Anti-cancer N-terminus Fc-conjugated immunotherapeutics

Abstract

A compound for preventing and treating cancer has a first binding peptide from a urokinase-type plasminogen receptor (uPAR) antagonist, the first binding peptide being operably linked to an antibody peptide, the antibody peptide being operably linked to a second binding peptide from an endostatin or plasminogen-derived peptide sequence.

Description

Technical Field

[0001] The present invention relates to compounds useful for preventing and treating cancer.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 318,225, filed on March 9, 2022.

[0004] Cross-references to related literature

[0005] This application is filed simultaneously with a sequence listing entitled SEQUENCE LISTING 56219-3003, the number of which complies with WIPO standards.

[0006] background

[0007] Cancer is the leading cause of death worldwide after cardiovascular disease. While current treatments work well for leukemias (blood-borne cancers) and melanomas (skin cancers) in most cases, solid tumors are unfortunately less effective. Mortality from solid tumors is therefore a major concern for oncologists and represents a major unmet medical need.

[0008] For solid tumors, two fundamental processes are important, in particular angiogenesis and metastasis.

[0009] Angiogenesis is the process by which new blood vessels grow to deliver more nutrients, but this natural process can be exploited in many solid tumor cancers for tumor growth. Therefore, it is a particularly important target for cancer treatment of solid tumors. However, in normal, non-pathological conditions, the body can use endogenous molecules such as endostatin and plasminogen to prevent uncontrolled angiogenesis.

[0010] Cancer metastasis is the main cause of cancer morbidity and mortality, and may account for 90% of cancer deaths. Although cancer survival rates have increased significantly over the years, this increase is mainly due to early diagnosis and inhibition of cancer growth. There has been limited progress in the treatment of cancer metastasis. Although a new generation of anticancer drugs (mainly growth factor neutralizing antibodies and small molecule kinase inhibitors) have an indirect effect on cancer metastasis in addition to a direct effect on cancer growth, the current treatment of cancer metastasis is mainly chemotherapy and radiotherapy.

[0011] In particular, currently limited treatment options include drugs and their use that limit tumor angiogenesis and limit tumor metastasis of solid tumors. SUMMARY OF THE INVENTION

[0013] 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, and have additional anti-angiogenic activity conferred by an endostatin and / or plasminogen-derived peptide sequence fused to the C-terminus of the same Fc fusion protein.

[0014] IgG can be a fragment of an Fc fusion peptide, such as a human Fc fusion peptide. The Fc fusion peptide can be an IgG protein. The Fc fusion protein can be selected from an IgG1 isotype Fc peptide, an IgG2 isotype Fc peptide, an IgG3 isotype Fc peptide, and an IgG4 isotype Fc peptide.

[0015] The first binding peptide can be an N-terminal fragment from uPAR. The first binding peptide can be only the heavy chain of the first binding peptide. The first binding peptide can be selected from EGF-like domain G, which is the N-terminal part of ATF from urokinase-type plasminogen receptor (uPAR) antagonist, antibody 2G10, antibody ATN-658, antibody 8B12. The first binding peptide can be a fragment of the heavy chain and light chain sequence of an antibody that blocks uPAR signaling.

[0016] 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 to 5 of plasminogen, Kringle domain 5 of plasminogen. The second binding peptide may be a fragment of endostatin and a fragment of plasminogen.

[0017] 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.

[0018] The compound may be a peptide encoded by the sequence 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 the 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.

[0019] The compound can be selected from G-Fc-Endostatin (P125A), ATN658-Fc-K5, Herceptin-Fc-ATF, Avastin-Fc-ATF, human IgG1 Fc-Linker-ATF, ATF-Linker-hIgG4 Fc-Klinger domain K5, ATF-Linker-hIgG4Fc-Endostatin (P125A), ATF-Linker-hIgG4 Fc-Endostatin (P125A), Herceptin-Fc-K5, ATF-Fc(IgG4)-Linker-Klinger domain 1-3, Herceptin-Inhibitor, ATF-huIgG4 Fc-Inhibitor and ATN658-Endo.

[0020] Compounds that are substantially homologous to the above compounds are also taught.

[0021] Nucleotides encoding chemical compounds are also taught.

[0022] Also taught is Herceptin operably linked to an inhibitory protein.

[0023] These compounds can exhibit anti-cancer activity. Methods of treating cancer are also taught, which include administering a composition comprising a compound of the invention, and the use of the compound for treating cancer. The cancer can be a solid cancer. These methods can inhibit metastases.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Block diagrams are provided for the fusion of uPAR inhibitors to the N-terminus of the IgG Fc domain and the fusion of anti-angiogenic sequences derived from endostatin and / or plasminogen to the C-terminus of the Fc domain. Linkers are optionally added between the different domains.

[0026] Figure 2 Chromatogram traces of G-Fc-endostatin (P125A) under reducing and non-reducing conditions are provided.

[0027] Figure 3A The effects of G-Fc-endostatin (P125A, also referred to as Construct 1) on tumor volume and metastasis in a zebrafish model compared to the anticancer agent Avastin (positive control) are provided.

[0028] Figure 3B The effect of G-Fc-endostatin (P125A) on angiogenesis in an independent experiment is presented.

[0029] Figure 4 SDS-PAGE analysis of ATN658-Fc-K5 is shown.

[0030] Figure 5 SDS-PAGE analysis of Herceptin-Fc-ATF is shown.

[0031] Figure 6 SDS-PAGE analysis of Avastin-Fc-ATF is shown.

[0032] Figure 7 SDS-PAGE analysis of human IgG1 Fc-linker-ATF is shown.

[0033] Figure 8 SDS-PAGE analysis of ATF-linker-hlgG4 Fc-Klinger domain K5 is shown.

[0034] Fig. 9 SDS-PAGE analysis of ATF-linker-hIgG4 Fc-endostatin (P125A) is shown.

[0035] Fig.10 SDS-PAGE analysis of ATF-linker-hIgG4 Fc-endostatin (P125A) is shown.

[0036] Fig.11 SDS-PAGE analysis of Herceptin-Fc-K5 is shown.

[0037] Fig.12 SDS-PAGE analysis of ATF-Fc(IgG4)-linker-Klinger domains 1-3 is shown.

[0038] Fig.13 SDS-PAGE analysis of Herceptin-arrestin is shown.

[0039] Fig.14 SDS-PAGE analysis of ATF-huIgG4-Fc-inhibitrin is shown.

[0040] Fig.15 SDS-PAGE analysis of ATN658-Endo is shown.

