Improved prostate apoptosis response-4 (PAR-4) polypeptide and methods of production and use thereof

Abstract

The present application discloses polypeptide molecules having prostate apoptosis response-4 (Par-4) pro-apoptotic activity and enhanced biological half-life in cancer cells, and related methods. Specifically, the present disclosure provides a composition comprising a Par-4 polypeptide fusion protein, and a method of inducing apoptosis of cancer cells using the composition, wherein the cancer cells are metastatic.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 832,155, filed on April 10, 2019, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The subject matter disclosed in the present application generally relates to polypeptide molecules having prostate apoptosis response-4 (Par-4) pro-apoptotic activity and enhancing biological half-life in cancer cells. The subject matter disclosed in the present application also relates to nucleic acid molecules encoding such polypeptide molecules, methods for preparing recombinant polypeptide molecules, compositions comprising such polypeptide molecules, methods for inducing apoptosis in cancer cells, and methods for treating cancer.

[0004] Introduction

[0005] Prostatic apoptosis response-4 (Par-4) is a tumor suppressor protein that is ubiquitously expressed in several tissues. In 1994, the Par-4 gene was first discovered as an early apoptotic gene in a rat prostate cancer cell line that was incubated with ionomycin to induce apoptotic cell death. (l,2) Overexpression of Par-4 has been shown to be sufficient to induce apoptosis in most cancer cells. (3) Consistent with this observation, the Par-4 gene has been reported to be mutated in endometrial cancer, (4) and is significantly downregulated in many different types of cancer, including renal cell carcinoma, (5) breast cancer, (6,7) gastric and pancreatic cancer, (8) glioblastoma, (9) and neuroblastoma. (10) Downregulation of Par-4 is associated with tumor recurrence and decreased patient survival. (7)

[0006] The core domain of Par-4 (amino acid residues 145-204 in human Par-4; 137-195 in rat Par-4), known as the selective apoptosis of cancer cells (SAC), serves as the effector domain for its pro-apoptotic activity. (11) Remarkably, this domain is 100% conserved among the mouse, rat, and human homologs, implying that Par-4 plays a key role in tumor surveillance. (11) In fact, both the mature Par-4 protein and its SAC domain can induce apoptosis through both the intrinsic pathway (activated by intrinsic stimuli such as biochemical stress or DNA damage and regulated primarily by Bcl-2 and Bax) (12) and the extrinsic pathway (activated in response to external stimuli such as Fas ligand). (13)

[0007] Initially, it was believed that Par-4 protein was located and functioned only in the cytoplasm and nucleus to induce apoptosis. (1,14) However, subsequent studies have found that Par-4 protein can be secreted into the extracellular space to exert its effects. (2) After the extracellular Par-4 protein binds to stress response proteins expressed on the surface of cancer cells, such as glucose-regulated protein 78 (GRP78), it can induce apoptosis through the activation of FADD, caspase-8, and -3. (2)

[0008] Studies have also shown that exposure to purified recombinant Par-4 protein can not only induce apoptosis in multiple types of cancer cells, but also inhibit tumor growth in vivo. (3,15) Therefore, previous drug discovery efforts related to Par-4 have focused on developing small molecule drugs that can promote the secretion of Par-4 from normal cells to achieve Par-4-dependent inhibition of tumor growth. Arylquin1 (16) and the antimalarial drug chloroquine (CQ) (17) have been found to be strong inducers of Par-4 secretion in normal cells or cancer cells.

[0009] Recombinant wild-type Par-4 remains in the circulation of mice for only a short time. Therefore, the limited persistence of Par-4 in serum in vivo may weaken the tumor suppressive activity of secreted Par-4. It is well known that the actual efficacy of protein drugs can be greatly increased by improving their pharmacokinetic (PK) profile. (18-24)

[0010] Therefore, there is still a need in the art for a composition having an improved in vivo biological half-life while maintaining the beneficial biological activity of Par-4.

[0011] Brief description of the invention

[0012] For those skilled in the art, after studying the information provided herein, it will be apparent that the subject matter of the present application satisfies some or all of the above needs.

[0013] The Summary of the Invention describes several embodiments of the subject matter of the present application and in many cases lists variations and permutations of these embodiments. The Summary of the Invention is merely an exemplary illustration of many different embodiments. Reference to representative features of one or more specific embodiments is also exemplary. Such an embodiment may generally exist with or without the one or more features; similarly, these features may also be applied to other embodiments of the subject matter of the present application, whether or not the embodiments are listed in the Summary of the Invention. To avoid excessive repetition, the Summary of the Invention does not list or suggest all possible combinations of such features.

[0014] The subject matter of the present application includes polypeptide molecules having prostate apoptosis response-4 (Par-4) activity and enhancing biological half-life. The subject matter of the present application also includes nucleic acid molecules encoding such polypeptide molecules, methods for preparing recombinant polypeptide molecules, compositions including such polypeptide molecules, methods for inducing apoptosis of cancer cells, and methods for treating cancer.

[0015] The polypeptide molecules disclosed in the present application have Par-4 activity and enhanced biological half-life relative to wild-type Par-4. In some embodiments, the polypeptide molecule includes a Par-4 polypeptide portion provided by a fusion protein and a second polypeptide molecule.

[0016] In some embodiments, the Par-4 polypeptide portion is a polypeptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide portion is a polypeptide comprising a modification relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide portion is a polypeptide comprising a deletion relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide portion is a polypeptide comprising an insertion relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide portion is a polypeptide comprising a substitution relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0017] In some embodiments, the Par-4 polypeptide composition is a polypeptide comprising a fragment of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide composition is a polypeptide comprising a variant of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0018] In some embodiments, the second polypeptide molecule comprises a polypeptide of an immunoglobulin G1 crystallizable fragment (Fc) containing the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule is a polypeptide comprising a modification relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule is a polypeptide comprising a deletion relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule is a polypeptide comprising an insertion relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule is a polypeptide comprising a substitution relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0019] In some embodiments, the second polypeptide molecule is a fragment of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the second polypeptide molecule is a variant of SEQ ID NO: 3 or SEQ ID NO: 4.

[0020] The subject matter of the present application also includes a method of inducing apoptosis in a cancer cell, the method involving contacting the cancer cell with a polypeptide molecule of the present application. In some embodiments, the cancer cell is metastatic.

[0021] The subject matter of the present application also includes a method of treating cancer in a subject, the method involving administering to a subject in need thereof a polypeptide molecule disclosed herein.

[0022] The subject matter of the present application further includes nucleic acid molecules. The nucleic acid molecules disclosed in the present application include nucleotide sequences encoding the above-mentioned polypeptide molecules or their complementary sequences. In this regard, the polypeptide molecules encoded by the nucleic acid molecules have the activity of Par-4 and enhanced biological half-life relative to wild-type Par-4. The nucleic acid molecule includes a sequence encoding the Par-4 polypeptide disclosed in the present application, and the sequence is operably connected to a sequence encoding a second polypeptide molecule disclosed in the present application. In some embodiments, the polypeptide molecule encoded by the nucleic acid molecule includes a Par-4 polypeptide portion provided as a fusion protein and a second polypeptide molecule.

[0023] The subject matter of the present application further includes a vector containing the nucleic acid molecules disclosed in the present application. The subject matter of the present application further includes a certain cell, wherein the cell includes a vector containing the nucleic acid molecules disclosed in the present application. The subject matter of the present application further includes a method for preparing a polypeptide molecule disclosed in the present application using the nucleic acid molecules disclosed in the present application.

[0024] The subject matter of the present application further includes compositions, such as pharmaceutical compositions, containing the polypeptide molecules disclosed in the present application, which have Par-4 activity and enhanced biological half-life relative to wild-type Par-4. The composition may include a pharmaceutically acceptable carrier known to those of ordinary skill in the art for the stability of the polypeptide molecules.

[0025] The present subject matter also includes methods of inducing apoptosis in cancer cells, the methods involving contacting the cancer cells with a composition disclosed herein. In some embodiments, the cancer cells are metastatic.

