Anti-cancer N-terminal Fc conjugate immunotherapy

Abstract

The compounds for the prevention and treatment of cancer have a first binding peptide from a urokinase-type plasminogen receptor (uPAR) antagonist operably linked to an antibody peptide operably linked to a second binding peptide from a peptide sequence derived from endostatin or plasminogen.

Description

[Technical field]

[0001] The present invention relates to compounds for the prevention and treatment of cancer.

[0002] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority from U.S. Provisional Patent Application No. 63 / 318,225, filed March 9, 2022.

[0003] [Cross-references to related literature] This application is being filed concurrently with a sequence listing, entitled SEQUENCE LISTING 56219-3003, which has been coded to comply with WIPO standards. [Background technology]

[0004] Cancer is the leading cause of death worldwide, second only to cardiovascular disease. While leukemia (blood-born cancer) and melanoma (skin cancer) are generally well-treated by current treatments, solid tumors are unfortunately poorly treated. Thus, the proportion of deaths from solid tumors is a major concern for oncologists and a major unmet medical need.

[0005] Two fundamental processes are important with respect to solid tumors: angiogenesis and metastasis.

[0006] Angiogenesis is the process by which new blood vessels are extended to deliver more nutrients, however this natural process can be exploited to feed tumor growth in many solid tumor cancers. As a result, angiogenesis is a particularly important target for cancer treatment in solid tumors. However, under normal, non-pathological conditions, the body can use endogenous molecules, such as endostatin and plasminogen, to prevent uncontrolled angiogenesis.

[0007] Cancer metastasis is the leading cause of cancer morbidity and mortality, and may account for up to 90% of cancer deaths. Cancer survival rates have improved significantly over the years, but the improvement is mainly due to early diagnosis and inhibition of cancer growth. Progress in the treatment of cancer metastasis has been limited. Although newer generations of anticancer drugs (mainly neutralizing antibodies for growth factors and small molecule kinase inhibitors) have indirect effects on cancer metastasis in addition to direct effects on cancer growth, current treatments for cancer metastasis are mainly chemotherapy and radiotherapy.

[0008] In particular, there are currently limited options for treatments, including drugs and their use, that limit tumor angiogenesis and limit tumor metastasis of solid tumors. Summary of the Invention [Means for solving the problem]

[0009] The compounds for the prevention and treatment of cancer have a first binding peptide from a urokinase-type plasminogen receptor (uPAR) antagonist operably linked to the N-terminus of an Fc domain, with additional anti-angiogenic activity conferred by peptide sequences derived from endostatin and / or plasminogen fused to the C-terminus of the same Fc fusion protein.

[0010] The IgG may be an Fc fusion peptide, such as a fragment of a human Fc fusion peptide. The Fc fusion peptide may be an IgG protein. The Fc fusion protein may be selected from the group consisting of an IgG1 isotype Fc peptide, an IgG2 isotype Fc peptide, an IgG3 isotype Fc peptide, and an IgG4 isotype Fc peptide.

[0011] The first binding peptide may be an N-terminal fragment from uPAR. The first binding peptide may be only the heavy chain of the first binding peptide. The first binding peptide may be selected from the group consisting of EGF-like domain G, the N-terminal part of ATF from a urokinase-type plasminogen receptor (uPAR) antagonist, antibody 2G10, antibody ATN-658, antibody 8B12. The first binding peptide may be a fragment of the heavy and light sequences of an antibody that blocks uPAR signaling.

[0012] The second binding peptide may be selected from the group consisting of a fragment of endostatin, endostatin, a fragment of plasminogen, a P125 mutation, a kringle domain of plasminogen, kringle domains 1-5 of plasminogen, and kringle domain 5 from plasminogen. The second binding peptide may be a fragment of endostatin and a fragment of plasminogen.

[0013] There may be a linker peptide between the antibody peptide and the first binding peptide. There may be a linker peptide between the antibody peptide and the second binding peptide.

[0014] The compound may be a peptide encoded by a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. The compound may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a peptide encoded by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0015] The compound may be selected from the group consisting of G-Fc-Endostatin(P125A), ATN658-Fc-K5, Herceptin-Fc-ATF, Avastin-Fc-ATF, human IgG1 Fc-linker-ATF, ATF-linker-hIgG4 Fc-Kringle domain K5, ATF-linker-hIgG4 Fc-Endostatin(P125A), ATF-linker-hIgG4 Fc-Endostatin(P125A), Herceptin-Fc-K5, ATF-Fc(IgG4)-linker-Kringle domain 1-3, Herceptin-arrestin, ATF-huIgG4 Fc-arrestin, and ATN658-Endo.

[0016] Also taught are compounds that are substantially homologous to the compounds described above.

[0017] Also taught are nucleotide sequences that code for said compounds.

[0018] Also taught is a herceptin operably linked to an arrestin.

[0019] The compound may exhibit anti-cancer activity. Thus, a method of treating cancer comprising administering a composition comprising the compound of the present invention, and the use of the compound for treating cancer are also taught. The cancer may be a solid cancer. The method may inhibit metastasis. [Brief description of the drawings]

[0020] [Figure 1] A block diagram of a fusion of a uPAR inhibitor fused to the N-terminus of an IgG Fc domain and an anti-angiogenic sequence derived from endostatin and / or plasminogen fused to the C-terminus of the Fc domain is presented. A linker is optionally added between the different domains. [Diagram 2] Trace chromatograms of G-Fc-Endostatin (P125A) under reduced and non-reduced conditions are presented. [Diagram 3]Figure 3A) Presents the effect of G-Fc-Endostatin (P125A), also referred to as Construct 1, on tumor volume and metastasis compared to the anti-cancer drug Avastin (positive control) in a zebrafish model. Figure 3B) Presents the effect of G-Fc-Endostatin (P125A) on angiogenesis in another example. [Figure 4] SDS-PAGE analysis of ATN658-Fc-K5 is shown. [Diagram 5] 1 shows SDS-PAGE analysis of Herceptin-Fc-ATF. [Figure 6] 1 shows SDS-PAGE analysis of Avastin-Fc-ATF. [Figure 7] 1 shows SDS-PAGE analysis of human IgG1 Fc-linker-ATF. [Figure 8] 1 shows SDS-PAGE analysis of ATF-linker-hIgG4 Fc-Kringle domain K5. [Figure 9] FIG. 1 shows SDS-PAGE analysis of ATF-linker-hIgG4 Fc-Endostatin(P125A). [Figure 10] FIG. 1 shows SDS-PAGE analysis of ATF-linker-hIgG4 Fc-Endostatin(P125A). [Figure 11] 1 shows SDS-PAGE analysis of Herceptin-Fc-K5. [Figure 12] 1 shows SDS-PAGE analysis of ATF-Fc(IgG4)-linker-Kringle domains 1-3. [Figure 13] 1 shows SDS-PAGE analysis of Herceptin-arrestin. [Figure 14] 1 shows SDS-PAGE analysis of ATF-huIgG4-Fc-arrestin. [Figure 15] SDS-PAGE analysis of ATN658-endo is shown. [Figure 16] Graph showing normalized tumor size induced by various Avastin and Herceptin concentrations, and graph showing number of metastases induced by various Avastin and Herceptin concentrations. [Figure 17] FIG. 1 is a graph showing normalized tumor size produced by various ATF constructs. [Figure 18] FIG. 1 is a graph showing normalized tumor size produced by various Avastin and Herceptin concentrations, as well as a graph showing normalized tumor size produced by various constructs. [Figure 19] FIG. 1 is a graph showing normalized tumor size produced by various constructs. [Figure 20] FIG. 1 is a graph showing normalized tumor size produced by various constructs. [Figure 21] FIG. 1 is a graph showing the number of metastases induced by various constructs. [Figure 22] FIG. 1 is a graph showing the normalized tumor volume reduction conferred by various constructs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention relates to the combination of a urokinase-type plasminogen receptor (uPAR) antagonist with additional anti-angiogenic activity conferred by sequences derived from endostatin and / or plasminogen in the same Fc fusion protein.