[0041] Fig.16 is a graph showing normalized tumor size affected by various concentrations of Avastin and Herceptin, and a graph showing the number of metastases affected by various concentrations of Avastin and Herceptin.

[0042] Fig.17 is a graph showing normalized tumor size affected by various ATF constructs.

[0043] Fig.18 is a graph showing the normalized tumor size affected by various concentrations of Avastin and Herceptin, and a graph showing the normalized tumor size affected by various constructs.

[0044] Fig.19 is a graph showing normalized tumor size affected by various constructs.

[0045] Fig. 20 is a graph showing normalized tumor size affected by various constructs.

[0046] Fig.21 is a graph showing the number of metastases affected by various constructs.

[0047] Fig. 22 is a graph showing the normalized tumor volume reduction affected by various constructs.

[0048] Details

[0049] The present invention relates to the combination of a urokinase-type plasminogen receptor (uPAR) antagonist with endostatin and / or plasminogen derived sequences conferring additional anti-angiogenic activity in the same Fc fusion protein.

[0050] The present invention uses a combination of endostatin or plasminogen derived sequences with inhibitors of the uPA-uPAR interaction and proteins that target the uPAR / vitronectin interaction to enhance the anticancer activity of molecules that inhibit only uPAR activity.

[0051] The present invention utilizes the Klinger domain of plasminogen fused to the C-terminus of an antibody.

[0052] The proteins used in the present invention are entirely endogenous sequences and therefore are less likely to produce deleterious immunogenicity to the therapeutic, meaning that the proteins should be able to be administered repeatedly, even as mRNA.

[0053] Therefore, the present invention also provides treatment schemes based on DNA, RNA and mRNA. DNA can be administered to reach damaged cells, enter cells and express the protein of the present invention. A variety of delivery techniques can be used. For example, DNA can be introduced into engineered viruses to deliver DNA into chromosomes. Naked DNA methods can also be used. Alternatively, genes that cause the expression of the desired protein can be administered.

[0054] In somatic cell gene therapy (SCGT), the therapeutic gene is transferred into any cell other than a gamete, germ cell, gametocyte, or undifferentiated stem cell. Therapeutic DNA (integrated into the genome or as an external episome or plasmid) is used to treat disease.

[0055] In germline gene therapy (GGT), germ cells (sperm or egg cells) are modified by introducing a functional gene into their genome. Modifying the germ cells results in all cells of the organism containing the modified gene. Therefore, the change is heritable and passed on to future generations.

[0056] In in vivo gene therapy, a vector (usually a virus) is introduced into the patient, which then achieves the desired biological effect by delivering genetic material (e.g., 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 (thus, ex vivo) using a vector that expresses a specific protein, such as a chimeric antigen receptor.

[0057] An advantage of the fusion protein constructs of the present invention is that they are single chain, unlike antibodies which have two chains (heavy and light), which means that only one mRNA needs to be delivered.

[0058] The present invention relates to combining endostatin or plasminogen derived sequences (or fragments thereof) with a uPAR binding domain and an intermediate Fc domain to produce novel anticancer agents.

[0059] The N-terminal uPAR binding domain can be derived from a sequence that blocks the binding of uPA to uPAR (such as a peptide ligand), from an amino terminal fragment of uPA (or a fragment thereof), or from the sequence responsible for antibody 2G10. The uPAR binding domain can also be derived from the heavy and light chain sequences of an antibody that blocks uPAR signaling, such as the heavy and light chain sequences found in antibodies ATN-658 or 8B12.

[0060] Optionally a linker region is added between the domain and the antibody Fc domain.

[0061] The isotype of the Fc domain (i.e., IgG1, IgG2, IgG3, or IgG4) can be selected according to the desired preference for improving half-life and / or ADCC and complement activity. For example, in some cases, the IgG1 isotype may be preferred to improve the anti-cancer activity of the antibody by increasing ADCC. In other cases, for example, the IgG4 isotype may be preferred to reduce ADCC, which may make toxicity less and thereby allow higher dosages. In some cases, the IgG3-Fc with ADCC activity between IgG1 or IgG4 may be preferred. Note that the Fc domain forms a natural homodimer.

[0062] The C-terminal anti-angiogenic domain is derived from endostatin (or a fragment thereof) and a plasminogen Klinger domain sequence, preferably a Klinger 5 domain. It is also contemplated that a combination of endostatin and plasminogen sequences are used together in the same molecule.

[0063] Various protein-based therapeutics have been used to block angiogenesis with known endogenous sequences of endostatin and plasminogen.

[0064] Endostatin is a 20 kDa carboxyl-terminal fragment of type XVIII collagen, which is present in the vascular wall and basement membrane and plays an important role in endothelial cell adhesion and cytoskeletal organization. Endogenous endostatin inhibits endothelial cell migration and induces apoptosis, inhibits tumor growth, and impairs vascular maturation in wound healing. It is thought to interfere with the proangiogenic effects of growth factors such as basic fibroblast growth factor and VEGF and is known to inhibit at least 65 different tumor types. In addition, a study measuring changes in gene expression in human dermal microvascular cells after endostatin treatment showed downregulation of several proangiogenic pathways and upregulation of many antiangiogenic genes.

[0065] Endostatin (trade name Endostatin) is a protein that specifically inhibits endothelial cell proliferation and effectively inhibits angiogenesis and tumor growth.

[0066] In 2005, a new recombinant human endostatin, Endostatin, was approved by the China State Food and Drug Administration (SFDA) for the treatment of non-small cell lung cancer. It therefore has a long history of human use but surprisingly has never been approved in the United States or Europe.

[0067] The half-life of this protein in the body is only 10 hours. Therefore, Endovir is administered intravenously once a day for 14 days, and the administration lasts for 4 hours. This pharmacokinetic characteristic inevitably brings inconvenience, affects the quality of life, and may reduce patient compliance.

[0068] A more active form of endostatin (P125A) has been identified. In addition, it is reported that the N-terminal fragment of endostatin has a large part of the activity of endostatin. It is reported that for wild-type endostatin, 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 KA 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).

[0069] Endostatin (P125A) has been fused to the C-terminus of other proteins, such as Herceptin, a drug approved to block growth factor signaling, which enhances 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).

[0070] Plasminogen is an endogenous protein that can be cleaved by urokinase-activated plasmin and contains five Klinger (K) domains, each of approximately 80 residues. Endogenous angiostatin is a 38 kDa amino-terminal fragment of plasminogen, and studies using recombinant angiostatin have shown that tumor suppressor activity resides in domains K1-3. 8 Angiostatin was originally isolated from tumor-bearing mice. 6 It also has potent anti-angiogenic and anti-proliferative activities against endothelial cells and cancer cells. 7 Larger plasminogen fragments containing all five Klinger domains (K1-5) may be even more active than angiostatin alone (K1-3) (Cao R, Wu HL, Ki 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).