[0026] The present subject matter also includes methods of treating cancer in a subject, the methods involving administering to a subject in need thereof a composition disclosed herein. Sequence Listing <110> University of Kentucky Research Foundation <120> Improved prostate apoptosis response-4 (PAR-4) polypeptides and methods of producing and using the same <130> 13177N / 2354US <150> US 62 / 832,155 <151> 2019-04-10 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 332 <212> PRT <213> Roof rat (Rattus rattus) <400> 1 Met Ala Thr Gly Gly Tyr Arg Ser Ser Gly Ser Thr Thr Asp Phe Leu 1 5 10 15 Glu Glu Trp Lys Ala Lys Arg Glu Lys Met Arg Ala Lys Gln Asn Pro 20 25 30 Val Gly Pro Gly Ser Ser Gly Gly Asp Pro Ala Ala Lys Ser Pro Ala 35 40 45 Gly Pro Leu Ala Gln Thr Thr Ala Ala Gly Thr Ser Glu Leu Asn His 50 55 60 Gly Pro Ala Gly Ala Ala Ala Pro Ala Ala Pro Gly Pro Gly Ala Leu 65 70 75 80 Asn Cys Ala His Gly Ser Ser Ala Leu Pro Arg Gly Ala Pro Gly Ser 85 90 95 Arg Arg Pro Glu Asp Glu Cys Pro Ile Ala Ala Gly Ala Ala Gly Ala 100 105 110 Pro Ala Ser Arg Gly Asp Glu Glu Glu Pro Asp Ser Ala Pro Glu Lys 115 120 125 Gly Arg Ser Ser Gly Pro Ser Ala Arg Lys Gly Lys Gly Gln Ile Glu 130 135 140 Lys Arg Lys Leu Arg Glu Lys Arg Arg Ser Thr Gly Val Val Asn Ile 145 150 155 160 Pro Ala Ala Glu Cys Leu Asp Glu Tyr Glu Asp Asp Glu Ala Gly Gln 165 170 175 Lys Glu Arg Lys Arg Glu Asp Ala Ile Thr Gln Gln Asn Thr Ile Gln 180 185 190 Asn Glu Ala Ala Ser Leu Pro Asp Pro Gly Thr Ser Tyr Leu Pro Gln 195 200 205 Asp Pro Ser Arg Thr Val Pro Gly Arg Tyr Lys Ser Thr Ile Ser Ala 210 215 220 Pro Glu Glu Glu Ile Leu Asn Arg Tyr Pro Arg Thr Asp Arg Ser Gly 225 230 235 240 Phe Ser Arg His Asn Arg Asp Thr Ser Ala Pro Ala Asn Phe Ala Ser 245 250 255 Ser Ser Thr Leu Glu Lys Arg Ile Glu Asp Leu Glu Lys Glu Val Leu 260 265 270 Arg Glu Arg Gln Glu Asn Leu Arg Leu Thr Arg Leu Met Gln Asp Lys 275 280 285 Glu Glu Met Ile Gly Lys Leu Lys Glu Glu Ile Asp Leu Leu Asn Arg 290 295 300 Asp Leu Asp Asp Met Glu Asp Glu Asn Glu Gln Leu Lys Gln Glu Asn 305 310 315 320 Lys Thr Leu Leu Lys Val Val Gly Gln Leu Thr Arg 325 330 <210> 2 <211> 339 <212> PRT <213> Homo sapiens <400> 2 Ala Thr Gly Gly Tyr Arg Thr Ser Ser Gly Leu Gly Gly Ser Thr Thr 1 5 10 15 Asp Phe Leu Glu Glu Trp Lys Ala Lys Arg Glu Lys Met Arg Ala Lys 20 25 30 Gln Asn Pro Pro Gly Pro Ala Pro Pro Gly Gly Gly Ser Ser Asp Ala 35 40 45 Ala Gly Lys Pro Pro Ala Gly Ala Leu Gly Thr Pro Ala Ala Ala Ala 50 55 60 Ala Asn Glu Leu Asn Asn Asn Leu Pro Gly Gly Ala Pro Ala Ala Pro 65 70 75 80 Ala Val Pro Gly Pro Gly Gly Val Asn Cys Ala Val Gly Ser Ala Met 85 90 95 Leu Thr Arg Ala Ala Pro Gly Pro Arg Arg Ser Glu Asp Glu Pro Pro 100 105 110 Ala Ala Ser Ala Ser Ala Ala Pro Pro Pro Gln Arg Asp Glu Glu Glu 115 120 125 Pro Asp Gly Val Pro Glu Lys Gly Lys Ser Ser Gly Pro Ser Ala Arg 130 135 140 Lys Gly Lys Gly Gln Ile Glu Lys Arg Lys Leu Arg Glu Lys Arg Arg 145 150 155 160 Ser Thr Gly Val Val Asn Ile Pro Ala Ala Glu Cys Leu Asp Glu Tyr 165 170 175 Glu Asp Asp Glu Ala Gly Gln Lys Glu Arg Lys Arg Glu Asp Ala Ile 180 185 190 Thr Gln Gln Asn Thr Ile Gln Asn Glu Ala Val Asn Leu Leu Asp Pro 195 200 205 Gly Ser Ser Tyr Leu Leu Gln Glu Pro Pro Arg Thr Val Ser Gly Arg 210 215 220 Tyr Lys Ser Thr Thr Ser Val Ser Glu Glu Asp Val Ser Ser Arg Tyr 225 230 235 240 Ser Arg Thr Asp Arg Ser Gly Phe Pro Arg Tyr Asn Arg Asp Ala Asn 245 250 255 Val Ser Gly Thr Leu Val Ser Ser Ser Thr Leu Glu Lys Lys Ile Glu 260 265 270 Asp Leu Glu Lys Glu Val Val Arg Glu Arg Gln Glu Asn Leu Arg Leu 275 280 285 Val Arg Leu Met Gln Asp Lys Glu Glu Met Ile Gly Lys Leu Lys Glu 290 295 300 Glu Ile Asp Leu Leu Asn Arg Asp Leu Asp Asp Ile Glu Asp Glu Asn 305 310 315 320 Glu Gln Leu Lys Gln Glu Asn Lys Thr Leu Leu Lys Val Val Gly Gln 325 330 335 Leu Thr Arg <210> 3 <211> 234 <212> PRT <213> Homo sapiens <400> 3 Met Gln Glu Pro Lys Cys Ser Asp Lys Thr His Thr Cys Pro Pro Cys 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro 20 25 30 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 65 70 75 80 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90 95 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 130 135 140 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 145 150 155 160 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 4 <211> 234 <212> PRT <213> Artificial Sequence <400> 4 Met Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro 20 25 30 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 65 70 75 80 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90 95 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 130 135 140 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 145 150 155 160 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 5 <211> 1701 <212> DNA <213> Artificial Sequence <400> 5 atgcaggagc ctaagtcctc cgacaaaact cacacatccc caccgtcccc agcacctgaa 60 ctcctggggg gatcctcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420 tcccgggacg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcacga ggctctgcac 660 aaccactaca cgcagaagag cctctccctg tctccgggta aaatggcgac cggcggctat 720 780 cgcgccaagc agaaccccgt gggcccgggt tcgagcggcg gggatccagc cgccaagtcc 840 cctgcgggac cgctcgccca gactacggcc gcgggggacct cggaactcaa ccacggcccc 900 gccggcgcgg ccgcacctgc cgccccccggg ccgggcgcccc tgaactgcgc tcacggctcg 960 tccgcgctgc cccgcggggc tcccggctcc cggcggccgg aggacgagtg tcctattgcc 1020 gctggggccg cgggagcacc cgcgtcccgg ggagacgagg aggagccgga tagcgccccg 1080 gagaagggcc gcagctcggg gcccagcgcc aggaaggca aagggcagat cgagaagagg 1140 aagctgcggg agaagcgccg ctccaccggc gtggtcaaca tccccgcggc ggagtgctta 1200 gatgagtacg aagatgacga agcaggacag aagcaacgga agcgagaga tgctatcaca 1260 caggaagaa ccatccagaa tgaagctgcg agcctcccag atccaggaac ctcctacctg 1320 cccagagcacc cgtcgagaac agtcccaggc agatacaaaa gcacaatcag tgccccagaa 1380 1440 gataccagtg cgcctgctaa cttcgcttca agtagcacct tggaaaagag aattgaagat 1500 cttgagaagg aagtcttgag agaaaggcaa gaaaaccttc gacttacgag gctgatgcaa 1560 gataaagaag aaatgattgg aaaactcaag gaagagattg atttgttaaa tagagacctc 1620 gatgacatgg aagacgaaaa cgagcaacta aagcaggaaa ataaaactct tttgaaagtt 1680 gttgggcagc tgacaaggta g 1701 <210> 6 <211> 566 <212> PRT <213> Artificial Sequence <400> 6 Met Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro 20 25 30 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 65 70 75 80 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90 95 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 130 135 140 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 145 150 155 160 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Met Ala Thr Gly Gly Tyr 225 230 235 240 Arg Ser Ser Gly Ser Thr Thr Asp Phe Leu Glu Glu Trp Lys Ala Lys 245 250 255 Arg Glu Lys Met Arg Ala Lys Gln Asn Pro Val Gly Pro Gly Ser Ser 260 265 270 Gly Gly Asp Pro Ala Ala Lys Ser Pro Ala Gly Pro Leu Ala Gln Thr 275 280 285 Thr Ala Ala Gly Thr Ser Glu Leu Asn His Gly Pro Ala Gly Ala Ala 290 295 300 Ala Pro Ala Ala Pro Gly Pro Gly Ala Leu Asn Cys Ala His Gly Ser 305 310 315 320 Ser Ala Leu Pro Arg Gly Ala Pro Gly Ser Arg Arg Pro Glu Asp Glu 325 330 335 Cys Pro Ile Ala Ala Gly Ala Ala Gly Ala Pro Ala Ser Arg Gly Asp 340 345 350 Glu Glu Glu Pro Asp Ser Ala Pro Glu Lys Gly Arg Ser Ser Gly Pro 355 360 365 Ser Ala Arg Lys Gly Lys Gly Gln Ile Glu Lys Arg Lys Leu Arg Glu 370 375 380 Lys Arg Arg Ser Thr Gly Val Val Asn Ile Pro Ala Ala Glu Cys Leu 385 390 395 400 Asp Glu Tyr Glu Asp Asp Glu Ala Gly Gln Lys Glu Arg Lys Arg Glu 405 410 415 Asp Ala Ile Thr Gln Gln Asn Thr Ile Gln Asn Glu Ala Ala Ser Leu 420 425 430 Pro Asp Pro Gly Thr Ser Tyr Leu Pro Gln Asp Pro Ser Arg Thr Val 435 440 445 Pro Gly Arg Tyr Lys Ser Thr Ile Ser Ala Pro Glu Glu Glu Ile Leu 450 455 460 Asn Arg Tyr Pro Arg Thr Asp Arg Ser Gly Phe Ser Arg His Asn Arg 465 470 475 480 Asp Thr Ser Ala Pro Ala Asn Phe Ala Ser Ser Ser Thr Leu Glu Lys 485 490 495 Arg Ile Glu Asp Leu Glu Lys Glu Val Leu Arg Glu Arg Gln Glu Asn 500 505 510 Leu Arg Leu Thr Arg Leu Met Gln Asp Lys Glu Glu Met Ile Gly Lys 515 520 525 Leu Lys Glu Glu Ile Asp Leu Leu Asn Arg Asp Leu Asp Asp Met Glu 530 535 540 Asp Glu Asn Glu Gln Leu Lys Gln Glu Asn Lys Thr Leu Leu Lys Val 545 550 555 560 Val Gly Gln Leu Thr Arg 565 <210> 7 <211> 1725 <212> DNA <213> Artificial Sequence <400> 7 atgcaggagc ctaagtcctc cgacaaaact cacacatccc caccgtcccc agcacctgaa 60 ctcctggggg gatcctcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 120 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 180 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 240 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 300 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 360 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 420 tcccgggacg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 480 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 540 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 600 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcacga ggctctgcac 660 aaccactaca cgcagaagag cctctccctg tctccgggta aaatggcgac cggtggctac 720 cggaccagca gcggcctcgg cggcagcacc acagacttcc tggaggagtg gaaggcgaaa 780 cgcgagaaga tgcgcgccaa gcagaacccc ccgggcccgg cccccccggg agggggcagc 840 agcgacgccg ctgggaagcc ccccgcgggg gctctgggca ccccggcggc cgccgctgcc 900 aacgagctca acaacaacct cccgggcggc gcgccggccg cacctgccgt ccccggtccc 960 gggggcgtga actgcgcggt cggctccgcc atgctgacgc gggcggcccc cggcccgcgg 1020 cggtcggagg acgagccccc agccgcctct gcctcggctg caccgccgcc ccagcgtgac 1080 gaggaggagc cggacggcgt cccagagaag ggcaagagct cgggccccag tgccaggaaa 1140 ggcaaggggc agatcgagaa gaggaagctg cgggagaagc ggcgctccac cggcgtggtc 1200 aacatccctg ccgcagagtg cttagatgag tacgaagatg atgaagcagg gcagaaagag 1260 cggaaacgag aagatgcaat tacacaacag aacactattc agaatgaagc tgtaaactta 1320 ctagatccag gcagttccta tctgctacag gagccaccta gaacagtttc aggcagatat 1380 aaaagcacaa ccagtgtctc tgaagaagat gtctcaagta gatattctcg aacagataga 1440 agtgggttcc ctagatataa cagggatgca aatgtttcag gtactctggt ttcaagtagc 1500 acactggaaa agaaaattga agatcttgaa aaggaagtag taagagaaag acaagaaaac 1560 ctaagacttg tgagactgat gcaagataaa gaggaaatga ttggaaaact caaagaagaa 1620 attgatttat taaatagaga cctagatgac atagaagatg aaaatgaaca gctaaagcag 1680 gaaaataaaa ctcttttgaa agttgtgggt cagctgacca ggtag 1725 <210> 8 <211> 574 <212> PRT <213> Artificial Sequence <400> 8 Met Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro 20 25 30 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 65 70 75 80 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90 95 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 130 135 140 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 145 150 155 160 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Met Ala Thr Gly Gly Tyr 225 230 235 240 Arg Thr Ser Ser Gly Leu Gly Gly Ser Thr Thr Asp Phe Leu Glu Glu 245 250 255 Trp Lys Ala Lys Arg Glu Lys Met Arg Ala Lys Gln Asn Pro Pro Gly 260 265 270 Pro Ala Pro Pro Gly Gly Gly Ser Ser Asp Ala Ala Gly Lys Pro Pro 275 280 285 Ala Gly Ala Leu Gly Thr Pro Ala Ala Ala Ala Ala Asn Glu Leu Asn 290 295 300 Asn Asn Leu Pro Gly Gly Ala Pro Ala Ala Pro Ala Val Pro Gly Pro 305 310 315 320 Gly Gly Val Asn Cys Ala Val Gly Ser Ala Met Leu Thr Arg Ala Ala 325 330 335 Pro Gly Pro Arg Arg Ser Glu Asp Glu Pro Pro Ala Ala Ser Ala Ser 340 345 350 Ala Ala Pro Pro Pro Gln Arg Asp Glu Glu Glu Pro Asp Gly Val Pro 355 360 365 Glu Lys Gly Lys Ser Ser Gly Pro Ser Ala Arg Lys Gly Lys Gly Gln 370 375 380 Ile Glu Lys Arg Lys Leu Arg Glu Lys Arg Arg Ser Thr Gly Val Val 385 390 395 400 Asn Ile Pro Ala Ala Glu Cys Leu Asp Glu Tyr Glu Asp Asp Glu Ala 405 410 415 Gly Gln Lys Glu Arg Lys Arg Glu Asp Ala Ile Thr Gln Gln Asn Thr 420 425 430 Ile Gln Asn Glu Ala Val Asn Leu Leu Asp Pro Gly Ser Ser Tyr Leu 435 440 445 Leu Gln Glu Pro Pro Arg Thr Val Ser Gly Arg Tyr Lys Ser Thr Thr 450 455 460 Ser Val Ser Glu Glu Asp Val Ser Ser Arg Tyr Ser Arg Thr Asp Arg 465 470 475 480 Ser Gly Phe Pro Arg Tyr Asn Arg Asp Ala Asn Val Ser Gly Thr Leu 485 490 495 Val Ser Ser Ser Thr Leu Glu Lys Lys Ile Glu Asp Leu Glu Lys Glu 500 505 510 Val Val Arg Glu Arg Gln Glu Asn Leu Arg Leu Val Arg Leu Met Gln 515 520 525 Asp Lys Glu Glu Met Ile Gly Lys Leu Lys Glu Glu Ile Asp Leu Leu 530 535 540 Asn Arg Asp Leu Asp Asp Ile Glu Asp Glu Asn Glu Gln Leu Lys Gln 545 550 555 560 Glu Asn Lys Thr Leu Leu Lys Val Val Gly Gln Leu Thr Arg 565 570