[0022] The present invention uses sequences derived from endostatin or plasminogen in combination with uPA-uPAR interaction inhibitors, and proteins that target the uPAR / vitronectin interaction to improve the anti-cancer activity of molecules that simply inhibit uPAR activity.

[0023] The present invention uses the kringle domain of plasminogen fused to the C-terminus of an antibody.

[0024] The proteins used in the present invention are completely endogenous sequences and therefore there is low potential for unwanted immunogenicity to the treatment, which means that the proteins can be given repeatedly, even as mRNA.

[0025] Thus, the present invention also provides DNA, RNA and mRNA based treatments. DNA may be administered to reach and enter the damaged cells and express the protein of the present invention. Various delivery techniques may be used. For example, DNA may be placed into an artificial virus to deliver the DNA into a chromosome. Naked DNA approaches may also be used. Alternatively, a gene may be administered that expresses the required protein.

[0026] In somatic cell gene therapy (SCGT), therapeutic genes are introduced into any cell other than gametes, germ cells, gametocytes, or undifferentiated stem cells. Therapeutic DNA (either integrated into the genome or as an exogenous episome or plasmid) is used to treat disease.

[0027] In germline gene therapy (GGT), germ cells (sperm or eggs) are modified by the introduction of a functional gene into their genome. Modifying one of the germ cells can result in all living cells containing the modified gene. The change is therefore heritable and can be transmitted to future generations.

[0028] In in vivo gene therapy, a vector (typically a virus) is introduced into the patient, which then achieves the desired biological effect by delivering genetic material (e.g., for a therapeutic protein) into the patient's cells. In ex vivo gene therapy, such as CAR-T therapy, the patient's own cells (autologous) or healthy donor cells (allogeneic) are modified outside the body (hence ex vivo) with vectors expressing a particular protein, e.g., a chimeric antigen receptor.

[0029] The fusion protein constructs of the present invention have the advantage of being single-chain, as opposed to antibodies which have two chains (heavy and light chains), i.e. only one mRNA needs to be delivered.

[0030] The invention described herein relates to the combination of endostatin or plasminogen derived sequences (or fragments thereof) with an interposed Fc domain and a uPAR binding domain, resulting in novel anti-cancer drugs.

[0031] The N-terminal uPAR binding domain may consist of a sequence that blocks the binding of uPA to uPAR, such as a peptide ligand, an amino-terminal fragment from uPA (or a fragment thereof), or the responsible sequence of antibody 2G10. The uPAR binding domain may also consist of heavy and light sequences of an antibody that blocks uPAR signaling, such as those found in antibodies ATN-658 or 8B12.

[0032] A linker region is optionally added between the domain and the antibody Fc domain.

[0033] The isotype of the Fc domain (i.e., IgG1, 2, 3, or 4) may be selected if improved half-life and / or ADCC and complement activity is desired. For example, in some situations, an IgG1 isotype may be selected to increase the anti-cancer activity of the antibody by increasing ADCC. In other situations, an IgG4 isotype may be selected to decrease ADCC, which may allow for less toxicity, thereby allowing for higher doses. In some cases, an IgG3 Fc may be selected, which is intermediate in ADCC activity relative to either IgG1 or IgG4. Note that the Fc domain forms a natural homodimer.

[0034] The C-terminal anti-angiogenic domain is derived from endostatin (or a fragment thereof) and plasminogen kringle domain sequences, preferably the kringle 5 domain. The use of a combination of endostatin and plasminogen sequences together in the same molecule is also within the scope of the invention.

[0035] A variety of protein-based therapeutics have been utilized to block angiogenesis with the known endogenous sequences of endostatin and plasminogen.

[0036] Endostatin is a 20 kDa carboxyl-terminal fragment of collagen type XVIII, which is present in the walls of blood vessels and basement membranes and plays an important role in endothelial cell adhesion and cytoskeletal organization. Endogenous endostatin inhibits migration and induces apoptosis in endothelial cells, inhibits tumor growth, and impairs vascular maturation in wound healing. Endogenous endostatin is thought to counteract the pro-angiogenic activity of growth factors such as basic fibroblast growth factor and VEGF, which are known to inhibit at least 65 different tumor types. Moreover, studies measuring changes in gene expression in human dermal microvascular cells after treatment with endostatin have demonstrated a decrease in several pro-angiogenic pathways and an increase in many angiogenic genes.

[0037] Endostatin (trade name Endostar) is a protein that specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth.

[0038] In 2005, a novel recombinant human endostatin, Endostar, was approved by the Chinese State Food and Drug Administration (SFDA) for the treatment of non-small cell lung cancer. Thus, Endostar has a long history of human use, but surprisingly has never been approved in the United States or Europe.

[0039] The half-life of the protein in the body is only 10 hours. Therefore, Endostar is administered intravenously over 4 hours once per day for 14 days or more. This pharmacokinetic property necessarily causes inconvenience, affects quality of life, and may reduce patient compliance.

[0040] A more active form of endostatin (P125A) has been identified. Furthermore, it has been reported that the N-terminal fragment of endostatin possesses a significant portion of endostatin's activity. Along with wild-type endostatin, it has been reported that most of the effects of endostatin can be attributed to the N-terminal sequence (1-27) (Sjin RMTT, Satchi-Fainaro R, Birsner AE, Ramanujam VMS, Folkman J, Javaherian K. A 27-amino-acid synthetic peptide corresponding to the NH 2-terminal zinc-binding domain of endostatin is responsible for its antitumor activity. Cancer Res. 2005;65(9):3656-3663. doi:10.1158 / 0008-5472.CAN-04-1833).

[0041] Endostatin (P125A) has also been fused to the C-terminus of other proteins, such as Herceptin, and is an approved drug that blocks growth factor signals, improving the activity of the original Herceptin (Shin SU, Cho HM, Merchan J, et al. Targeted delivery of an antibody-mutant human endostatin fusion protein results in enhanced antitumor efficacy. Mol Cancer Ther. 2011;10(4):603-614. doi:10.1158 / 1535-7163.MCT-10-0804).