[0071] Recent evidence supports the dual anti-tumor mechanism of plasminogen derivatives, one affecting angiogenesis and the other directly targeting tumor cells (Sun Q, Xu Q, Dong X, et al. A hybrid protein comprising ATFdomain 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).

[0072] Klinger domain 5 (K5), like angiostatin, is a byproduct of 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).

[0073] HGF contains a Klinger motif and promotes angiogenesis by stimulating the tyrosine kinase receptor Met. In addition to its potent angiostatic effects, K5 can direct potent antitumor responses through its ability to recruit tumor-associated neutrophils and natural killer T cells.

[0074] Thus, plasminogen has multiple Klinger domains that appear to act alone (angiostatin (K1-4) and K5) or in combination. Thus, plasminogen-derived sequences, particularly the K5 domain, have been reported for use with other anticancer proteins (Wang H, Yang Z, Gu J. Therapeutic targeting of angiogenesis with arecombinant CTT peptide-endostatin mimic-Klinger domain 5 protein. Mol Cancer Ther. 2014; 13(11): 2674-2687. doi: 10.1158 / 1535-7163. MCT-14-0266).

[0075] One of the major events leading to metastasis is the proteolytic degradation of the extracellular matrix (ECM) to facilitate tumor cell invasion, migration, and homing to distant organs. Although several protease systems are involved in this process, a large body of evidence indicates that the urokinase-type plasminogen activator receptor (uPAR) system is a central player mediating proteolysis during cancer invasion and metastasis. uPA-mediated ECM degradation is also critical for the initiation of angiogenesis.

[0076] uPAR is a GPI-anchored cell membrane receptor that localizes the proteolytic activity of urokinase (uPA) to the cell surface. Its main function is to concentrate the proteolytic activity of urokinase (uPA) responsible for the degradation of ECM components on the cell surface. uPAR expression is increased in many human cancers and is associated with poor prognosis and early invasion and metastasis, and has been reported in up to 80% of tumors. At a certain point, the uPA-UPAR complex is usually internalized to disassemble the complex, and the unoccupied UPAR can be recycled to the surface.

[0077] There are two broad mechanisms for inhibiting UPAR function: (1) uPA binding to uPAR; and (2) prevention of cellular signaling by UPAR, which involves uPA binding and its subsequent binding to co-receptors such as integrins.

[0078] The binding of uPA to uPAR facilitates the activation of plasminogen to plasmin, which in turn initiates a series of proteolytic cascades to degrade components of the extracellular matrix and thereby lead to the migration of tumor cells from the primary site of origin to distant secondary organs.

[0079] uPA is usually 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: epidermal growth factor (EGF)-like domain and Klinger domain. The "G" domain is the EGF-like region, and the "K" is the Klinger domain. Most of the binding occurs only in the single EGF-like domain, but the whole ATF region including the EGF-like domain and the Klinger domain does have greater binding than the single EGF-like domain. In addition, a short peptide sequence unrelated to the natural ATF sequence has been identified, which also competes with uPA for 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 U S A. 1994; 91 (15): 7129-7133. doi: 10.1073 / pnas.91.15.7129). In fact, ATF-Fc fusion (Chinese Patent No. CN101050237) has 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).

[0080] An antibody (2G10) that prevents uPA from binding to uPAR was identified from a phage display library, which has been reported to prevent uPA from binding 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). In addition, non-natural small peptides that inhibit uPA / uPAR interaction were identified from combinatorial libraries.

[0081] The second region contains the enzymatic activity of uPA. It is initially an inactive proenzyme that, when activated, enzymatically cleaves plasminogen and activates the enzyme to plasmin, which mediates the degradation of the extracellular matrix.

[0082] Once uPA binds to UPAR, the complex of these two proteins is also able to interact with other proteins (co-receptors) on the surface of cancer cells, such as integrins. When UPAR interacts with these co-receptors primarily through domain 3 of its three domains, this leads to the activation of cell signaling pathways that accelerate cancer progression, including metastasis.

[0083] Unlike preventing uPA from binding to UPAR (where therapeutic proteins "compete" with uPA for binding to occupy UPAR receptors), the signaling aspect of targeting UPAR function is "non-competitive" with uPA binding to UPAR, and metastasis can be more effectively prevented by blocking cell signaling events that drive cancer cell metastasis. An example of this is the binding sequence (CDR) of antibody ATN-658 (Mahmood N, Arakelian A, Khan HA, Tanvir I, Mazar AP, Rabbani SA. uPARantibody (huATN-658) and Zometa reduce breast cancer growth and skeletallesions. Bone Res. 2020; 8 (1). doi: 10.1038 / S41413-020-0094-3). Another antibody (8B12) reportedly targets conformation-specific uPAR to reduce the binding of uPA to vitronectin and 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).

[0084] There are reports of the use of uPA-uPAR inhibitors (such as ATF) in combination with inhibitors such as protease inhibitor domains and the anti-angiogenic functional domain of angiostatin. Similarly, there are reports of endostatin (P125A) fused to the C-terminus of other antibodies (such as Herceptin).

[0085] The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0086] The terms "polypeptide", "peptide", and "protein" are often used interchangeably herein to refer to a polymer of amino acid residues. Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-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 having the same biological function or activity.

[0087] In some embodiments, polymeric molecules (e.g., polypeptide sequences or nucleic acid sequences) are considered "homologous" to each other 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.

[0088] 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 of a protein encoded by the open reading frame nucleotide sequence (as defined herein).

[0089] As used herein, the term "nucleic acid fragment" refers to a nucleic acid sequence having a deletion. In some embodiments, the 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 nucleotide sequence of the open reading frame.

[0090] The term "construct" refers to a nucleic acid sequence encoding a protein operably linked to a promoter and / or other regulatory sequences.

[0091] The term "genomic sequence" refers to a sequence with discontinuous open reading frames, wherein introns interrupt the protein coding regions.

[0092] As used herein, when the term "encoding", "coding" or "encoded" is used in the context of a particular nucleic acid, it means that the nucleic acid may be the necessary information to direct the translation of a nucleotide sequence into a particular protein. The information encoding a protein is specified by using codons. A protein-encoding nucleic acid may contain non-translated sequences (e.g., introns) within the translated region of the nucleic acid, or may lack such inserted non-translated sequences (e.g., in cDNA).

[0093] In the context of nucleic acid sequences, the term "percent sequence identity" or "identical" refers to the same residues that are aligned when two sequences are maximally aligned. 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).