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The novel features of the present invention are particularly described in the appended claims. The features and advantages of the present invention can be better understood by referring to the following detailed description of illustrative embodiments using the principles of the present invention and the following drawings:

[0029] Fig. 1 shows that the serum persistence of recombinant Par-4 protein in mice is limited. (A) SDS-PAGE of purified TRX-fused Par-4 or hexa-histidine-tagged Par-4 (TRX-Par-4 and 6xHis-Par-4, respectively). Mice were intravenously injected with 5mg / kg TRX-Par-4 or 6xHis-Par-4. The relative serum concentrations of TRX-Par-4 (B) and 6xHis-Par-4 (C) were assessed by Western blotting. Recombinant Par-4 protein was detected with antibodies against Par-4 and observed by chemiluminescence. The light chain of mouse IgG1 was used as an internal loading control. Two figures are representative blots.

[0030] Figure 2 shows the preparation of Par-4Ex. (A) Schematic diagram of Par-4Ex or Fc(Ml)-Par-4. (B) Western blot analysis of Par-4 protein in bacterial extracts transformed with pET-22b(+) / 6xhis-Par-4 and induced with IPTG. Prior to immunoblotting, the soluble portion (S) of the bacterial extract was separated from the insoluble portion (I) by centrifugation. (C) SDS-PAGE of purified Par-4Ex protein. The soluble Par-4Ex protein was separated by protein A chromatography (Protein A), followed by an additional ion exchange chromatography step (Ion exchanger) to achieve the desired purity.

[0031] Figure 3 shows apoptosis induced by recombinant Par-4 and Par-4Ex proteins in E0771 (mouse breast cancer cell line) cells. (A) SDS-PAGE of purified Par-4Ex. (B) Cells were treated with vehicle or purified Par-4 (6xHis-Par-4) or Par-4Ex (100 nM each). 24 hours after treatment, apoptosis was scored by immunocytochemistry (ICC) for caspase 3 activity. The results represent the average of three samples, and the values ​​are expressed as mean ± SD. Asterisks (*) indicate that the differences are statistically significant (P < 0.05) by Student's t test.

[0032] Figure 4 The serum concentration (%) of recombinant Par-4 and Par-4Ex proteins in mice is shown as a function of time. E. coli (E.Coli)-derived Par-4Ex (■) or 6xHis-Par-4 (□) was administered by intravenous (iv) injection at a dose of 5 mg / kg, and serum protein concentration was determined by ELISA. The results represent the mean of three samples per group and are expressed as mean ± standard error.

[0033] FIG5 shows that the recombinant Par-4 protein inhibits the metastatic growth of tumor (E0771). 5cells) were injected into the tail vein of B6C3H mice (n=5). (A) Five hours after administration, vehicle or 250 μg purified Par-4 (6xHis-Par-4) or Par-4Ex was injected through the tail vein every other day for 12 days (a total of 1500 μg protein / mouse). (B) Five hours after administration, vehicle or 150 or 75 μg purified Par-4Ex was injected through the tail vein every other day for 12 days (a total of 900 and 450 μg of protein / mouse). Four weeks later, the mice were euthanized and the number of lung nodules was counted. Data are expressed as mean ± SEM. Single and double asterisks indicate P<0.05 and P<0.01, respectively.

[0034] Brief description of sequence listing

[0035] SEQ ID NO: 1 comprises the amino acid sequence of rat Par-4.

[0036] SEQ ID NO: 2 comprises the amino acid sequence of human Par-4.

[0037] SEQ ID NO: 3 comprises the amino acid sequence of an Fc polypeptide.

[0038] SEQ ID NO:4 comprises the amino acid sequence of a modified Fc polypeptide.

[0039] SEQ ID NO:5 comprises a nucleic acid sequence encoding a modified rat Par-4 (Par-4Ex) polypeptide.

[0040] SEQ ID NO:6 comprises the amino acid sequence of a modified rat Par-4 (Par-4Ex) polypeptide.

[0041] SEQ ID NO:7 comprises a nucleic acid sequence encoding a modified human Par-4 (Par-4Ex) polypeptide; and

[0042] SEQ ID NO: 8 comprises the amino acid sequence of a modified human Par-4 (Par-4Ex) polypeptide.

[0043] Description of Exemplary Embodiments

[0044] The details of one or more embodiments of the subject matter disclosed in this application are set forth herein. A person of ordinary skill in the art will understand modifications of the embodiments described herein and other embodiments after studying the information provided herein. The information provided in this document, especially the specific details of the exemplary embodiments, is primarily intended to provide clarity of understanding and no unnecessary limitations should be understood therefrom. In the event of a conflict, the specification herein, including definitions, shall prevail.

[0045] The subject matter of the present application includes polypeptide molecules having prostate apoptosis response-4 (Par-4) activity and enhancing biological half-life. The subject matter of the present application also includes nucleic acid molecules encoding such polypeptide molecules, methods for preparing recombinant polypeptide molecules, compositions containing such polypeptide molecules, methods for inducing apoptosis of cancer cells, and methods for treating cancer.

[0046] Peptide molecules

[0047] The polypeptide molecules disclosed herein have Par-4 activity and enhanced biological half-life relative to wild-type Par-4. In some embodiments, the polypeptide molecule includes a Par-4 polypeptide portion provided by a fusion protein and a second polypeptide molecule.

[0048] In some embodiments, the Par-4 polypeptide portion is a polypeptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide portion is a polypeptide comprising a modification relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide portion is a polypeptide comprising a deletion relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide portion is a polypeptide comprising an insertion relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide portion is a polypeptide comprising a substitution relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0049] In some embodiments, the Par-4 polypeptide composition is a polypeptide comprising a fragment of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide composition is a polypeptide comprising a variant of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0050] As used herein, "modification" refers to modification of the amino acid sequence of a polypeptide or the nucleotide sequence of a nucleic acid molecule, including deletion, insertion and substitution of amino acids and nucleotides, respectively. Methods for modifying polypeptides are routine to those skilled in the art, such as by using recombinant DNA methods or direct synthesis.

[0051] As used herein, "deletion" when referring to a nucleic acid molecule or a polypeptide refers to the deletion of one or more nucleotides from either end of a nucleic acid molecule, or the deletion of one or more amino acids from either end of a polypeptide, compared to a reference sequence, such as a wild-type sequence.

[0052] As used herein, "insertion" when referring to a nucleic acid molecule or polypeptide describes the inclusion of one or more additional nucleotides in a nucleic acid molecule, or one or more amino acids in a polypeptide, in a reference sequence, such as a wild-type sequence. Thus, a molecule containing one or more insertions contains one or more additional residues within the linear length of the sequence compared to the wild-type sequence.

[0053] As used herein, "addition" to nucleic acid molecules and polypeptides refers to the addition of nucleotides or amino acids at either terminus compared to a reference nucleic acid molecule or polypeptide.

[0054] As used herein, "substitution" refers to replacing one or more nucleotides or amino acids in a reference sequence with alternative nucleotides or amino acids without changing the length (described by the number of residues) of the molecule. Thus, one or more substitutions in a molecule will not change the number of amino acid residues or nucleotides of the molecule.

[0055] When used in reference to a polypeptide, the term "polypeptide fragment" or "fragment" refers to a polypeptide in which amino acid residues have been deleted compared to the reference polypeptide itself. A fragment may also be a "functional fragment", in which case the fragment retains some or all of the activity of the reference polypeptide described herein.

[0056] The term "polypeptide variant" refers to an amino acid sequence that differs from a reference polypeptide, for example, by substitution of one or more amino acids. Variants of a reference polypeptide also refer to variants of fragments of a reference polypeptide, for example, fragments in which one or more amino acids are substituted relative to the reference polypeptide. Variants may also be "functional variants," wherein the variant retains some or all of the activity of the reference protein described herein.

[0057] As used herein, the "activity" of a polypeptide refers to any activity exhibited by wild-type Par-4, such as binding to the stress response protein glucose-regulated protein 78 (GRP78), apoptotic activity in cancer cells, and tumor inhibition activity. The activity of a modified polypeptide can be any percentage level of the activity of an unmodified polypeptide, including but not limited to 50% activity, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more activity compared to an unmodified polypeptide.