[0042] Plasminogen is an endogenous protein that can be cleaved by urokinase to become activated into plasmin, and contains five kringle (K) domains of ~80 residues each. Endogenous angiostatin is a 38 kDa amino-terminal fragment of plasminogen, and studies using recombinant angiostatin have demonstrated that tumor-inhibitory activity is conserved in the K1-3 domains. 8Angiostatin was first isolated from tumor-bearing mice and 6 , which has potent antiangiogenic and antiproliferative activity against endothelial cells and cancer cells 7 A larger fragment of plasminogen containing all five kringle domains (K1-5) may be more active than angiostatin alone (K1-3) (Cao R, Wu HL, Veitonmaki N, et al. Suppression of angiogenesis and tumor growth by the inhibitor K1-5 generated by plasmin-mediated proteolysis. Proc Natl Acad Sci US A. 1999;96(10):5728-5733. doi:10.1073 / pnas.96.10.5728).

[0043] Recent evidence supports two antitumor mechanisms for plasminogen derivatives, one affecting angiogenesis and the other directly targeting tumor cells (Sun Q, Xu Q, Dong X, et al. A hybrid protein comprising ATF domain of pro-UK and VAS, an angiogenesis inhibitor, is a potent candidate for targeted cancer therapy. Int J cancer. 2008;123(4):942-950. doi:10.1002 / IJC.23537).

[0044] Kringle 5 (K5), like angiostatin, is a by-product of the proteolytic cleavage of plasminogen. In a recent study, Ansell et al. demonstrated the function of K5 as a competitive antagonist of hepatocyte growth factor (HGF) (Cho HM, Rosenblatt JD, Kang YS, et al. Enhanced inhibition of murine tumor and human breast tumor xenografts using targeted delivery of an antibody-endostatin fusion protein. Mol Cancer Ther. 2005;4(6):956-967. doi:10.1158 / 1535-7163.MCT-04-0321).

[0045] HGF contains a kringle motif and promotes angiogenesis by stimulating the tyrosine kinase receptor Met. In addition to its potent angiogenic role, K5 may direct a potent antitumor response capable of recruiting tumor-associated neutrophils and natural killer T cells.

[0046] Thus, plasminogen has various kringle domains that appear to be active alone (angiostatin (K1-4) and K5) as well as in combination. Thus, the use of plasminogen-derived sequences, particularly the K5 domain, together with other anti-cancer proteins has been reported (Wang H, Yang Z, Gu J. Therapeutic targeting of angiogenesis with a recombinant CTT peptide-endostatin mimic-kringle 5 protein. Mol Cancer Ther. 2014;13(11):2674-2687. doi:10.1158 / 1535-7163.MCT-14-0266).

[0047] One of the major phenomena underlying metastasis is the proteolysis of the extracellular matrix (ECM), which promotes tumor cell invasion, migration, and homing to distant organs. Although several protease systems have been implicated in this process, a growing body of evidence has identified the urokinase-type plasminogen receptor (uPAR) system as a central player in mediating proteolysis from cancer invasion to metastasis. uPAR-mediated degradation of ECM is also crucial for the initiation of angiogenesis.

[0048] uPAR is a GPI-anchored cell membrane receptor that localizes the proteolytic activity of urokinase (uPA) to the cell surface. Its primary function is to focus the proteolytic activity of urokinase (uPA) on the cell surface, which is responsible for the degradation of ECM components. uPAR expression is increased in many human cancers, correlates with poor prognosis and early invasion and metastasis, and has been reported in as many as 80% of tumors. At some point, the uPA-UPAR complex is often internalized to degrade the complex, and unoccupied uPAR may be recycled back to the cell surface.

[0049] There are two broad mechanisms for inhibiting the function of UPAR; (1) uPA binding to uPAR and (2) preventing the cell signaling that can occur through UPAR, which involves both uPA binding and subsequent binding to co-receptors such as integrins.

[0050] Binding of uPA to uPAR contributes to the activation of plasminogen to plasmin, which in turn initiates a proteolytic cascade that degrades components of the extracellular matrix, thereby triggering the migration of tumor cells from the primary lesion to distant secondary organs.

[0051] uPA is often divided into two regions. The first region responsible for the binding of uPA to UPAR is the amino-terminal fragment (ATF). ATF can be further divided into two domains: the epidermal growth factor (EGF)-like domain and the kringle domain. The "G" domain is the EGF-like domain and "K" is the kringle domain. Although the majority of the binding occurs with the EGF-like domain alone, the entire ATF region, including both the EGF-like domain and the kringle domain, has stronger binding than the EGF-like domain alone. Additionally, short peptide sequences unrelated to the native ATF sequence have been identified that also compete for uPA binding to UPAR (Goodson RJ, Doyle M V., Kaufman SE, Rosenberg S. High-affinity urokinase receptor antagonists identified with bacteriophage peptide display. Proc Natl Acad Sci US A. 1994;91(15):7129-7133. doi:10.1073 / pnas.91.15.7129). Indeed, ATF-Fc fusions (Chinese Patent No. CN101050237) have been shown to reduce tumor growth and metastasis (Hu XW, Duan HF, Gao LH, et al. Inhibition of tumor growth and metastasis by ATF-Fc, an engineered antibody targeting urokinase receptor. Cancer Biol Ther. 2008;7(5):651-659. doi:10.4161 / cbt.7.5.5643).

[0052] An antibody (2G10) was identified from a phage display library that prevents the association of uPA with uPAR and has been reported to actually block the binding of uPAR to uPA (Duriseti S, Goetz DH, Hostetter DR, LeBeau AM, Wei Y, Craik CS. Antagonistic anti-urokinase plasminogen activator receptor(uPAR)antibodies significantly inhibit uPAR-mediated cellular signaling and migration. J Biol Chem. 2010;285(35):26878-26888. doi:10.1074 / jbc.M109.077677). Smaller unnatural peptides that inhibit uPA / uPAR interactions have been identified from combinatorial libraries as well.

[0053] The second domain contains the enzymatic activity of uPA, which is initially an inactive proenzyme and, upon activation, enzymatically cleaves plasminogen, activating it to plasmin, which mediates the degradation of the extracellular matrix.

[0054] When uPA binds to UPAR, the complex of the two proteins can also interact with other proteins (co-receptors) on the surface of cancer cells, such as integrins. The interaction of UPAR with these co-receptors, mostly through domain 3 of the three domains of UPAR, leads to the activation of cell signaling pathways that promote cancer progression, including metastasis.

[0055] Targeting the signaling aspect of UPAR function may be more effective in preventing metastasis by blocking the cell signaling events that drive cancer cells to metastasize, as opposed to therapeutic proteins that “compete” with uPA for occupancy of the UPAR receptor and prevent uPA binding to UPAR, and are “non-competitive” with uPA binding to UPAR. One example of this would be the binding sequences (CDRs) of the antibody ATN-658 (Mahmood N, Arakelian A, Khan HA, Tanvir I, Mazar AP, Rabbani SA. uPAR antibody(huATN-658)and Zometa reduce breast cancer growth and skeletal lesions. Bone Res. 2020;8(1). doi:10.1038 / S41413-020-0094-3). Another antibody (8B12), directed against conformation-specific uPAR, has been reported to reduce uPA binding to vitronectin, which can reduce tumor volume (Yuan C, Guo Z, Yu S, Jiang L, Huang M. Development of inhibitors for uPAR: blocking the interaction of uPAR with its partners. Drug Discov Today. 2021; 26(4):1076-1085. doi:10.1016 / j.drudis.2021.01.016).