[0094] The terms "substantially homologous" or "substantially similar" when referring to nucleic acids or fragments thereof refer to 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 appropriate nucleotide insertions or deletions of another nucleic acid (or its complementary strand) as determined by any known sequence identity algorithm (e.g., BLAST, described above).

[0095] As used herein, a "heterologous nucleic acid sequence" is any sequence placed in a location that does not normally occur in the genome. In some embodiments, the heterologous nucleic acid sequence is a natural phage sequence, albeit from a different phage.

[0096] A specific nucleic acid sequence also includes conservatively modified variants thereof (such as degenerate codon substitutions) and complementary sequences, as well as sequences that are clearly indicated. Therefore, nucleic acid sequences encoding protein sequences disclosed herein also include modified variants thereof as described herein. Substantially similar nucleic acid fragments of the present invention can also be characterized by the percentage identity of their encoded amino acid sequences to the amino acid sequences disclosed herein, as determined by algorithms commonly used by those skilled in the art.

[0097] An "operably linked" or "effectively linked" expression control sequence refers to an expression control sequence that is adjacent to a target coding sequence to control the expression of the target coding sequence, as well as an expression control sequence that acts in trans or remotely to control the expression of a coding sequence.

[0098] A "coding sequence" or "open reading frame" is a nucleotide sequence that encodes a polypeptide or protein. The endpoints of a coding sequence are a start codon and a stop codon. The present disclosure also includes natural, isolated or recombinant nucleic acid sequences encoding proteins, and vectors and / or (host) cells comprising the protein coding sequence.

[0099] The term "Fc domain", "Fc peptide" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of a 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 human IgG heavy chain Fc region is generally defined as extending 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 known 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 includes the C-terminal constant region of an immunoglobulin heavy chain, which is capable of stably self-association. For example, the subunit of an IgG Fc domain includes the IgG CH2 and IgG CH3 constant domains.

[0100] Fused or linked means that the components (eg, Fab molecule and Fc domain subunit) are linked directly by peptide bonds or through one or more peptide linkers.

[0101] The present invention also includes fragments and variants of the disclosed nucleotide sequences and proteins encoded therefrom. "Fragment" refers to a portion of a nucleotide sequence or a portion of an amino acid sequence and the protein encoded thereby. A fragment of a nucleotide sequence can encode a protein fragment that retains the biological activity of the native protein. Therefore, the present disclosure relates to any nucleic acid fragment comprising a nucleotide sequence encoding all or most of the amino acid sequence encoded thereby.

[0102] 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.

[0103] For the purpose of illustrating and describing the present invention, the above description of the present invention and some representative embodiments and details are provided. It is not intended to be exhaustive or limit the present invention to the exact form disclosed. It is obvious to those skilled in the art that modifications and variations may be made thereto without departing from the scope of the present invention. Example

[0104] Example 1 - Construction of G-Fc-endostatin (P125A)

[0105] The construct used the N-terminal portion of ATF from uPA (UniProt P00749), specifically the EGF-like domain "G" (residues 21-69, in bold), after the signal export peptide, which was fused to human Fc (IgG3 isotype) via a linker (GGGGAGGGG), and then further linked (GGGAGGGG) to the endostatin protein (UniProt P39060, residues 1572-1754, P125A mutation in italics). The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture, yielding a pure target molecule after protein A purification ( Figure 2 ). The sequence is listed in SEQ ID No. 1 as shown below.

[0106]

[0107]

[0108] Cloning, expression and purification are performed by methods known in the art.

[0109] Figure 2 The trace chromatograms of G-Fc-endostatin (P125A) under reducing and non-reducing conditions are shown. Under reducing conditions, the interaction between the two polypeptides is destroyed. However, under non-reducing conditions, the interaction is retained. Therefore, the two conditions allow us to identify a protein-protein interaction. The single peak under these two conditions indicates a stable single compound.

[0110] Example 2 - In vivo anticancer testing using a zebrafish model

[0111] The zebrafish model is a very short but predictive model for screening anticancer agents and has been widely accepted as a useful in vivo model.

[0112] Zebrafish Tumor Xenograft (ZTX) Model

[0113] Transgenic Tg(fli1:EGFP)y1 zebrafish embryos were maintained in E3 embryo medium containing 0.2 mM 1-phenyl-2-thiourea (also known as PTU) at 28°C for 48 hours. Unfertilized eggs or larvae that looked unhealthy or exhibited any obvious developmental defects were excluded before treatment began.

[0114] PC3 cancer cells were labeled with Dil red fluorescent dye. Approximately 700 (efficacy study) or 2500 (angiogenesis study) Dil-labeled PC3 cancer cells (red) ± the given concentration of the test article were implanted subcutaneously into the perivitelline space of 2-day-old larvae.

[0115] Larvae that had inadvertently injected tumor cells into the circulation or that had mistakenly implanted tumors into the yolk instead of the perivitelline space were excluded from the study. Photos of primary tumors were taken immediately after injection. Tumor-bearing embryos were incubated in E3 / PTU medium at 36°C for 72 h. After incubation, photos of primary tumors and CVP were taken using a red fluorescence filter. Larvae that died or were lost for other reasons during the study were excluded from the final analysis. Embryos that survived 3 days after treatment were fixed, and images of primary tumors and intratumoral vasculature were taken using an upright Zeiss confocal LSM700.

[0116] Tumor growth inhibition and metastasis analysis

[0117] Images acquired after implantation (day 0) and after 72 h of incubation (day 3) were analyzed using in-house developed software. Tumor growth regression was calculated and normalized to the corresponding controls. Metastases were assessed in the software and counted manually.

[0118] Tumor angiogenesis analysis

[0119] The primary tumor images acquired in the red and green channels were analyzed using ImageJ v1.53f51. The primary tumor and intratumoral vascular areas were measured. Tumor angiogenesis was calculated by normalizing the vascular area to the corresponding primary tumor area.

[0120] Avastin (bevacizumab) is a drug used to treat a variety of cancers. It is a monoclonal antibody that acts as an angiogenesis inhibitor. It was approved for medical use in the United States in 2004.

[0121] Figure 3A Provided is the effect of G-Fc-endostatin (P125A), also known as construct 1, on tumor volume and metastasis compared to the anticancer agent 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.

[0122] Figure 3B The effect of G-Fc-endostatin (P125A) on angiogenesis in a separate experiment is presented. Construct 1 was shown to be a better inhibitor of angiogenesis compared to vehicle and Avastin.

[0123] Significance ( * p < 0.05) was determined using a two-tailed Student's t test.

