430 microliter) was used for each lane. See results in Figure 12. Western blotting showed that highly purified MOL608 composition-resultant proteins of the instant disclosure had been obtained (IFTR: F1-F4 lanes of Figure 17).EXAMPLE 6. MOL608 COMPOSITION-RESULTANT PROTEIN IS A POTENT INHIBITOR OF EPITHELIAL OVARIAN AND PANCREATIC CANCER CELL GROWTH

[0111] Cancer cells were seeded in 96-well plates (1 x 104cells / well in triplicate). The cells were cultured in serum-free media alone, or were exposed to a MOL608 composition-resultant protein of the instant disclosure at varying doses (0, 200, 400, 600, 800, 1000 nmol) for 24 hours. Following treatment incubation, murine ovarian cancer cells (ID8 cells) and murine pancreatic cancer cells (KPC cells) were exposed to crystal violet dye that binds to proteins and DNA of living cells. The dye binds to living cells, not to dead cells. After binding, the dye creates a colorimetric reaction and the intensity of the color is read on a plate reader to determine a percentage of cell viability (See C. Sanjai, S.S. Hakimane, B.R. Guru, S.L. Gaonkar, A Comprehensive Review on Anticancer Evaluation Techniques, Biorganic Chemistry, 142: 2024). After a 4 hour incubation, the colorimetric reaction was read at excitation and emission wavelengths of 560 nm and 590 nm, respectively. Exemplary images of murine ovarian cancer (ID8 cells) cells and murine pancreatic cancer cells (KPC cells) which were exposed to crystal violet dye after MOL608 compositionresultant protein exposure at different concentrations (0, 200, 400, 600, 800, 1000 nmol) are provided as Figures 14 and 16. This assay was repeated in triplicate. Data was graphed as a percent of cell viability following treatment as depicted in Figures 13 and 15. As reflected in Figure 13, the MOL608 composition-resultant protein was shown to be cytotoxic to ID8 cells in vitro, with cancer cell cytotoxicity increasing in a dose dependent manner, wherein the half maximal inhibitory concentration (IC50) was noted at 457 nM, in this embodiment. A concentration of approximately 500 nM MOL608 composition-resultant protein yielded approximately 60% cytotoxicity in ID8 cells, whereas a concentration of approximately 1000 nM MOL608 composition-resultant protein yielded approximately 90% cytotoxicity in ID8 cells with a high statistical significance, as shown at *p<0.05, 211420P-ACP-W001and ****p<0.0001 in Figure 13. As reflected in Figure 15, the MOL608 composition-resultant protein was shown to be cytotoxic to KPC cells in vitro, with cancer cell cytotoxicity increasing in a dose dependent manner wherein an IC50 was noted at400 nM in this embodiment. A concentration of approximately 500 nM MOL608 composition-resultant protein yielded approximately 70% cytotoxicity in KPC cells, whereas a concentration of approximately 1000 nM MOL608 compositionresultant protein yielded approximately 100% cytotoxicity in KRC cells with a high statistical significance, as shown at *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 in Figure 15. For further clarity, in both Figures 15 and 16, the asterixis reflect the calculated p-values for the examples, with * reflecting p<0.05, ** reflecting p<0.01, *** reflecting p<0.001 and **** reflecting p<0.0001. Accordingly, the MOL608 compositions as disclosed herein have been shown to be stably transfected into various cell types and produce MOL608 composition-resultant protein.

[0112] The MOL608 composition-resultant proteins as disclosed herein have been further shown to be effective in inducing cancerous cell cytotoxicity and may provide useful agents and methods for the amelioration and treatment of various type of cancers.EXAMPLE 7: MOL608 COMPOSITION-RESULTANT PROTEIN IS POTENT INHIBITOR OF CELL GROWTHIN VARIOUS SOLID TUMOR CELL LINES

[0113] Various solid tumor cancer cells were seeded in 96-well plates (1 x 104cells / well in triplicate). The cells were cultured in serum-free media alone, or were exposed to a MOL608 composition-resultant protein of the instant disclosure at varying doses (0, 200, 400, 600, 800, 1000, 1200 nmol) for 24 hours. Similar to the experiments of Example 6, following treatment incubation the cells were exposed to crystal violet dye that binds to proteins and DNA of living cells. This assay was repeated in triplicate for each of the various cell lines. Data was graphed as a percent of cell viability following treatment as depicted in Figures 17 to 21. As reflected in Figures 17 to 21, the MOL608 composition-resultant protein was shown to be cytotoxic solid tumor cancer cell line in vitro, with cancer cell cytotoxicity increasing in a dose dependent manner.

[0114] The cytotoxic effects of MOL608 composition-resultant protein were tested in the following diverse solid tumor cell lines: canine primary lung adenocarcinoma cells (HDC cells), canine primary lung adenocarcinoma cells (LuBi cells), canine melanoma cells (CML1 cells), canine melanoma cells (CML6M cells), and canine osteosarcoma cells (DHARMA cells). The results of the experiments of these embodiments are shown in Figures 17 to 21.221420P-ACP-W001

[0115] Figures 17 and 18 show the dose-dependent effects of in vitro treatment with MOL608 composition-resultant protein on canine primary lung adenocarcinoma cells (HDC cells) and canine primary lung adenocarcinoma cells (LuBi cells). The MOL608 composition-resultant protein, as shown in the graph of Figure 17, is cytotoxic to HDC cells in a dose-dependent manner with an IC50 of 464.50 nM and is cytotoxic to LuBi cells in a dose-dependent manner with an IC50 of 333.33 nM as shown in the graph of the embodiment of Figure 18.