[0056] There have been reports of combining inhibitors of uPA-uPAR (e.g., ATF) with inhibitors such as the protease inhibitor domain and the antiangiogenic functional domain of vasostatin, as well as other antibodies such as endostatin (P125A) fused to the C-terminus of Herceptin. The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art and, unless otherwise indicated, as described in the various general and more specialized references cited and discussed throughout this specification.

[0057] The terms "polypeptide," "peptide," and "protein" are typically used interchangeably herein to refer to a polymer of amino acid residues. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Each protein or polypeptide has a unique function. The present invention includes polypeptides and functional fragments thereof, as well as mutants and variants that have the same biological function or activity.

[0058] In some embodiments, polymer molecules (e.g., polypeptide or nucleic acid sequences) are considered to be "homologous" to one another if their sequences are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical.

[0059] In some embodiments, a fragment of a nucleic acid sequence is a fragment of an open reading frame sequence. In some embodiments, such a fragment encodes a polypeptide fragment (as defined herein) of a protein encoded by the open reading frame nucleotide sequence.

[0060] The term "nucleic acid fragment" as used herein refers to a nucleic acid sequence having a deletion. In some embodiments, a fragment of a nucleic acid sequence is a fragment of an open reading frame sequence. In some embodiments, such a fragment encodes a polypeptide fragment (as defined herein) of a protein encoded by the open reading frame nucleotide sequence.

[0061] The term "construct" refers to a nucleic acid sequence encoding a protein operably linked to a promoter and / or other control sequences.

[0062] The term "genomic sequence" refers to a sequence having non-contiguous open reading frames in which introns separate protein coding regions.

[0063] As used herein, the terms "encode" or "encoded," when used in the context of a particular nucleic acid, means that the nucleic acid may be the information necessary to direct the translation of a nucleotide sequence into a particular protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid that encodes a protein may contain non-translated sequences (e.g., introns) within the translated region of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA).

[0064] The term "percent sequence identity" or "identical" in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. For example, polynucleotide sequences can be compared using the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410(1990); Gish and States, Nature Genet. 3:266-272(1993)).

[0065] The terms "substantial homology" or "substantial similarity," when referring to a nucleic acid or a fragment thereof, indicate nucleotide sequence identity of at least about 70%, 80%, 85%, or at least about 90%, or at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases when optimally aligned with another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, as measured by any well-known algorithm of sequence identity, such as BLAST, discussed above.

[0066] As used herein, a "heterologous nucleic acid sequence" is any sequence located at a site in a genome that is not normally present. In some embodiments, the heterologous nucleic acid sequence is a native phage sequence, albeit from a different phage.

[0067] A particular nucleic acid sequence also encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequences explicitly indicated. Thus, a nucleic acid sequence encoding a protein sequence disclosed herein also encompasses its modified variants as described herein. Substantially similar nucleic acid fragments of the present invention may be characterized by the percent identity of the amino acid sequences they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly used by those skilled in the art.

[0068] "Operably linked" or "operably linked" expression control sequences refer to the association of expression control sequences in close proximity to a coding sequence of interest to control expression of the coding sequence of interest, and expression control sequences acting in trans or at a distance to control expression of the coding sequence.

[0069] A "coding sequence" or "open reading frame" is a sequence of nucleotides that encodes a polypeptide or protein. The ends of a coding sequence are denoted by a start codon and a stop codon. The present disclosure also includes natural, isolated, or recombinant nucleic acid sequences encoding a protein, as well as vectors and / or (host) cells comprising the coding sequence for that protein.

[0070] The term "Fc domain", "Fc peptide", or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the Fc region of a human IgG heavy chain is usually defined to extend from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that contains the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. For example, a subunit of an IgG Fc domain contains the IgG CH2 and IgG CH3 constant domains.

[0071] Fused or linked means that the components (eg, a Fab molecule and an Fc domain subunit) are linked by a peptide bond, either directly or via one or more peptide linkers.

[0072] Fragments and variants of the disclosed nucleotide sequences and the proteins encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and thus the protein encoded thereby. A fragment of a nucleotide sequence may encode a protein fragment that retains the biological activity of the native protein. Thus, the present disclosure relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequence encoded thereby.

[0073] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0074] The foregoing description and specific representative embodiments and details of the invention have been presented for purposes of illustration and description of the present invention. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. It will be apparent to those skilled in the art that modifications and variations may be made in the description without departing from the scope of the invention. EXAMPLES

[0075] Example 1 - Constructing G-Fc-Endostatin (P125A) The construct uses the N-terminal part of the ATF from uPA (UniProt P00749), specifically the EGF-like domain "G" (residues 21-69, in bold) fused to human Fc (of IgG3 isotype) via a linker (GGGGAGGGG) and then further linked (GGGAGGGG) to the endostatin protein (UniProt P39060, residues 1572-1754, in italics, with the P125A mutation). The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture to produce pure target molecule after protein A purification (Figure 2). The sequence is presented below in SEQ ID NO: 1.

[0076] [ka]

[0077] Cloning, expression and purification were carried out by methods known in the art.

[0078] Figure 2 shows the trace chromatograms of G-Fc-Endostatin (P125A) under reduced and non-reduced conditions. Under reduced conditions, the interaction between the two polypeptides is destroyed. However, under non-reduced conditions, the interaction is preserved. Thus, the two conditions can reveal protein-protein interactions. A single peak under both conditions indicates a stable single compound.

[0079] Example 2 - In vivo anti-cancer test using zebrafish model The zebrafish model is a remarkably simple yet predictive model for screening anticancer drugs and has gained widespread acceptance as a useful in vivo model.

[0080] (Zebrafish tumor xenograft (ZTX) model) Transgenic Tg(fli1:EGFP)y1 zebrafish embryos were cultured in E3 embryo medium containing 0.2 mM 1-phenyl-2-thiourea, also known as PTU, for 48 hours at 28° C. Unfertilized eggs or larvae that appeared unhealthy or showed any obvious developmental defects were excluded before the start of the procedure.

[0081] PC3 cancer cells were labeled with Dil red fluorescent stain. Approximately 700 (efficacy study) or 2500 (angiogenesis study) Dil-labeled PC3 cancer cells (red) were implanted subcutaneously into the perivitelline space of 2-day-old larvae ± test article at the indicated concentrations.

[0082] Larvae with inadvertent injection of tumor cells into the circulation or with incorrect implantation of tumors in the yolk rather than the perivitelline space were excluded from the study. Photographs of the primary tumors were taken immediately after injection. Tumor-containing embryos were cultured in E3 / PTU medium for 72 hours at 36°C. After incubation, photographs of the primary tumors and CVP were taken by using a red fluorescent filter. Larvae that died or were otherwise lost during the study were excluded from the final analysis. Embryos that survived 3 days after treatment were mounted, and images of the primary tumors and intratumoral blood vessels were taken using an upright Zeiss confocal LSM700.