[0124] Example 3 - Construction of ATN658-Fc-K5

[0125] The fusion protein uses heavy chain ATF658 to bind to a linker and then to K5. The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein is transiently transfected and expressed in mammalian cells using a chemically defined culture medium. The protein is purified by protein A affinity chromatography, dialyzed in PBS, concentrated (if necessary), and then sterilized with 0.2 microns to obtain a high-purity stock solution. The sequence is listed in SEQ ID No. 2, as shown below.

[0126] Heavy chain (ATN 658)-Linker-K5

[0127]

[0128] Light chain (ATN658)

[0129]

[0130] Purity determined by SEC-HPLC is 99.6835%. Endotoxin: <1EU / mg. Concentration is 1.2mg / mL. Formulation buffer is PBS, pH 7.4.

[0131] like Figure 4 As shown, SDS-PAGE analysis of ATN658-Fc-K5 under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The different reduced proteins (heavy chain and light chain) migrated at approximately 63 kDa and 25 kDa, respectively. The non-reduced protein migrated at approximately 180 kDa.

[0132] Example 4 - Construction of Herceptin-Fc-ATF

[0133] The fusion protein uses the heavy chain Herceptin combined with an Fc linker and then combined with ATF. The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein is transiently transfected and expressed in mammalian cells using a chemically defined medium. The protein is purified by protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then sterile filtered at 0.2 microns to obtain a high-purity bulk. The sequence is listed in SEQ ID No. 3, as shown below.

[0134] Heavy Chain (Herceptin)-Linker-ATF

[0135]

[0136] Light chain (Herceptin)

[0137]

[0138] The purity determined by SEC-HPLC is 99.6621%. Endotoxin: <1EU / mg. Concentration is 2mg / mL. The formulation buffer is PBS, pH 7.4.

[0139] like Figure 5 As shown, SDS-PAGE analysis of the construct under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The results showed that the different reduced proteins (heavy chain and light chain) migrated about 63kD to 75kDa and 25kDa, respectively. The non-reduced protein migrated about 180kDa.

[0140] Example 5 - Construction of Avastin-Fc-ATF

[0141] The fusion protein uses heavy chain Avastin to bind to the Fc linker and then to ATF. The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein is transiently transfected and expressed in mammalian cells using a chemically defined culture medium. The protein is purified by protein A affinity chromatography, dialyzed in PBS, concentrated (if necessary), and then sterilized with 0.2 microns to obtain a high-purity stock solution. The sequence is listed in SEQ ID No. 4, as shown below.

[0142] Heavy Chain (Avastin)-Linker-ATF

[0143]

[0144] Light chain (Avastin)

[0145]

[0146] Purity determined by SEC-HPLC is 98.2435%. Endotoxin: <1EU / mg. Concentration is 2mg / mL. Formulation buffer is PBS, pH 7.4.

[0147] like Figure 6 As shown, SDS-PAGE analysis of the construct under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The different reduced proteins (heavy chain and light chain) migrated about 63kD to 75kDa and 25kDa, respectively. The non-reduced protein migrated about 180kDa.

[0148] Example 6 - Construction of human IgG1 Fc-linker-ATF

[0149] This fusion protein uses human IgG1 Fc-linker-ATF. The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein is transiently transfected and expressed in mammalian cells using chemically defined culture media. The protein is purified by protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then sterile filtered at 0.2 microns to obtain a high purity stock solution. The sequence is listed in SEQ ID No.5, as shown below.

[0150] Human IgG1 Fc-Linker-ATF

[0151]

[0152]

[0153] The purity determined by SEC-HPLC is 97.8344%. Endotoxin: <1EU / mg. Concentration is 2mg / mL. The formulation buffer is PBS, pH 7.4.

[0154] like Figure 7 As shown, SDS-PAGE analysis of the constructs under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The different reduced proteins migrated from about 63 kDa to 48 kDa, respectively. The non-reduced proteins migrated from about 75 kDa to 100 kDa.

[0155] Example 7 - Construction of ATF-Linker-hIgG4 Fc-Klinger Domain K5

[0156] The fusion protein is bound to the linker using ATF, then to Fc, and then to the Klinger domain K5. The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein is transiently transfected and expressed in mammalian cells using a chemically defined culture medium. The protein is purified by protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then sterile filtered at 0.2 microns to obtain a high purity stock solution. The sequence is listed in SEQ ID No.6, as shown below.

[0157] ATF-Linker-hIgG4 Fc-Klinger domain K5

[0158]

[0159] Purity determined by SEC-HPLC is 98.6250%. Endotoxin: <1EU / mg. Concentration is 2mg / mL. Formulation buffer is PBS, pH 7.4.

[0160] like Figure 8 As shown, SDS-PAGE analysis of the constructs under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The different reduced proteins migrated from about 48 kDa to 63 kDa. The non-reduced proteins migrated from about 135 kDa to 180 kDa.

[0161] Example 8 - Construction of ATF-Linker-hIgG4 Fc-Endostatin (P125A)

[0162] The fusion protein is combined with a linker using heavy chain ATF, then with Fc and then with endostatin (P125A). The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein is transiently transfected and expressed in mammalian cells using a chemically defined culture medium. The protein is purified by protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then sterile filtered at 0.2 microns to obtain a high-purity stock solution. The sequence is listed in SEQ ID No.7, as shown below.

[0163] ATF-Linker-hIgG4 Fc-Endostatin (P125A)

[0164]

[0165] Purity determined by SEC-HPLC is 97.6569%. Endotoxin: <1EU / mg. Concentration is 2mg / mL. Formulation buffer is PBS, pH 7.4.

[0166] like Fig. 9 As shown, SDS-PAGE analysis of the constructs under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The different reduced proteins migrated from about 63 kDa to 48 kDa. The non-reduced protein migrated about 135 kDa.

[0167] Example 9 - Construction of ATF-Linker-hIgG4 Fc-Endostatin (P125A)

[0168] The fusion protein is bound to a linker using ATF, bound to Fc and then bound to endostatin (P125A). The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein is transiently transfected and expressed in mammalian cells using a chemically defined culture medium. The protein is purified by protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then sterile filtered at 0.2 microns to obtain a high-purity stock solution. The sequence is listed in SEQ ID No. 8, as shown below.

[0169] ATF-Linker-hIgG4 Fc-Endostatin (P125A)

[0170]

[0171] Purity determined by SEC-HPLC is 99.6569%. Endotoxin: <1EU / mg. Concentration is 2mg / mL. Formulation buffer is PBS, pH 7.4.