[0058] In some embodiments, the Par-4 polypeptide portion includes a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the Par-4 polypeptide portion included in the polypeptide molecule contains a fragment of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the fragment has 100% homology with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in a region including the selective apoptosis (SAC) core domain of cancer cells. In some embodiments, the Par-4 polypeptide portion included in the polypeptide molecule contains a fragment of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues are deleted relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0059] Sequence "identity" or "homology" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid molecules or polypeptide molecules or regions thereof can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide, or along a region of a molecule. Although there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0060] Sequence identity along the full length of two polynucleotides or polypeptides refers to the percentage of identical nucleotides or amino acid residues along the full length of the molecule. For example, if a polypeptide A has 100 amino acids and polypeptide B has 95 amino acids, which are identical to 1-95 amino acids of polypeptide A, then when the sequence identity is compared along the full length of polypeptide A with the full length of polypeptide B, polypeptide B has 95% identity. In addition, the sequence identity of polypeptide A and polypeptide B can be compared along a region of each polypeptide, such as a similar region of 20 amino acids. In this case, if polypeptides A and B have 20 identical amino acids along the region, the sequence identity of the region is 100%. For example, in some embodiments, the Par-4 polypeptide portion of the polypeptide molecule described herein can have 100% identity with the Par-4 core domain of wild-type Par-4 (amino acid residues 145-204 in human Par-4; and 137-195 in rat Par-4), which is called selective apoptosis of cancer cells (SAC).

[0061] As described above, in addition to the Par-4 polypeptide portion, the polypeptide molecule also includes a second polypeptide molecule that forms a fusion protein with the Par-4 polypeptide portion.

[0062] In some embodiments, the second polypeptide molecule includes a polypeptide of an immunoglobulin G1 crystallizable fragment (Fc) comprising the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule is a polypeptide comprising a modification relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule is a polypeptide comprising a deletion relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule is a polypeptide comprising an insertion relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule is a polypeptide comprising a substitution relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0063] In some embodiments, the second polypeptide molecule is a fragment of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the second polypeptide molecule is a variant of SEQ ID NO: 3 or SEQ ID NO: 4.

[0064] In some embodiments, the second polypeptide molecule comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule comprises a fragment of the sequence of SEQ ID NO:3 or SEQ ID NO:4, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues are deleted relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule comprises a variant of SEQ ID NO:3 or SEQ ID NO:4, wherein the cysteine ​​residue of the polypeptide has been mutated relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule comprises a variant of a fragment of SEQ ID NO:3 or SEQ ID NO:4, wherein the cysteine ​​residue of the polypeptide has been mutated relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4, and wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues have been deleted relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the second polypeptide molecule comprises a variant of SEQ ID NO:3 or SEQ ID NO:4, wherein the variant comprises a substitution of C6S, C12S and C15S. In some embodiments, the second polypeptide molecule comprises a variant of a fragment of SEQ ID NO:3 or SEQ ID NO:4, wherein the variant comprises a substitution of C6S, C12S and C15S, and wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues have been deleted relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0065] In some embodiments, the polypeptide molecule comprises an embodiment of a second polypeptide molecule disclosed in the present application, wherein the second polypeptide molecule is located near the N-terminus of a partial embodiment of a Par-4 polypeptide disclosed in the present application.

[0066] The terms "amino terminus" or "N-terminus" and "carboxyl terminus" or "C-terminus" are used herein to denote positions within a polypeptide. Where the context permits, these terms refer to proximity or relative positions with respect to a particular sequence or portion of a polypeptide. For example, a sequence within a polypeptide that is located at the carboxyl terminus of a reference sequence is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the entire polypeptide.

[0067] In some embodiments, the polypeptide molecules disclosed in the present application include the sequence of SEQ ID NO: 6. In some embodiments, the polypeptide molecules disclosed in the present application include the sequence of SEQ ID NO: 8.

[0068] The subject matter disclosed in the present application also includes a method of inducing apoptosis in a cancer cell, the method involving contacting the cancer cell with a polypeptide molecule disclosed in the present application. In some embodiments, the cancer cell is metastatic.

[0069] The presently disclosed subject matter also includes methods of treating cancer in a subject, the methods involving administering to a subject in need thereof a polypeptide molecule disclosed herein.

[0070] As used herein, the term "treatment" or "treating" refers to any treatment of cancer, including but not limited to prophylactic treatment to reduce the severity of cancer, as well as therapeutic treatment. In this regard, it is understood that treatment can involve curing cancer, or substantially curing cancer, or ameliorating at least one symptom of cancer, or reducing the severity of cancer.

[0071] As used herein, the term "subject" refers to an object to which administration is given. The object of the method disclosed in the present application can be a vertebrate, such as a mammal. The object of the method disclosed in the present application can be a human or a non-human. Therefore, according to the subject matter disclosed in the present application, a veterinary therapeutic use is provided.

[0072] Nucleic acid molecules

[0073] The subject matter disclosed in the present application further includes nucleic acid molecules. The nucleic acid molecules disclosed in the present application include nucleotide sequences encoding the above-mentioned polypeptide molecules or their complementary sequences. In this regard, the polypeptide molecules encoded by the nucleic acid molecules have the activity of Par-4 and an enhanced biological half-life relative to wild-type Par-4. The nucleic acid molecules include a sequence encoding the Par-4 polypeptide disclosed in the present application, and the sequence is operably connected to a sequence encoding a second polypeptide molecule disclosed in the present application. In some embodiments, the polypeptide molecule encoded by the nucleic acid molecule includes a Par-4 polypeptide portion provided by a fusion protein and a second polypeptide molecule.

[0074] The term "complementary" refers to two nucleotide sequences (a sequence and its complementary sequence), the two nucleotide sequences comprising antiparallel nucleotide sequences in which complementary base residues form hydrogen bonds that can pair with each other. As is well known in the art, the nucleic acid sequences of the two complementary chains are reverse complementary to each other when viewed from the 5' to 3' direction of each.

[0075] As used herein, reference to "operably linked" to nucleic acid sequences refers to nucleic acid sequences that are functionally related to each other. For example, a first nucleic acid molecule encoding a first polypeptide can be operably linked to a second nucleic acid molecule encoding a second polypeptide, whereby the nucleic acid molecules can be transcribed and translated to express a functional fusion protein.

[0076] In some embodiments, the nucleic acid molecule includes a sequence encoding a polypeptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid molecule includes a sequence encoding a polypeptide comprising a modification relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid molecule includes a sequence encoding a polypeptide comprising a deletion relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid molecule includes a sequence encoding a polypeptide comprising an insertion relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid molecule includes a sequence encoding a polypeptide comprising a substitution relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0077] In some embodiments, the nucleic acid molecule comprises a sequence encoding a polypeptide comprising a fragment of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the nucleic acid molecule comprises a sequence encoding a polypeptide comprising a variant of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0078] In some embodiments, the nucleic acid molecule comprises a sequence encoding a second polypeptide molecule, the second polypeptide molecule comprising a polypeptide of an immunoglobulin G1 crystallizable fragment (Fc) comprising the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the nucleic acid molecule comprises a sequence encoding a polypeptide comprising a modification relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the nucleic acid molecule comprises a sequence encoding a polypeptide comprising a deletion relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the nucleic acid molecule comprises a sequence encoding a polypeptide comprising an insertion relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the nucleic acid molecule comprises a sequence encoding a polypeptide comprising a substitution relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0079] In some embodiments, the nucleic acid molecule comprises a sequence encoding a fragment of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the nucleic acid molecule comprises a sequence encoding a variant of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0080] In some embodiments, the nucleic acid molecule comprises a sequence encoding a fragment of SEQ ID NO:3 or SEQ ID NO:4, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues are deleted relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the nucleic acid molecule comprises a sequence encoding a variant of SEQ ID NO:3 or SEQ ID NO:4, wherein the cysteine ​​residue of the polypeptide has been mutated relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the nucleic acid molecule comprises a sequence encoding a variant of a fragment of SEQ ID NO:3 or SEQ ID NO:4, wherein the cysteine ​​residue of the polypeptide has been mutated relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4, and wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues have been deleted relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the nucleic acid molecule comprises a sequence encoding a variant of SEQ ID NO:3 or SEQ ID NO:4, wherein the variant comprises a substitution of C6S, C12S and C15S. In some embodiments, the nucleic acid molecule comprises a sequence encoding a variant of a fragment of SEQ ID NO:3 or SEQ ID NO:4, wherein the variant comprises a substitution of C6S, C12S and C15S, and wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues have been deleted relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0081] In some embodiments, the nucleic acid molecule comprises a sequence encoding an embodiment of a second polypeptide molecule disclosed herein, and the second polypeptide molecule is located proximal to the N-terminus of a sequence encoding a partial embodiment of a Par-4 polypeptide disclosed herein.