[0083] (Analysis of tumor growth inhibition and metastasis) Images obtained immediately after implantation (day 0) and after 72 hours of culture (day 3) were analyzed by using in-house developed software. Tumor growth regression was calculated and normalized to the respective controls. Metastases were assessed by the software and counted manually.

[0084] (Analysis of tumor angiogenesis) Primary tumor images acquired in the red and green channels were analyzed using ImageJ v1.53f51. The areas of primary tumors and intratumoral blood vessels were measured. Tumor angiogenesis was calculated by normalizing the blood vessel area to the respective primary tumor area.

[0085] Avastin (bevacizumab) is a drug used to treat many types of cancer. It is a monoclonal antibody that functions as an angiogenesis inhibitor. It was approved in the United States for medical use in 2004.

[0086] Figure 3A presents the effect of G-Fc-Endostatin (P125A), also referred to as Construct 1, on tumor volume and metastasis compared to the anti-cancer drug Avastin (positive control) in a zebrafish model. Construct 1 was shown to be a better inhibitor of tumor volume and metastasis compared to vehicle and Avastin.

[0087] Figure 3B presents the effect of G-Fc-Endostatin (P125A) on angiogenesis in another example. Construct 1 was shown to be a more potent inhibitor of angiogenesis compared to vehicle and Avastin.

[0088] Significance (*p<0.05) was determined using a two-tailed Student's t-test.

[0089] Example 3 -Construction of ATN658-Fc-K5 The fusion protein uses the heavy chain ATF658 linked to a linker and then to K5. The molecule was made using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein was transiently transfected and expressed in mammalian cells with a chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain the bulk at high purity. The sequence is presented below in SEQ ID NO:2.

[0090] [ka]

[0091] Purity was determined by SEC-HPLC to be 99.6835%. Endotoxin: <1 EU / mg. Concentration was 1.2 mg / mL. Formulation buffer was PBS, pH 7.4.

[0092] Shown in Figure 4 is an SDS-PAGE analysis of ATN658-Fc-K5 under β-mercaptoethanol-reduced (lane 1) and non-reduced (lane 2) conditions. The gel was stained with Coomassie Blue for 30 min. As a result, the different reduced proteins (heavy and light chains) migrate similarly at around 63 kDa and 25 kDa, respectively. The non-reduced protein migrates at around 180 kDa.

[0093] Example 4 - Constructing Herceptin-Fc-ATF The fusion protein uses heavy chain Herceptin linked to an Fc linker and then to ATF. The molecule was made using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein was transiently transfected and expressed in mammalian cells with a chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain the bulk at high purity. The sequence is presented below in SEQ ID NO:3.

[0094] [ka]

[0095] Purity was determined by SEC-HPLC to be 99.6621%. Endotoxin: <1 EU / mg. Concentration was 2 mg / mL. Formulation buffer was PBS, pH 7.4.

[0096] Shown in Figure 5 is an SDS-PAGE analysis of the construct under β-mercaptoethanol-reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Blue for 30 min. As a result, the different reduced proteins (heavy and light chains) migrate similarly at around 63-75 kDa and around 25 kDa, respectively. The non-reduced protein migrates around 180 kDa.

[0097] Example 5 - Constructing Avastin-Fc-ATF The fusion protein uses the heavy chain Avastin linked to an Fc linker and then to ATF. The molecule was made using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein was transiently transfected and expressed in mammalian cells with a chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed with PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain the bulk at high purity. The sequence is presented below in SEQ ID NO: 4.

[0098] [ka]

[0099] Purity was determined by SEC-HPLC to be 98.2435%. Endotoxin: <1 EU / mg. Concentration was 2 mg / mL. Formulation buffer was PBS, pH 7.4.

[0100] Shown in Figure 6 is an SDS-PAGE analysis of the construct under β-mercaptoethanol-reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Blue for 30 min. As a result, the different reduced proteins (heavy and light chains) migrate similarly at around 63-75 kDa and around 25 kDa, respectively. The non-reduced protein migrates around 180 kDa.

[0101] Example 6 - Constructing human IgG1 Fc-linker-ATF The fusion protein uses a human IgG1 Fc-linker-ATF combination. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein was transiently transfected and expressed in mammalian cells with a chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain a high purity bulk. The sequence is presented below in SEQ ID NO:5.

[0102] [ka]

[0103] Purity was determined by SEC-HPLC to be 97.8344%. Endotoxin: <1 EU / mg. Concentration was 2 mg / mL. Formulation buffer was PBS, pH 7.4.

[0104] Shown in Figure 7 is an SDS-PAGE analysis of the constructs under β-mercaptoethanol-reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie blue for 30 min. As a result, the different reduced proteins migrate similarly, around 63-48 kDa, respectively. The non-reduced proteins migrate around 75-100 kDa.

[0105] <Example 7 - Constructing ATF-Linker-hIgG4 Fc-Kringle domain K5> The fusion protein uses ATF linked to a linker, then Fc, then kringle domain K5. The molecule was made using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein was transiently transfected and expressed in mammalian cells with a chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed with PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain a bulk with high purity. The sequence is presented below in SEQ ID NO:6.

[0106] [ka]

[0107] Purity was determined by SEC-HPLC to be 98.6250%. Endotoxin: <1 EU / mg. Concentration was 2 mg / mL. Formulation buffer was PBS, pH 7.4.

[0108] Shown in Figure 8 is an SDS-PAGE analysis of the constructs under β-mercaptoethanol-reduced (lane 1) and non-reduced (lane 2) conditions. The gel was stained with Coomassie Blue for 30 min. As a result, the different reduced proteins migrate similarly around 48-63 kDa. The non-reduced proteins migrate around 135-180 kDa.

[0109] Example 8 - Constructing ATF-linker-hIgG4 Fc-Endostatin (P125A) The fusion protein uses the heavy chain ATF linked to a linker, then to Fc, then to endostatin (P125A). The molecule was made using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein was transiently transfected and expressed in mammalian cells with chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain the bulk at high purity. The sequence is presented below in SEQ ID NO: 7.

[0110] [ka]

[0111] Purity was determined by SEC-HPLC to be 97.6569%. Endotoxin: <1 EU / mg. Concentration was 2 mg / mL. Formulation buffer was PBS, pH 7.4.

[0112] Shown in Figure 9 is an SDS-PAGE analysis of the constructs under β-mercaptoethanol-reduced (lane 1) and non-reduced (lane 2) conditions. The gel was stained with Coomassie Blue for 30 min. As a result, the different reduced proteins migrate similarly around 63-48 kDa. The non-reduced protein migrates around 135 kDa.