[0172] like Fig.10 As shown, SDS-PAGE analysis of the constructs under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The different reduced proteins migrated to about 48 kDa to 63 kDa. The non-reduced protein migrated to about 135 kDa.

[0173] Example 10 - Construction of Herceptin-Fc-K5

[0174] The fusion protein uses the heavy chain Herceptin to bind to Fc and then to K5. The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein is transiently transfected and expressed in mammalian cells using a chemically defined medium. The protein is purified by protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then sterile filtered at 0.2 microns to obtain a high purity stock solution. The sequence is listed in SEQ ID No.9, as shown below.

[0175] Heavy Chain (Herceptin)-Linker-K5

[0176]

[0177] Light chain (Herceptin)

[0178]

[0179] Purity determined by SEC-HPLC is 99.0715%. Endotoxin: <1EU / mg. Concentration is 2mg / mL. Formulation buffer is PBS, pH 7.4.

[0180] like Fig.11 As shown, SDS-PAGE analysis of the construct under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The different reduced proteins (heavy and light chains) migrated to about 63 kDa to 75 kDa, respectively. The non-reduced proteins migrated to about 180 kDa to 245 kDa.

[0181] Example 11 - Construction of ATF-Fc (IgG 4)-Linker-Klinger Domains 1-3

[0182] The fusion protein uses ATF to bind to Fc, to a linker, and to Klinger domains 1-3. The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein is transiently transfected and expressed in mammalian cells using a chemically defined culture medium. The protein is purified by protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then sterile filtered at 0.2 microns to obtain a high purity stock solution. The sequence is listed in SEQ ID No. 10, as shown below.

[0183] ATF-Fc(IgG4)-Linker-Klinger domain 1-3

[0184]

[0185] Purity determined by SDS-PAGE was greater than 90%. Endotoxin: <1 EU / mg. Concentration was 1.3 mg / mL. Formulation buffer was PBS, pH 7.4.

[0186] like Fig.12 As shown, SDS-PAGE analysis of the constructs under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The different reduced proteins migrated to about 63 kDa to 75 kDa, respectively. The non-reduced proteins migrated to about 135 kDa to 180 kDa.

[0187] Example 12 - Construction of Herceptin-Inhibitor Protein

[0188] The fusion protein uses heavy chain Herceptin to bind to a linker and then to an inhibitory protein. The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein is transiently transfected and expressed in mammalian cells using a chemically defined culture medium. The protein is purified by protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then sterile filtered at 0.2 microns to obtain a high-purity stock solution. The sequence is listed in SEQ ID No. 11, as shown below.

[0189] Heavy chain (Herceptin)-Linker-Inhibitor protein

[0190]

[0191] Light chain (Herceptin)

[0192]

[0193] Purity determined by SDS-PAGE was greater than 90%. Endotoxin: <1 EU / mg. Concentration was 1.5 mg / mL. Formulation buffer was PBS, pH 7.4.

[0194] like Fig.13 As shown, SDS-PAGE analysis of the constructs under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The different reduced proteins (heavy and light chains) migrated to approximately 63 kDa to 75 kDa.

[0195] Example 13 - Construction of ATF-huIgG4 Fc-inhibitory protein

[0196] The fusion protein uses ATF to bind to the Fc linker and then to the inhibitor protein. The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector of the fusion protein is transiently transfected and expressed in mammalian cells using a culture medium with a clear chemical composition. The protein is purified by protein A affinity chromatography, dialyzed by PBS, concentrated (if necessary), and then sterilized by 0.2 micron filtration to obtain a highly purified stock solution. The sequence is listed in SEQ ID No. 12, as shown below.

[0197] ATF-huIgG4 Fc-Inhibitory Protein

[0198]

[0199] The purity determined by SEC-HPLC is 95.2801%. Endotoxin: <1EU / mg. Concentration is 2mg / mL. The formulation buffer is PBS, pH 7.4.

[0200] like Fig.14 As shown, SDS-PAGE analysis of the constructs under β-mercaptoethanol reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The different reduced proteins migrated to about 63 kDa to 75 kDa, respectively. The non-reduced proteins migrated to about 180 kDa and 180 kDa.

[0201] Example 15 - Construction of anti-PLAUR recombinant antibody (clone ATN-658) - endostatin P125A

[0202] The fusion protein uses ATN-658 to bind to endostatin P125A. The molecule is produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vector for the fusion protein is transiently transfected and expressed in mammalian cells using a chemically defined culture medium. The protein is purified by protein A affinity chromatography, dialyzed against PBS, concentrated (if necessary), and then sterile filtered at 0.2 microns to obtain a high-purity stock solution. The sequence is listed in SEQ ID No. 13, as shown below.

[0203] Heavy chain sequence:

[0204]

[0205] Light chain sequence:

[0206]

[0207] Purity determined by SEC-HPLC was 97.26%. Endotoxin: <1 EU / mg. Concentration was 1 mg / mL. Formulation buffer was PBS, pH 7.4.

[0208] like Fig.15 As shown, the SDS-PAGE analysis of the construct under β-mercaptoethanol reduction (lane 1) and non-reduction (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 minutes. The theoretical molecular weights of the heavy chain and light chain connected to the endostatin P125A mutant protein are 70.29 kDa and 23.97 kDa, respectively. Here, in the SDS-PAGE reduced by β-mercaptoethanol, the heavy chain connected to the endostatin P125A mutant protein migrates to about 63 kDa to 75 kDa, and the light chain migrates to about 25 kDa.

[0209] Example 16 - In vivo anticancer testing using a zebrafish model

[0210] The zebrafish model described in Example 2 was used, except that DU145 cells were labeled with Dil red fluorescent dye. Approximately 700 Dil-labeled cancer cells were co-implanted into the perivitelline space of 2-day-old larvae with 2 mg / mL of Avastin, Avastin-ATF, Herceptin, Herceptin-ATF, Herceptin-K5, and Herceptin-Arrestin and 1 mg / mL of ATN658, ATN658 K5, ATN658 Endostatin (P125A), ATF-Fc, ATF-Fc-Klinger 1-3, ATF-Fc (IgG 4)-Endostatin (P125A), ATF-Fc-K5, and ATF-Fc-Arrestin.

[0211] Data are shown as mean ± SEM, and D'Agostino-Pearson 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 acquired / analyzed using GraphPad Prism v9.0.2.

[0212] In summary, the results showed that treatment with all the different constructs and Herceptin significantly reduced the size of the primary tumors 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. ATF-Fc(IgG4)-Endostatin(P125A) and ATF-Fc-K5 constructs showed the most significant reduction in primary tumor size compared to the negative control compared to the other tested compounds.