[0082] In some embodiments, the nucleic acid molecules disclosed in the present application include the sequence of SEQ ID NO: 5, or its complementary sequence. In some embodiments, the nucleic acid molecules disclosed in the present application include the sequence of SEQ ID NO: 7, or its complementary sequence.

[0083] The subject matter disclosed in the present application further includes vectors containing nucleic acid molecules disclosed in the present application. The subject matter disclosed in the present application further includes cells containing vectors containing nucleic acid molecules disclosed in the present application. The subject matter disclosed in the present application further includes methods for preparing polypeptide molecules disclosed in the present application using nucleic acid molecules disclosed in the present application.

[0084] As used herein, a "vector" is a replicable nucleic acid molecule that, when transformed into an appropriate host cell, can express one or more heterologous proteins from the nucleic acid molecule. A "host cell" is a cell that is used to receive, maintain, propagate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a vector.

[0085] Composition

[0086] The subject matter disclosed in the present application further includes compositions, such as pharmaceutical compositions, containing the polypeptide molecules disclosed in the present application, which have Par-4 activity and enhanced biological half-life relative to wild-type Par-4. The composition may include a pharmaceutically acceptable carrier known to those of ordinary skill in the art and provide stability of the polypeptide molecule.

[0087] The composition comprises a polypeptide molecule comprising a Par-4 polypeptide portion and a second polypeptide molecule provided by a fusion protein.

[0088] In some embodiments, the composition includes a polypeptide molecule, wherein the Par-4 polypeptide portion is a polypeptide comprising a sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the composition includes a polypeptide molecule, wherein the Par-4 polypeptide portion is a polypeptide comprising a modification relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the composition includes a polypeptide molecule, wherein the Par-4 polypeptide portion is a polypeptide comprising a deletion relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the composition includes a polypeptide molecule, wherein the Par-4 polypeptide portion is a polypeptide comprising an insertion relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the composition includes a polypeptide molecule, wherein the Par-4 polypeptide portion is a polypeptide comprising a substitution relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0089] In some embodiments, the composition comprises a polypeptide molecule, wherein the Par-4 polypeptide component is a polypeptide comprising a fragment of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the composition comprises a polypeptide molecule, wherein the Par-4 polypeptide component is a polypeptide comprising a variant of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0090] In some embodiments, the composition includes a polypeptide molecule, wherein the Par-4 polypeptide portion includes a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the composition includes a polypeptide molecule, wherein the polypeptide molecule includes a Par-4 polypeptide portion containing a fragment of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein the fragment has 100% homology to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in a region including the selective apoptosis of cancer cells (SAC) core domain. In some embodiments, the composition includes a polypeptide molecule, wherein the polypeptide molecule includes a Par-4 polypeptide portion comprising a fragment of the sequence of SEQ ID NO:1 or SEQ ID NO:2, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues are deleted relative to the sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0091] As described above, in addition to the Par-4 polypeptide portion, the polypeptide molecule of the composition disclosed in the present application includes a second polypeptide molecule, which forms a fusion protein with the Par-4 polypeptide portion.

[0092] In some embodiments, the composition includes a polypeptide molecule, wherein the second polypeptide molecule includes a polypeptide of an immunoglobulin G1 crystallizable fragment (Fc) containing the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the composition includes a polypeptide molecule, wherein the second polypeptide molecule is a polypeptide comprising a modification relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the composition includes a polypeptide molecule, wherein the second polypeptide molecule is a polypeptide comprising a deletion relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the composition includes a polypeptide molecule, wherein the second polypeptide molecule is a polypeptide comprising an insertion relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the composition includes a polypeptide molecule, wherein the second polypeptide molecule is a polypeptide comprising a substitution relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0093] In some embodiments, the composition comprises a polypeptide molecule, wherein the second polypeptide molecule is a fragment of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the composition comprises a polypeptide molecule, wherein the second polypeptide molecule is a variant of SEQ ID NO: 3 or SEQ ID NO: 4.

[0094] In some embodiments, the composition comprises a polypeptide molecule, wherein the second polypeptide molecule comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the composition comprises a polypeptide molecule, wherein the second polypeptide molecule comprises a fragment of the sequence of SEQ ID NO:3 or SEQ ID NO:4, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues have been deleted relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the composition comprises a polypeptide molecule, wherein the second polypeptide molecule comprises a variant of SEQ ID NO:3 or SEQ ID NO:4, wherein the cysteine ​​residue of the polypeptide has been mutated relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the composition comprises a polypeptide molecule, wherein the second polypeptide molecule comprises a variant of a fragment of SEQ ID NO:3 or SEQ ID NO:4, wherein the cysteine ​​residue of the polypeptide has been mutated relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4, and wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues have been deleted relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the composition comprises a polypeptide molecule, wherein the second polypeptide molecule comprises a variant of SEQ ID NO:3 or SEQ ID NO:4 containing C6S, C12S and C15S substitutions. In some embodiments, the composition comprises a polypeptide molecule, wherein the second polypeptide molecule comprises a variant of SEQ ID NO:3 or SEQ ID NO:4 containing C6S, C12S and C15S substitutions, and wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues have been deleted relative to the sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0095] In some embodiments, the composition includes a polypeptide molecule disclosed herein, wherein a second polypeptide molecule disclosed herein is located proximal to the N-terminus of an embodiment of a Par-4 polypeptide portion disclosed herein.

[0096] In some embodiments, the composition includes a polypeptide molecule, wherein the polypeptide molecule disclosed in the present application includes the sequence of SEQ ID NO: 6. In some embodiments, the polypeptide molecule disclosed in the present application includes the sequence of SEQ ID NO: 8.

[0097] The presently disclosed subject matter also includes methods of inducing apoptosis in cancer cells, the methods involving contacting the cancer cells with a composition disclosed herein. In some embodiments, the cancer cells are metastatic.

[0098] The presently disclosed subject matter also includes methods of treating cancer in a subject, the methods involving administering to a subject in need thereof a composition disclosed herein.

[0099] Although the terms used herein are believed to be understood by one of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the subject matter disclosed herein.

[0100] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0101] Unless otherwise indicated, all patents, patent applications, published applications and publications, GenBank sequences, databases, websites, and other published materials mentioned throughout the disclosure of this application are incorporated herein by reference in their entirety.

[0102] Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and specific information on the Internet can come and go, but equivalent information can be found by searching the Internet. Reference to information evidences the availability and public dissemination of such information.

[0103] As used herein, any abbreviations for protective groups, amino acids, and other compounds, unless otherwise indicated, conform to their commonly used, recognized abbreviations, or the IUPAC-IUB Biochemical Nomenclature Commission (see Biochem. (1972) 11(9): 1726-1732).

[0104] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are described herein.

[0105] In some cases, the nucleotides and polypeptides disclosed in this application are included in publicly available databases, e.g. and SWISSPROT. The information, including sequences, and other information relating to such nucleotides and polypeptides contained in such public databases are expressly incorporated herein by reference. Unless otherwise stated or apparent, references to these publicly available databases refer to the latest version of the database as of the filing date of this application.

[0106] In accordance with long-standing patent law practice, the terms "a," "an," and "the" when used in this application, including the claims, mean "one or more." Thus, for example, reference to "a cell" includes a plurality of such cells, and so forth.

[0107] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the subject matter disclosed herein.

[0108] As used herein, the term "about" when referring to a value or amount of mass, weight, time, volume, concentration, or percentage is meant to include variations from the particular amount of ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, ±0.1% in some embodiments, ±0.01% in some embodiments, and ±0.001% in some embodiments, as such variations are suitable for practicing the disclosed methods.