[0113] Example 9 - Constructing ATF-Linker-hIgG4 Fc-Endostatin (P125A) The fusion protein uses ATF linked to a linker, linked to Fc, and then linked to one of endostatin (p125A). The molecule was made using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein was transiently transfected and expressed in mammalian cells with a chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain a high purity bulk. The sequence is presented below in SEQ ID NO:8.

[0114] [ka]

[0115] Purity was determined by SEC-HPLC to be 99.6569%. Endotoxin: <1 EU / mg. Concentration was 2 mg / mL. Formulation buffer was PBS, pH 7.4.

[0116] Shown in Figure 10 is an SDS-PAGE analysis of the constructs under β-mercaptoethanol-reduced (lane 1) and non-reduced (lane 2) conditions. The gel was stained with Coomassie Blue for 30 min. As a result, the different reduced proteins migrate similarly around 48-63 kDa. The non-reduced protein migrates around 135 kDa.

[0117] Example 10 - Constructing Herceptin-Fc-K5 The fusion protein uses heavy chain Herceptin linked to Fc and then to K5. The molecule was made using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein was transiently transfected and expressed in mammalian cells with chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain the bulk at high purity. The sequence is presented below in SEQ ID NO: 9.

[0118] [ka]

[0119] Purity was determined by SEC-HPLC to be 99.0715%. Endotoxin: <1 EU / mg. Concentration was 2 mg / mL. Formulation buffer was PBS, pH 7.4.

[0120] Shown in Figure 11 is an SDS-PAGE analysis of the constructs under β-mercaptoethanol-reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie blue for 30 min. As a result, the different reduced proteins (heavy and light chains) migrate similarly, around 63-75 kDa, respectively. The non-reduced proteins migrate around 180-245 kDa.

[0121] <Example 11 - Constructing ATF-Fc(IgG4)-Linker-Kringle domain 1-3> The fusion protein uses ATF linked to Fc linked to a linker and kringle domains 1-3. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein was transiently transfected and expressed in mammalian cells with a chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain a bulk with high purity. The sequence is presented below in SEQ ID NO: 10.

[0122] [ka]

[0123] Purity was determined by SEC-HPLC and was greater than 90%. Endotoxin: <1 EU / mg. Concentration was 1.3 mg / mL. Formulation buffer was PBS, pH 7.4.

[0124] Shown in Figure 12 is an SDS-PAGE analysis of the constructs under β-mercaptoethanol-reduced (lane 1) and non-reduced (lane 2) conditions. The gel was stained with Coomassie Blue for 30 min. As a result, the different reduced proteins migrate similarly, around 63-75 kDa, respectively. The non-reduced proteins migrate around 135-180 kDa.

[0125] Example 12 - Constructing Herceptin-Arrestin The fusion protein uses heavy chain Herceptin linked to a linker and then to arrestin. The molecule was made using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein was transiently transfected and expressed in mammalian cells with a chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain the bulk at high purity. The sequence is presented below in SEQ ID NO: 11.

[0126] [ka]

[0127] Purity was greater than 90% as determined by SDS-PAGE. Endotoxin: <1 EU / mg. Concentration was 1.5 mg / mL. Formulation buffer was PBS, pH 7.4.

[0128] Shown in Figure 13 is an SDS-PAGE analysis of the constructs under β-mercaptoethanol-reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Blue for 30 min. As a result, the different reduced proteins (heavy and light chains) migrate similarly around 63-75 kDa.

[0129] Example 13 - Constructing ATF-huIgG4 Fc-arrestin The fusion protein uses ATF linked to an Fc linker and then to arrestin. The molecule was made using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein was transiently transfected and expressed in mammalian cells with a chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed with PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain a high purity bulk. The sequence is presented below in SEQ ID NO: 12.

[0130] [ka]

[0131] Purity was determined by SEC-HPLC to be 95.2801%. Endotoxin: <1 EU / mg. Concentration was 2 mg / mL. Formulation buffer was PBS, pH 7.4.

[0132] Shown in Figure 14 is an SDS-PAGE analysis of the constructs under β-mercaptoethanol-reduced (lane 1) and non-reduced (lane 2) conditions. The gel was stained with Coomassie Blue for 30 min. As a result, the different reduced proteins migrate similarly, around 63-75 kDa, respectively. The non-reduced protein migrates around 180 kDa.

[0133] Example 15 - Constructing anti-PLAUR recombinant antibody (clone ATN-658)-endostatin P125A The fusion protein uses ATN-658 conjugated with endostatin P125A. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein was transiently transfected and expressed in mammalian cells with a chemically defined culture medium. The protein was purified by Protein A affinity chromatography, dialyzed with PBS, concentrated (if necessary), and then 0.2 micron sterile filtered to obtain a high purity bulk. The sequence is presented below in SEQ ID NO: 13.

[0134] [ka]

[0135] Purity was determined by SEC-HPLC to be 97.26%. Endotoxin: <1 EU / mg. Concentration was 1 mg / mL. Formulation buffer was PBS, pH 7.4.

[0136] Shown in Figure 15 is the SDS-PAGE analysis of the construct under β-mercaptoethanol-reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie blue for 30 minutes. The theoretical molecular weights of the heavy and light chains associated with the endostatin P125A mutant protein are 70.29 kDa and 23.97 kDa, respectively. Here, in β-mercaptoethanol-reducing SDS-PAGE, the heavy chain associated with the endostatin P125A mutant protein migrates at around 63-75 kDA, and the light chain migrates at around 25 kDA.

[0137] Example 16 - In vivo anti-cancer test using zebrafish model The zebrafish model described in Example 2 was used, except that DU145 cells labeled with Dil red fluorescent stain were used. Approximately 700 Dil-labeled cancer cells were subcutaneously co-implanted with 2 mg / mL Avastin, Avastin-ATF, Herceptin, Herceptin-ATF, Herceptin-K5, and Herceptin-arrestin, and 1 mg / mL ATN658, ATN658 K5, ATN658 endostatin (P125A), ATF-Fc, ATF-Fc-kringle 1-3, ATF-Fc (IgG4)-endostatin (P125A), ATF-Fc-K5, and ATF-Fc-arrestin into the perivitelline space of 2-day-old larvae.

[0138] Data are presented as mean ± SEM and D'Agostino-Pearson omnibus normality test was performed followed by Kruskal-Wallis test, two-tailed Student's t test, or Mann-Whitney test as appropriate. Graphs and experimental data were obtained / analyzed by using GraphPad Prism v9.0.2.

[0139] In summary, the results showed that treatment with all the different constructs and Herceptin significantly reduced the primary tumor size compared to the negative control after 3 days of treatment. Avastin treatment showed a reduction in primary tumor size; however, this effect did not reach mathematical significance. The ATF-Fc(IgG4)-Endostatin(P125A) and ATF-Fc-K5 constructs showed the most significant reduction in primary tumor size compared to the negative control, more so than the other tested compounds.

[0140] The results also showed that the Avastin and Herceptin constructs had higher antitumor efficacy than Avastin and Herceptin treatment alone. Furthermore, the ATF-Fc(IgG4)-endostatin(P125A) and ATF-Fc-K5 constructs had higher antitumor efficacy than ATF-Fc alone.