[0213] The results also showed that the Avastin and Herceptin constructs had higher anti-tumor efficacy than Avastin and Herceptin treatment alone. In addition, the ATF-Fc(IgG4)-endostatin(P125A) and ATF-Fc-K5 constructs had higher anti-tumor efficacy than ATF-Fc alone.

[0214] Study Design

[0215] This study was divided into two parts:

[0216] Part 1. Positive Control Test

[0217] Part 2. Efficacy evaluation of constructs

[0218] DU-145 cells were used to evaluate the efficacy of the compounds. The study consisted of 16 groups and was conducted over two days. DU-145 cells were chosen because of their high expression of UPAR, HER2, and VEGF-A, and provide a good basis for positive controls to be able to evaluate new constructs. The DU-145 cell line also had good engraftment in the zebrafish model, with approximately 50% of the tumor remaining 3 days after implantation, with an average of 10 to 15 disseminated tumor cells per fish.

[0219] Dosage options:

[0220] Cancer cells will be co-injected with the test compound. The injection volume is generally 2nL to 5nL per embryo (usually around 3nL). To calculate the dose / embryo, use the following formula:

[0221] 3x 10^-9L xc(g / L)=ng / embryo

[0222] Avastin

[0223] Weekly Avastin treatment (25 mg / kg) in mice was highly effective against DU-145 cells (Cancer Chemother Pharmacol (2009) 65:191-195).

[0224] In a zebrafish study using the A549 lung cancer cell line and Avastin and Endostatin (wild-type endostatin, not the more active P125A mutant), bevacizumab was used at doses of 28 ng / embryo, 83 ng / embryo, and 250 ng / embryo; Endostatin was used at doses of 11 ng / embryo, 33 ng / embryo, and 99 ng / embryo. In this study, significant inhibition of tumor fluorescence was observed at all doses of both agents, but the 33 ng dose of Endostatin was not as effective as the two-sided dose (Scientific Reports (2018) 8: 15837). U-shaped dose curves have been reported in other studies using Endostatin (Cancer Gene Therapy (2006) 13: 619-627).

[0225] In previous Example 2, a 0.25 mg / mL Avastin solution did not show improvement in tumor volume reduction, but did affect angiogenesis for PC3 cells. Therefore, the dose in the current experiment will use a higher dose of 0.5 mg / mL for DU-145 cells before implantation, equivalent to 1.5 ng / embryo.

[0226] Herceptin

[0227] Almost weekly Herceptin treatment (10 mg / kg) in mice showed a general (not statistically significant) reduction in tumor volume using DU-145 cells (Cancer Biol. & Therapy (2020) 21:463-475).

[0228] Because DU-145 cells are less sensitive to Herceptin than Avastin, Herceptin was administered at a slightly higher level (1 mg / mL) than Avastin. This dose is equivalent to 1.8 ng / embryo.

[0229] ATN-658

[0230] This unapproved antibody has not been studied as extensively as Avastin or Herceptin; however, it has been tested in mice transplanted with PC3 cells and given twice weekly at a dose of 10 mg / kg, resulting in significant reductions in tumor volume and metastases (Neoplasia (2010) 10:778-88).

[0231] When allometrically scaled for zebrafish, this mouse dosing guide uses a dose of 0.3 mg / mL (0.9 ng / embryo) when mixed with tumor cells prior to implantation.

[0232] ATF-Fc fusion

[0233] In Example 2, 0.125 mg / mL of G-Fc-endostatin (P125A) was the lowest and best active dose using PC3 cells. Therefore, the same dose was used for ATF-Fc fusion (0.375 ng / embryo).

[0234] ATF is a combination of an EGF-like domain (G) and a Klinger domain.

[0235] All compounds were co-injected with human tumor cells in zebrafish embryos. The size of the primary tumor was assessed directly after implantation and three days after implantation. Metastasis was assessed three days after implantation. Anticancer efficacy will be determined based on changes in primary tumor size (i.e., tumor growth or shrinkage) and the number of tumor cells that spread to the distal caudal venous plexus (CVP) three days after implantation.

[0236] Part 1. Positive Control Test

[0237] Prior to the work plan (Table 2), dose trials of Avastin and Herceptin were performed to confirm the efficacy of these positive controls (Table 1). In this study, DU-145 cells were tested with the planned doses as well as 10 ng / embryo. The higher dose was selected from doses previously used in zebrafish (Avastin) or mice (Herceptin).

[0238] Table 1. Doses of Avastin and Herceptin tested prior to construct testing.

[0239]

[0240] Part 2. Efficacy evaluation of constructs

[0241] Table 2. Description of experimental groups for compound efficacy analysis.

[0242]

[0243]

[0244] result

[0245] Part 1. Positive Control Test

[0246] Prior to the efficacy studies, dose trials of Avastin and Herceptin were conducted to confirm the efficacy of these positive controls and to determine the doses for the efficacy studies.

[0247] Results showed that treatment with Herceptin showed a borderline significant reduction in primary tumor size compared to the negative control at 3 mg / mL. In contrast, treatment with Avastin showed no effect on primary tumor size compared to the negative control ( Fig.16 ).

[0248] Fig.16 It also showed that 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 after three days of treatment; however, this effect did not reach mathematical significance. Fig.16 The anti-tumor and anti-metastatic efficacy of Avastin and Herceptin on DU145 cancer cells after 72 hours of exposure was evaluated. Cancer cells ± Avastin ± Herceptin were subcutaneously co-implanted into the perivitelline space of 2-day-old zebrafish embryos as described in the study plan. Data are presented as mean ± SEM, and two-tailed Student's t-test was performed.

[0249] Part 2. Efficacy evaluation of constructs

[0250] Following Part 1, the concentrations of positive controls and constructs tested in the efficacy evaluation were determined (see Table 2).

[0251] Primary tumor

[0252] The results are first presented by comparing the primary tumor size between the different compounds by grouping them according to the fusion construct (i.e. ATF construct, ATN construct, Avastin and Herceptin constructs and K5, Arrestin and P125 constructs). The various constructs can be found in multiple graphs. All constructs are compiled and presented in a single graph where the anti-tumor efficacy is compared to the negative control.

[0253] ATF construct

[0254] Results showed that ATF-Fc-K5 and ATF-Fc(IgG4)-endostatin(P125A) constructs showed significant reduction in primary tumor size compared to the rest of the ATF constructs. Meanwhile, Avastin ATF, Herceptin ATF, ATF-Fc, ATF-Fc-inhibitory protein, and ATF-Fc-Klinger domains 1-3 showed similar, non-significantly different primary tumor sizes after three days of treatment ( Fig.17 ).