[0109] As used herein, ranges can be expressed as from one "approximately" particular value, and / or to another "approximately" particular value. It is also understood that some of the numerical values ​​disclosed herein, and in addition to the numerical values ​​themselves, each numerical value is also disclosed in this application as an "approximately" numerical value of that particular numerical value. For example, if the numerical value "10" is disclosed, then "about 10" is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0110] The subject matter disclosed in this application is further illustrated by the following specific but non-limiting examples. The following examples may include compilations of data that are representative of data collected at various times during the development and experimentation associated with the present invention. Example

[0111] Par-4Ex protein design. Several issues must be considered in designing an ideal Par-4Ex as a candidate therapeutic drug. For example, the molecular weight of an ideal Par-4Ex must be significantly greater than that of Par-4 (~40 kDa). For another example, the additional amino acid residues of the expansin may affect binding to GRP78, thereby rendering the expansin (Par-4Ex) inactive against cancer cells. In view of such issues, and considering that the SAC domain is closer to the C-terminus, the inventors added additional amino acid residues to the N-terminus of Par-4 to conduct the studies described in these embodiments.

[0112] In addition, in order to extend the biological half-life of Par-4, it is desirable to avoid the immunogenicity of the extended protein (Par-4Ex) to humans. To this end, the inventors selected additional amino acid residues from human protein fragments, but without the unnecessary biological functions of human proteins. The first candidate for the human protein fragment is the first 233 amino acid residues of human immunoglobulin G1 (IgG1), which is called the crystallizable fragment (Fc). In fact, although recent studies have shown that Fc fusion does not improve the biological half-life of candidate protein drugs expressed in Escherichia coli (27), protein fusion with the Fc region of human IgG1 (IgG1Fc) is one of the most commonly used strategies to extend the biological half-life of protein therapeutics (25, 26).

[0113] Wild-type IgGlFc will form dimers through intermolecular disulfide bonds. Therefore, the dimer of Par-4Ex may block the intermolecular binding interface between Par-4 and GRP78, and therefore may lose the binding affinity of Par-4 to GRP78. Taking this issue into consideration, the three cysteine ​​residues (#6, #12, and #15) in the Fc region were all changed to serine residues to avoid possible dimerization of Par-4Ex. For convenience, the mutant Fc is referred to as Fc (Ml), referring to the first mutant of Fc tested in this study (mutant 1 or Ml). Therefore, the first Par-4Ex protein tested in this study can also be expressed as Fc (Ml)-Par-4.

[0114] The Par-4 polypeptide sequence used for Par-4Ex fusion protein and the polypeptide sequence of immunoglobulin G1 crystallizable fragment (Fc) used for Par-4Ex fusion protein are listed in the sequence list. SEQ ID. NO5-8 show exemplary sequences of nucleotides and proteins that retain the beneficial function of binding to GRP78.

[0115] Cloning, expression, and purification of 6xHis-Par-4 and Par-4Ex. Bacterial expression constructs of TRX-Par-4, 6xHis-Par-4, and Par-4Ex were generated by subcloning the respective genes into the pET-22b(+) vector. The construct of TRX-Par-4 was prepared by subcloning the rat Par-4 sequence and the thioredoxin cDNA (TRX) expression cassette in the vector pThio-His (Invitrogen, Carlsbad, CA) as previously reported by Burikhanov et al. (2). The respective constructs were used to transform E. coli BL21(DE3)Star TM Cells were prepared from the culture medium (ThermoFisher Scientific, Waltham, MA) and induced with 0.5 mM IPTG (Sigma-Aldrich, St. Louis, MO). Cells were harvested 10 hours after IPTG induction. Cells were then washed with Tris buffered saline (25 mM Tris base, pH 7.4, 138 mM NaCl, and 2.7 mM KCl) and centrifuged at 2,000 x g for 5 min at 4 °C. Cell homogenates were prepared by resuspending the cell pellet in 25 mM Tris-Cl, pH 7.4 and sonicated. To remove cell debris or unbroken cells, the whole cell homogenate was centrifuged at 10,000 x g for 20 minutes.

[0116] To purify TRX-Par-4 or 6xHis-Par-4 protein, the resulting supernatant was loaded onto HisPur TM Cobalt resin (Resin) (Thermo Fisher Scientific) was pre-equilibrated with wash buffer (25 mM Tris, pH 7.4, 500 mM NaCl, 0.05% Triton X-100 and 50 mM imidazole). After extensive washing with wash buffer, bound His-tagged proteins were eluted by stepwise gradient elution with imidazole at 150 mM NaCl.

[0117] To purify Par-4Ex protein, the resulting supernatant was loaded onto a rmp protein A Sepharose Fast Flow column (GE Healthcare Life Sciences) pre-equilibrated with 20 mM Tris-HCl (pH 7.4). The mixture was then loaded onto the column and washed with 5 column volumes (CV) of 20 mM Tris-HCl (pH 7.4) (containing 200 mM NaCl) until an OD of 0. 280<0.02. Then, the protein was eluted with 50mM sodium acetate (pH4.0) containing 200mM NaCl. For buffer exchange, the eluate was dialyzed in 20mM Tris-HCl (pH 7.4) using Millipore centrifugal filter units. The protein solution was then loaded onto a Q-Sepharose Fast Flow (GE Healthcare Life Sciences, Pittsburgh, Pennsylvania) pre-equilibrated with 20mM Tris-HCl (pH7.4) for a second round of chromatographic separation. Par-4Ex protein was eluted from the Q-Sepharose column with a stepwise gradient of NaCl (100-800mM). For buffer exchange, the eluate was dialyzed in a storage buffer (50mM Hepes, 20% sorbitol, 1M glycine, pH7.4) using a Millipore centrifugal filter unit. The entire purification process was carried out in a cold room at 8°C, and the purified protein was stored at -80°C until activity testing. Their purity was analyzed by SDS-PAGE on 4-12% NuPAGE Novex Bis-Tris gels (Life Technologies).

[0118] Western blotting. Recombinant proteins in bacterial extracts transformed with pET-22b(+) / 6xhis-Par-4 or pET-22b(+) / Par-4Ex were analyzed by Western blotting using goat anti-rat Par-4 IgG obtained from Santa Cruz Biotechnology (Dallas, Texas) (1:3000 dilution according to the manufacturer's instructions). Pre-absorbed HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology) was used as the secondary antibody at a ratio of 1:4000, and finally the SuperSignal WestDura Extended Duration Substrate from Pierce Biotechnology (Waltham, Massachusetts) was used to detect Par-4 protein by chemiluminescence.

[0119] Immunocytochemistry and apoptosis analysis. 100 nM purified Par-4Ex or 6xhis-Par-4 was exposed to cells on slides. After 24 h of treatment, cells were subjected to immunocytochemistry (ICC) analysis using the indicated anti-caspase 3 IgG and then stained with appropriate secondary antibodies conjugated to Alexa Fluor-488 (green fluorescence) or Alexa Fluor-594 (red fluorescence) (Molecular Probes). Apoptotic nuclei were identified by TUNEL assay, caspase 3 immunostaining, or 4,6-diamidino-2-phenylindole (DAPI) staining. Three independent experiments were performed, and approximately 300 cells in each experiment were scored for apoptosis under a fluorescence microscope as described previously. (28)

[0120] Pharmacokinetic studies in mice. Mice were injected with various recombinant proteins or saline at a dose of 5 mg / kg body weight via the tail vein. Blood samples (15-30 μL each) were collected from the saphenous vein into heparinized capillary tubes at various time points after intravenous (iv) injection of the proteins. Plasma was separated from the collected blood by centrifugation (15 min, 5,000 x g). 200 ng of plasma protein was immobilized in 96-well flat-bottom EIA plates (Corning) in 100 μL of 0.05 M PBS, pH 7.4, overnight at 4°C (or 2 h at 37°C). The liquid was poured from the plate and the rest was drained on a paper towel. The coated wells were blocked with blocking buffer (0.05 M PBS, pH 7.4, containing 1 mg / ml casein) (250 μL / well) for 30 min at room temperature (RT). After washing twice with wash buffer (0.05M PBS, pH 7.4) (250 μL / well), 100 μL of goat anti-rat Par-4 IgG (Santa Cruz Biotechnology) in blocking buffer at multiple concentration ranges was added to each well. The plate was then covered with adhesive plastic and incubated for 1 hour at room temperature with continuous shaking. After washing three times with wash buffer, the pre-adsorbed HRP-conjugated secondary antibody (anti-goat IgG-HRP) (70 μL / well) was diluted 1:30,000 with blocking buffer and added to each well and incubated on a shaker at room temperature for 1 hour. The wells were then washed three times with wash buffer (250 μL / well) and 250 μL of TMB substrate was added to the wells. The ELISA plate was kept in the dark until the desired color appeared. The reaction was stopped with 100 μL of 0.5M HCl. The absorbance was measured at 450 nm using a microplate reader (= displayed blue). All measurements were performed in triplicate or quadruplicate. The obtained PK data (time-dependent enzyme concentration) were analyzed using GraphPad Prism 5.01 software ([E] t ) is fitted to a double exponential equation (29):

[0121]

[0122] The equation takes into account both the distribution process (fast phase, related to k1) and the elimination process (slow phase, related to k2) of the protein in the animal body. 1 / 2 Known as biological 1 / 2 or eliminate t 1 / 2 .