[0141] [Research design] This study is divided into two parts: Part 1. Positive control testing Part 2. Construct validity assessment

[0142] DU-145 cells were used to evaluate the efficacy of the compounds. The study was assembled using 16 groups split over two days. DU-145 cells were selected due to their high expression of UPAR, HER2 and VEGF-A, providing an excellent basis for a positive control on which new constructs could be evaluated. The DU-145 cell line also had excellent engraftment in the zebrafish model, with around 50% tumor remaining 3 days after implantation and an average of 10-15 tumor cells spread per fish.

[0143] (Dose Selection) Cancer cells are co-injected with the test compound. The injection volume is typically 2-5 nL (usually around 3 nL) per embryo. To calculate the dose / embryo, the following formula is used: 3 x 10^-9 L x c(g / L) = ng / embryo

[0144] (Avastin) Weekly Avastin treatment (25 mg / kg) in mice was highly effective against DU-145 cells (Cancer Chemother Pharmacol (2009) 65:191-195).

[0145] In one zebrafish study using Avastin and Endostar (wild-type endostatin, but not the more active P125A mutant) with the A549 lung cancer cell line, bevacizumab was used at doses of 28 ng, 83 ng, and 250 ng per embryo; Endostar at doses of 11 ng, 33 ng, and 99 ng per embryo. In this study, significant inhibition of tumor fluorescence was observed at all doses for both agents, although the 33 ng dose of Endostar was less effective than adjacent doses (Scientific Reports (2018) 8: 15837). A U-shaped dosing curve has been reported in other studies using Endostar (Cancer Gene Therapy (2006) 13: 619-627).

[0146] In the previous Example 2, Avastin 0.25 mg / mL solution did not show any improvement in tumor volume reduction but did induce angiogenesis in PC3 cells. Therefore, the dose in this experiment is higher than 0.5 mg / mL in pre-implanted DU-145 cells, which corresponds to 1.5 ng / embryo.

[0147] (Herceptin) Nearly weekly Herceptin treatment (10 mg / kg) in mice showed a modest (not statistically significant) reduction in tumor volume using DU-145 cells (Cancer Biol. & Therapy (2020) 21: 463-475).

[0148] Herceptin is administered at a slightly higher level than Avastin (1 mg / mL) due to the decreased sensitivity of DU-145 cells to Herceptin compared to Avastin. This dose corresponds to 1.8 ng / embryo.

[0149] (ATN-658) This unapproved antibody has not been studied as intensively as Avastin or Herceptin; however, it has been tested in mice using PC3 xenograft cells and administered at 10 mg / kg twice a week - resulting in a significant reduction in tumor volume and metastases (Neoplasia (2010) 10:778-88).

[0150] When adjusted non-proportionally for zebrafish, this mouse dose would suggest using a 0.3 mg / mL dose (0.9 ng / embryo) when mixing with tumor cells prior to implantation.

[0151] (ATF-Fc fusion) In Example 2, 0.125 mg / mL of G-Fc-Endostatin (P125A) was the lowest and most potent dose for activity using PC3 cells, therefore this same dose was used for the ATF-Fc fusion (0.375 ng / embryo).

[0152] ATF is a combination of an EGF-like domain (G) and a kringle domain.

[0153] All compounds were co-injected with human tumor cells into zebrafish embryos. Primary tumor size was assessed immediately after implantation and 3 days after implantation. Metastasis was assessed 3 days after implantation. Anticancer efficacy was determined as the change in primary tumor size (i.e., tumor growth or reduction) 3 days after implantation and the number of tumor cells that spread to the distal caudal venous plexus (CVP).

[0154] Part 1. Positive control test Prior to this experimental design (Table 2), dose studies with Avastin and Herceptin were performed to confirm the validity of these positive controls (Table 1). In this study, DU-145 cells were tested at the planned doses and at 10 ng / embryo. Higher doses were selected from previous doses in zebrafish (Avastin) or mice (Herceptin).

[0155] [Table 1]

[0156] Part 2. Construct validity assessment

[0157] [Table 2]

[0158] [result] Part 1. Positive control test Prior to the efficacy study, dose studies with Avastin and Herceptin were conducted to confirm the efficacy of these positive controls and to determine the doses for the efficacy study.

[0159] The results showed that Herceptin treatment showed a significant reduction in borderline primary tumor size compared to the 3 mg / mL negative control. In contrast, Avastin treatment showed no effect on primary tumor size compared to the negative control (Figure 16).

[0160] Figure 16 also showed that after 3 days of treatment, the number of spread cancer cells was reduced in the presence of 3 mg / mL Avastin and 1 mg / mL Herceptin compared to the negative control; however, this effect did not reach mathematical significance. Figure 16 is an evaluation of the antitumor and antimetastatic efficacy of Avastin and Herceptin in DU145 cancer cells after 72 hours of exposure. Cancer cells were co-injected subcutaneously ± Avastin ± Herceptin into the perivitelline space of 2-day-old zebrafish embryos as described in this study design. Data are presented as mean ± SEM and two-tailed Student's t-test was performed.

[0161] Part 2. Construct validity assessment After Part 1, positive controls and concentrations of constructs to test in the efficacy evaluation were determined (see Table 2).

[0162] (Primary tumor) The results were first presented as a comparison of primary tumor size between different compounds by grouping them according to fusion constructs, i.e., ATF constructs, ATN constructs, Avastin and Herceptin constructs, and K5, Arrestin, and P125 constructs. Different constructs can be found in different graphs. All constructs were summarized and presented in a single graph where antitumor efficacy is compared to negative control.

[0163] (ATF Construct) The results showed that the ATF-Fc-K5 and ATF-Fc(IgG4)-endostatin(P125A) constructs showed a significant reduction in primary tumor size compared to other ATF constructs, while Avastin ATF, Herceptin ATF, ATF-Fc, ATF-Fc-arrestin, and ATF-Fc-kringle 1-3 showed similar, not significantly different, primary tumor size 3 days after treatment (FIG. 17).

[0164] Figure 17: Comparison of DU145 primary tumor size between different ATF constructs after 72 hours of exposure. Data are presented as mean ± SEM and D'Agostino-Pearson omnibus normality test was performed. Kruskal-Wallis test was performed followed by two-tailed Student's t-test or Mann-Whitney test as appropriate (p<0.0001). *p<0.05, **p<0.01, and ***p<0.001.

[0165] (Avastin and Herceptin Constructs) Figure 18 is a comparison of DU145 primary tumor size between different Avastin and Herceptin constructs after 72 hours of exposure. Data are presented as mean ± SEM.

[0166] (K5, arrestin, and P125A constructs) Primary tumor size after 3 days compared to K5 or arrestin construct treatment is shown in Figure 19. ATF-Fc(IgG4)-endostatin(P125A) showed significantly reduced primary tumor size after 3 days compared to ATN658endostatin(P125A) construct treatment (Figure 19). Data are presented as mean±SEM and D'Agostino-Pearson omnibus normality test was performed. Kruskal-Wallis test was performed followed by two-tailed Student's t-test or Mann-Whitney test as appropriate (p<0.0001). *p<0.05.