[0116] Figures 19 and 20 show the dose-dependent effects of in vitro treatment with MOL608 composition-resultant protein on canine melanoma cells (CML1 cells) and canine melanoma cells (CML6M cells). The MOL608 composition-resultant protein, as shown in the graph of the embodiment of Figure 19, is cytotoxic to CML1 cells in a dose-dependent manner with an IC50 of 1348.80 nM and is cytotoxic to CML6M cells in a dose-dependent manner with an IC50 of 1490.63 nM as shown in the graph of the embodiment of Figure 20.

[0117] Figure 21 shows the dose-dependent effects of in vitro treatment with MOL608 composition-resultant protein on canine osteosarcoma cells (DHARMA cells). The MOL608 composition-resultant protein, as shown in the graph of the embodiment of Figure 21, is cytotoxic to DHARMA cells in a dose-dependent manner with an IC50 of 987.76 nM.

[0118] Accordingly, Figures 17 to 21 show that the MOL608 composition-resultant protein is cytotoxic to various solid tumor cells.OTHER EMBODIMENTS

[0119] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[0120] While the present disclosure describes various embodiments for illustrative purposes, such description is not intended to be limited to such embodiments. On the contrary, the applicant's teachings described and illustrated herein encompass various alternatives, modifications, and equivalents, without departing from the embodiments, the general scope of which is defined in231420P-ACP-W001the appended claims. Except to the extent necessary or inherent in the processes themselves, no particular order to steps or stages of methods or processes described in this disclosure is intended or implied. In many cases the order of process steps may be varied without changing the purpose, effect, or import of the methods described.

[0121] Information as herein shown and described in detail is fully capable of attaining the above-described object of the present disclosure, the presently preferred embodiment of the present disclosure, and is, thus, representative of the subject matter which is broadly contemplated by the present disclosure. The scope of the present disclosure fully encompasses other embodiments which may become apparent to those skilled in the art, and is to be limited, accordingly, by nothing other than the appended claims, wherein any reference to an element being made in the singular is not intended to mean "one and only one" unless explicitly so stated, but rather "one or more." All structural and functional equivalents to the elements of the above-described preferred embodiment and additional embodiments as regarded by those of ordinary skill in the art are intended to be encompassed by the present claims. Moreover, no requirement exists for a system or method to address each and every problem sought to be resolved by the present disclosure, for such to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. However, that various changes and modifications in form, material, work-piece, and fabrication material detail may be made, without departing from the spirit and scope of the present disclosure, as set forth in the appended claims, as may be apparent to those of ordinary skill in the art, are also encompassed by the disclosure.241420P-ACP-W001

Claims

CLAIMSWhat is claimed is:

1. A deoxyribonucleic acid (DNA) construct comprising at least one of SEQ ID NO: 2 or SEQ ID NO: 4, or portion thereof.

2. A mammalian cell comprising the DNA construct of claim 1, having a DNA sequence comprising SEQ ID NO: 2 or SEQ ID NO: 4.

3. The mammalian cell of claim 2, wherein the DNA sequence is stably inserted into the genome of said mammalian cell at least 5 times.

4. The mammalian cell of claim 3, wherein the mammalian cell is a rodent cell, a primate cell, a canine cell, a feline cell, or a human cell.

5. The mammalian cell of claim 4, wherein a full-length protein resultant from is produced in the mammalian cell.

6. The mammalian cell of claim 5, wherein a majority of protein resultant from the DNA sequence is full-length protein.

7. The mammalian cell of claim 6, wherein substantially all of the protein resultant from the DNA sequence is full-length protein.

8. A protein produced using the DNA construct according to claim 1.

9. A protein produced using the mammalian cell according to any one of claims 2 to 7.

10. A deoxyribonucleic acid (DNA) construct comprising SEQ ID NO: 3.

11. Use of a composition comprising a protein resultant from a deoxyribonucleic acid (DNA) construct comprising at least one of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a portion thereof, as an anti -cancer agent.

12. The use of claim 11, wherein the cancer is at least one of ovarian cancer, pancreatic cancer, or a solid tumor cancer.251420P-ACP-W00113. Use of a protein resultant from a deoxyribonucleic acid (DNA) construct comprising at least one of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, for the manufacture of a medicament for the treatment of cancer.

14. The use of claim 13, wherein the cancer is at least one of ovarian cancer, pancreatic cancer, or a solid tumor cancer.

15. A pharmaceutical composition comprising: a MOL608 composition-resultant protein secreted from production cells transfected with numerous functional copies of a deoxyribonucleic acid (DNA) construct comprising at least one of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 being inserted into the genome of the production cells; and a pharmaceutically acceptable excipient.

16. A pharmaceutical composition comprising: a MOL608 composition deoxyribonucleic acid (DNA) construct vector comprising at least one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; and a pharmaceutically acceptable excipient.

17. A method of treating a solid tumor cancer, comprising administering to a patient an effective amount of a composition comprising a protein resultant from a deoxyribonucleic acid (DNA) construct comprising at least one of: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

18. The method of claim 17, wherein the solid tumor cancer is ovarian cancer, or pancreatic cancer.261420P-ACP-W001

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