[0123] Breast cancer lung metastasis. E0771 cells (1.5x10 5Cells) were injected into the tail vein of immunodeficient B6C3H mice (n=5). Five hours after administration, 6xHis-Par-4 or Par-4Ex was injected intravenously (iv) through the tail vein every other day for 12 days, with a dose of 250, 150, or 75 μg / injection per mouse (a total of 1500, 900, or 450 μg protein / mouse injected in 12 days). Four weeks later, the mice were euthanized and the lungs were photographed. The number of lung nodules was then counted.

[0124] Results. Pharmacokinetics of recombinant Par-4. In a previous report by Zhao et al. (15), it was demonstrated that intravenous administration of recombinant TRX-Par-4 or TRX-SAC proteins (prepared in Escherichia coli) in immunocompetent C57 / BL6 mice significantly inhibited the lung metastasis of LLC1 (Lewis lung carcinoma line 1) cells in mice, (15), which means that both extracellular Par-4 and SAC proteins can inhibit metastatic tumor growth in vivo. Thioredoxin (TRX) fusion proteins are frequently used tools that can increase the solubility and expression of mammalian proteins when they are heterologously expressed in Escherichia coli. (30) However, despite extensive in vitro and in vivo antitumor activity tests against various cancer cells, the pharmacokinetic characteristics of TRX-Par-4 and TRX-SAC proteins have not been determined. (2,15,17) Therefore, it was necessary to determine how long TRX-Par-4 or unfused Par-4 proteins could remain in the circulation of mice. To address this issue, Par-4 proteins fused to the C-terminus of 6xHis tag or TRX were prepared using a bacterial expression system (TRX-Par-4 and 6xHis-Par-4, respectively; see Figure 1A ), and then each purified protein was intravenously injected into mice via the tail vein at a dose of 5 mg / kg. Blood samples were collected at different time points after protein injection and analyzed by Western blot using anti-Par-4 antibody. The results showed that both TRX-Par-4 (~50 kDa) and 6xHis-Par-4 (~38 kDa) proteins were rapidly removed from the circulation ( Figure 1B and C). The concentration of TRX-Par-4 in vivo decreases more slowly than that of 6xHis-Par-4, with the former being observed up to 90 minutes after intravenous injection, while the signal of 6xHis-Par-4 was very low at 30 minutes at most. These findings suggest that the antitumor activity of Par-4 protein in vivo may be limited due to its short circulating half-life.

[0125] Par-4 and Par-4Ex proteins expressed in E. coli also induced apoptosis in cancer cells. In addition to the preparation of TRX-Par-4 and unfused Par-4 proteins, the fusion protein Par-4Ex, Fc(Ml)-Par-4 (~70 kDa), was also prepared using the E. coli expression system to obtain sufficient protein for in vivo characterization studies of the protein. Purification of the E. coli-derived soluble Par-4Ex protein was performed using protein A affinity chromatography, followed by an additional ion exchange chromatography step to obtain the purity required for the following in vivo characterization studies ( Figure 2B and C).

[0126] Before in vivo testing of purified recombinant Par-4 and Par-4Ex proteins, it was necessary to determine whether Par-4Ex retained the unique pro-apoptotic activity of Par-4, since Fc(Ml) fused to the N-terminus of Par-4 might prevent or interfere with the protein-protein interaction between Par-4 and GRP78. To address this critical question, E0771 (murine breast cancer cell line) cells were treated with 100 nM 6xHis-Par-4 or Par-4Ex proteins and then incubated for 24 h. Storage buffer (50 mM Hepes, 20% sorbitol, 1 M glycine, pH 7.4) was used as a vehicle control. It was observed that under specific treatment conditions, Par-4Ex and 6xHis-Par-4 proteins induced similar levels of apoptosis in E0771 cells (Figure 3), indicating that the anti-tumor activity of Par-4 protein was not altered by its N-terminal fusion of Fc(Ml). This observation of the anti-tumor activity of Par-4 protein itself is consistent with the results of Zhao et al. (15) using TRX-Par-4. The study further demonstrated that Par-4Ex was as active as Par-4 itself in cell-based anti-tumor activity assays.

[0127] Biological half-life of recombinant proteins. To investigate whether Par-4Ex actually has a longer biological half-life compared to Par-4, a pharmacokinetic (PK) study was performed in mice. In vivo data were obtained based on intravenous injections of each protein into test mice. The PK data generated were Figure 4 The results showed that the biological half-life of Par-4Ex was much longer (~20.3 hours) compared to the biological half-life of 6xHis-Par-4 protein (~3 hours).

[0128] In vivo efficacy in inhibiting metastatic tumor growth. To investigate whether longer exposure due to an extended biological half-life could enhance the in vivo antitumor activity of Par-4, the efficiency of Par-4Ex and 6xHis-Par-4 proteins in inhibiting lung metastasis of E0771 breast cancer cells was evaluated in immunocompetent C57 / BL6 mice. Cells (1.5x10 5 coli-derived Par-4Ex (250, 150, or 75 μg) or 6xHis-Par-4 (250 μg) were then intravenously administered every other day for 12 consecutive days (a total of 1500, 900, or 450 μg per mouse over 12 days). It was observed that both Par-4Ex and 6xHis-Par-4 proteins significantly inhibited the growth of metastatic tumors in vivo compared to vehicle-treated controls, but no statistical difference was observed between the two protein treatment groups under the specific treatment conditions ( Figure 5A ). However, considering that the molecular weight of Par-4Ex protein (~70 kDa) is approximately 1.8 times larger than that of 6xHis-Par-4 protein (~38 kDa), the molar concentrations of recombinant Par-4 protein used to treat mice were not the same. This may indicate that Par-4Ex is indeed more effective than 6xHis-Par-4 in inhibiting metastatic tumor growth in vivo, but that their difference in potency may become less obvious at high doses. To more appropriately address this issue, it was determined whether the in vivo antitumor activity of Par-4Ex would diminish with decreasing doses. Based on the data obtained ( Figure 5B ), a 70% reduction in the dose of Par-4Ex protein (75 μg / injection) also produced a substantial inhibition of lung metastasis induced by E0771 breast cancer cells in mice. In contrast, a previous study (15) used the same animal model to investigate the in vivo activity of various doses of Par-4 (TRX-Par-4) and found that 250 μg / injection was the minimum effective dose; at any tested dose below 250 μg / injection, Par-4 injection could not significantly reduce lung metastasis of E0771 breast cancer cells. In summary, Par-4Ex does have improved in vivo efficacy in inhibiting metastatic tumor growth than Par-4.

[0129] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference into this application to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference, including those listed in the following list:

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[0176] It is to be understood that various details of the subject matter disclosed in this application may be changed without departing from the scope of the subject matter disclosed in this application.In addition, the above description is for illustrative purposes only, not for limiting purposes.

Claims

1. A polypeptide molecule, wherein the polypeptide molecule is represented by the sequence of SEQ ID NO:

6.

2. Use of the polypeptide molecule according to claim 1 for preparing a drug for inducing apoptosis of breast cancer cells.

3. The use according to claim 2, wherein the cancer cells are metastatic.

4. A nucleic acid molecule, which is a nucleotide sequence encoding a fusion protein or its complementary sequence, and the fusion protein is represented by the sequence of SEQ ID NO:

6.

5. The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule is represented by the sequence of SEQ ID NO: 5, or its complementary sequence. A vector comprising the nucleic acid molecule according to any one of claims 4 to 5.

7. A cell comprising the vector according to claim 6.

Images