[0167] (All constructs) The results showed that treatment with all the different constructs and Herceptin significantly reduced the primary tumor size compared to the negative control after 3 days of treatment (Figure 20). On the other hand, Avastin treatment showed a reduction in the primary tumor size; however, this effect did not reach mathematical significance (Figure 20).

[0168] FIG. 20 shows that the ATF-Fc(IgG4)-Endostatin(P125A) and ATF-Fc-K5 constructs showed the most significant reduction in primary tumor size compared to the other tested compounds.

[0169] Figure 20 also shows that the Herceptin-Arrestin construct showed higher antitumor efficacy than Herceptin, Herceptin-ATF, and Herceptin-K5 constructs. Data are presented as mean ± SEM and D'Agostino-Pearson omnibus normality test was performed. Kruskal-Wallis test was performed followed by two-tailed Student's t-test or Mann-Whitney test as appropriate (p<0.0001). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0170] (Transfer) The results showed that, compared to the negative control, reduced metastatic spread of DU145 cells was observed in the presence of Avastin, Herceptin-K5, Herceptin-arrestin, ATF-Fc, and ATF-Fc-Kringle 1-3 treatment after 3 days, however, this effect did not reach mathematical significance (Figure 21).

[0171] (Conclusion) The Avastin and Herceptin constructs have higher antitumor efficacy than Avastin and Herceptin treatment alone. Furthermore, the ATF-Fc(IgG4)-Endostatin(P125A) and ATF-Fc-K5 constructs have higher antitumor efficacy than ATF-Fc alone.

[0172] All of the treatments evaluated reduce the spread of DU145.

[0173] Example 17 - In vivo anti-cancer test using zebrafish model The zebrafish model described in Example 16 was used.

[0174] Figure 22 shows that ATF-Fc-K5 and ATF-Fc-endostatin (P125A) constructs show improved normalized tumor volume reduction versus untreated controls. Data are presented as mean ± SEM and D'Agostino-Pearson omnibus normality test was performed. Kruskal-Wallis test was performed followed by two-tailed Student's t-test or Mann-Whitney test where appropriate (p<0.0001). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0175] While the present disclosure has been described, at least in part, in terms of a method, those skilled in the art will appreciate that the present disclosure is also directed to various components for implementing at least some of the aspects and features of the described method. The present disclosure may be embodied in other specific forms without departing from the subject matter of the claims. The described exemplary embodiments are to be considered in all respects merely illustrative and non-binding. Features selected from one or more of the above-described embodiments may be combined to create alternative embodiments not expressly described, and features suitable for such combinations are understood to be within the scope of the present disclosure.

Claims

1. a first binding peptide from a urokinase-type plasminogen receptor (uPAR) antagonist having uPAR antagonist activity, the first binding peptide being operably linked to the N-terminus of an immunoglobulin G (IgG) antibody peptide which is operably linked to a second binding peptide; - the second binding peptide from an endostatin-derived peptide sequence having endostatin activity or a plasminogen-derived peptide sequence having plasminogen activity, operably linked to the C-terminus of the immunoglobulin G (IgG) antibody peptide; A compound comprising:

2. The compound of claim 1 , wherein the IgG is a fragment of an Fc fusion peptide.

3. The compound of claim 1 , wherein the IgG is a fragment of a human Fc fusion peptide.

4. The compound of claim 2 , wherein the Fc fusion peptide is an IgG protein.

5. 3. The compound of claim 2, wherein the Fc fusion protein is selected from the group consisting of an IgG1 isotype Fc peptide, an IgG2 isotype Fc peptide, an IgG3 isotype Fc peptide, and an IgG4 isotype Fc peptide.

6. The compound according to any one of claims 1 to 5, wherein the first binding peptide comprises an N-terminal fragment of uPAR having uPAR activity.

7. The compound of claim 1 , wherein the first binding peptide comprises only the heavy chain of the first binding peptide.

8. The compound according to any one of claims 1 to 7, wherein the first binding peptide is selected from the group consisting of EGF-like domain G, the N-terminal part of ATF from a urokinase-type plasminogen receptor (uPAR) antagonist, antibody 2G10, antibody ATN-658, and antibody 8B12.

9. The compound according to any one of claims 1 to 8, wherein the first binding peptide comprises a fragment of the heavy and light sequences of an antibody that blocks uPAR signaling.

10. 10. The compound of any one of claims 1 to 9, wherein the second binding peptide is selected from the group consisting of a fragment of endostatin having endostatin activity, endostatin, a fragment of plasminogen having plasminogen activity, endostatin with a P125 mutation, the kringle domain of plasminogen, kringle domains 1 to 5 of plasminogen, and kringle domain 5 from plasminogen.

11. The compound of any one of claims 1 to 10, wherein the second binding peptide comprises a fragment of endostatin having endostatin activity and a fragment of plasminogen having plasminogen activity.

12. The compound of any one of claims 1 to 11, further comprising a linker peptide between the antibody peptide and the first binding peptide.

13. 13. The compound of claim 1, further comprising a linker peptide between the antibody peptide and the second binding peptide.

14. 14. The compound of any one of claims 1 to 13, comprising a peptide encoded by a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13.

15. A compound comprising herceptin operably linked to an arrestin.

16. A compound comprising ATN-658 operably linked to endostatin P125A.

17. A compound, including a compound that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a peptide encoded by a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13.

18. A compound selected from the group consisting of G-Fc-Endostatin (P125A), ATN658-Fc-K5, Herceptin-Fc-ATF, Avastin-Fc-ATF, human IgG1 Fc-linker-ATF, ATF-linker-hIgG4 Fc-Kringle domain K5, ATF-linker-hIgG4 Fc-Endostatin (P125A), ATF-linker-hIgG4 Fc-Endostatin (P125A), Herceptin-Fc-K5, ATF-Fc(IgG4)-linker-Kringle domain 1-3, Herceptin-arrestin, ATF-huIgG4 Fc-arrestin and ATN658-Endostatin (P125A).

19. A compound comprising a compound substantially homologous to a compound according to any one of claims 1 to 18 and having the activity of a compound according to any one of claims 1 to 18.

20. A nucleotide encoding a compound according to any one of claims 1 to 19.

21. 20. A compound according to any one of claims 1 to 19, exhibiting anti-cancer activity.

22. 22. A method of treating cancer comprising administering a composition comprising a compound according to any one of claims 1 to 19 and 21.

23. 22. Use of a compound according to any one of claims 1 to 19 and 21 for the treatment of cancer.

24. 22. A vector comprising a compound according to any one of claims 1 to 19 and 21.

25. 24. The method or use of claim 22 or 23, wherein the cancer is a solid cancer.

26. 22. A method for inhibiting metastasis in a subject with cancer, comprising administering to the subject an effective amount of a compound described in any one of claims 1 to 19 and 21.

27. 27. The method or use of any one of claims 22, 23, 25 and 26, further comprising one or more of the following steps: a) administering chemotherapy; b) administering radiation therapy; or c) administering one or more additional therapeutic agents.

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