[0255] Fig.17Comparison of DU145 primary tumor size between different ATF constructs after 72 hours of exposure. Data are presented as mean ± SEM and were tested for normality using the D'Agostino-Pearson test. Kruskal-Wallis test (p<0.0001) was performed followed by two-tailed Student's t-test or Mann-Whitney test as appropriate. *p<0.05, **p<0.01 and ***p<0.001.

[0256] Avastin and Herceptin constructs

[0257] Fig.18 Comparison of DU145 primary tumor size between different Avastin and Herceptin constructs after 72 hours of exposure. Data are presented as mean ± SEM.

[0258] K5, arrestin, and P125A constructs

[0259] Fig.19 Shown is the primary tumor size after 3 days of treatment comparing K5 or arrestin constructs. After 3 days, ATF-Fc(IgG4)-endostatin(P125A) showed a significantly reduced primary tumor size ( Fig.19 ). Data are presented as mean ± SEM and were subjected to the D'Agostino-Pearson normality test. Kruskal-Wallis test (p < 0.0001) was performed, followed by two-tailed Student's t-test or Mann-Whitney test (as appropriate). *p < 0.05.

[0260] All constructs

[0261] The results showed that treatment with all the different constructs and Herceptin significantly reduced the size of the primary tumors compared to the negative control after 3 days of treatment ( Fig. 20 ). Meanwhile, Avastin treatment showed a reduction in primary tumor size; however, this effect did not reach mathematical significance ( Fig. 20 ).

[0262] Fig. 20 It was shown that 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.

[0263] Fig. 20It was also shown that the Herceptin-arrestin construct exhibited higher antitumor efficacy than the Herceptin, Herceptin-ATF, and Herceptin-K5 constructs. Data are presented as mean ± SEM and a D'Agostino-Pearson normality test was performed. A Kruskal-Wallis test (p<0.0001) was performed followed by a two-tailed Student's t-test or Mann-Whitney test as appropriate. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0264] Transfer

[0265] Results showed that a reduction in metastatic spread of DU145 cells was observed after three days in the presence of Avastin, Herceptin-K5, Herceptin-Arrestin, ATF-Fc, and ATF-Fc-Klinger domains 1-3 treatment compared to the negative control. However, this effect did not reach mathematical significance ( Fig.21 ).

[0266] in conclusion

[0267] Avastin and Herceptin constructs had higher anti-tumor efficacy than Avastin and Herceptin treatment alone. In addition, ATF-Fc(IgG4)-endostatin(P125A) and ATF-Fc-K5 constructs had higher anti-tumor efficacy than ATF-Fc alone.

[0268] All evaluated treatments reduced the spread of DU145.

[0269] Example 17 - In vivo anticancer testing using a zebrafish model

[0270] The zebrafish model described in Example 16 was used.

[0271] Fig. 22 ATF-Fc-K5 and ATF-Fc-endostatin (P125A) constructs were shown to show improved normalized tumor volume reduction compared to untreated controls. Data are presented as mean ± SEM and D'Agostino-Pearson normality test was performed. Kruskal-Wallis test (p < 0.0001) was performed followed by two-tailed Student's t-test or Mann-Whitney test as appropriate. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0272] Although the present disclosure is described at least in part in terms of methods, it will be understood by those of ordinary skill in the art that the present disclosure is also directed to various components for performing at least some aspects and features of the methods. The present disclosure may be implemented in other specific forms without departing from the subject matter of the claims. The exemplary embodiments described are considered to be merely illustrative and non-restrictive in all respects. Selected features from one or more of the above-described embodiments may be combined to create alternative embodiments that are not explicitly described, and features suitable for these combinations should be understood to be within the scope of the present disclosure.

Claims

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

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

3. The compound according to claim 1, wherein the IgG is a fragment of human Fc fusion peptide. The compound according to claim 2 , wherein the Fc fusion peptide is an IgG protein.

5. The compound according to 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 the preceding claim, wherein the first binding peptide comprises an N-terminal fragment of uPAR having uPAR activity.

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

8. The compound according to the preceding claim, wherein the first binding peptide is selected from the group consisting of EGF-like domain G, the N-terminal portion of ATF from the urokinase-type plasminogen receptor (uPAR) antagonist, antibody 2G10, antibody ATN-658, antibody 8B12.

9. The compound of the preceding claim, wherein the first binding peptide comprises fragments of the heavy and light chain sequences of an antibody that blocks uPAR signaling.

10. The compound according to the preceding claim, wherein the second binding peptide is selected from an endostatin fragment having endostatin activity, endostatin, a plasminogen fragment having plasminogen activity, endostatin having a P125 mutation, a Klinger domain of plasminogen, Klinger domains 1 to 5 of plasminogen, Klinger domain 5 from plasminogen.

11. The compound according to the preceding claim, wherein the second binding peptide comprises an endostatin fragment having endostatin activity and a plasminogen fragment having plasminogen activity.

12. The compound according to the preceding claim, further comprising a linker peptide between the antibody peptide and the first binding peptide.

13. The compound according to the preceding claim, further comprising a linker peptide between the antibody peptide and the second binding peptide.

14. A compound according to the preceding claim, wherein the compound comprises 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 inhibitory protein.

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

17. A compound comprising 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 the peptide encoded by a sequence selected from 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-Klinger domain K5, ATF-linker-hIgG4 Fc-endostatin (P125A), ATF-linker-hIgG4 Fc-endostatin (P125A), Herceptin-Fc-K5, ATF-Fc(IgG4)-linker-Klinger domains 1-3, Herceptin-inhibitor, ATF-huIgG4 Fc-inhibitor and ATN658-endostatin (P125).

19. A compound comprising a compound substantially homologous to the compound of the preceding claim and having the activity of the compound of the preceding claim.

20. A nucleotide encoding a compound as claimed in any preceding claim.

21. The compound according to the preceding claim, wherein the compound exhibits anti-cancer activity.

22. A method of treating cancer comprising administering a composition comprising a compound according to the preceding claim.

23. Use of a compound as claimed in the preceding claims for the treatment of cancer.

24. A carrier comprising a compound as claimed in the preceding claim.

25. The method or use according to the preceding claim, wherein the cancer is a solid cancer.

26. A method of inhibiting metastasis in a subject with cancer, the method comprising administering to the subject an effective amount of a compound of the preceding claim.

27. The method or use of the preceding claim, further comprising one or more of: a) administering chemotherapy; b) administering radiation therapy; or c) administering one or more additional therapeutic agents.

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