TRAIL compositions with reduced immunogenicity

Abstract

Fusion polypeptides with modified multimerization domains have been developed that provide the fusion polypeptides with high expression, solubility, stability, and reduced immunogenicity. These fusion polypeptides exhibit improved physicochemical and biological properties and reduced immunogenicity in mammalian hosts relative to TRAIL compositions with unmodified multimerization domains. TRAIL compositions induce apoptosis in cancer cells and cancer-associated fibroblasts in vivo, thereby reducing tumor size.

Description

[0001] Statement Regarding Federally Funded Research

[0002] This invention was made with government support under Grant No. 4U44AA026111-03 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] About Sequence Listing

[0004] The sequence listing submitted on June 4, 2021, as a text file named “THER_116_PCT_ST25.txt,” created on June 4, 2021, and 60,827 bytes in size, is hereby incorporated by reference pursuant to 37 CFR §1.52(e)(5). Technical Field

[0005] The present invention generally relates to fusion polypeptides comprising tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that contain a modified isoleucine zipper (miLZ) domain. Background Art

[0006] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein in the TNF-α superfamily that shares sequence homology with TNF and FasL. TRAIL can be proteolytically cleaved from the cell surface and released in a soluble form. Soluble TRAIL is essentially a homotrimer that subsequently trimerizes upon binding to the TRAIL receptor. Five TRAIL receptors have been identified in humans, but only the TRAIL-R1 / DR4 and TRAIL-R2 / DR5 receptors initiate apoptosis similar to the Fas / FasL and TNF-R / TNF signaling pathways. TRAIL receptor binding stimulates the formation of the death-inducing signaling complex (DISC) with the recruitment of the adaptor protein, Fas-associated protein with a death domain (FADD). FADD recruits procaspases 8 and 10, and the DISC allows for the autoactivation of these caspases. Downstream of this signaling is the proteolytic cleavage and activation of caspases 3 / 6 / 7, leading to apoptosis. Another apoptotic pathway is to induce mitochondrial dysfunction and membrane permeabilization, leading to the release of cytochrome c, which activates caspase 9 and ultimately cleaves caspase 3 / 7, leading to apoptosis. TRAIL can also bind to its decoy receptors TRAIL-R3 / DcR1 and TRAIL-R4 / DcR2, but these receptors lack functional death domains and are unable to induce apoptosis.

[0007] A major difference between TRAIL and other members of the TNF superfamily (such as TNF and CD95L) is that TRAIL cannot induce cell death in normal tissues. A variety of medical or pharmaceutical applications have been attempted to use TNF and CD95L to induce cell death. Because TNF and CD95L proteins induce the death of normal cells as well as cancer cells and overactivated immune cells, their applicability is limited. In contrast, TRAIL induces apoptosis in a wide range of cancer cells and overactivated immune cells, while having little effect on normal cells. This is due to the differential expression of TRAIL receptors between cell types.

[0008] Five TRAIL receptors have been identified. Among them, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) are representative cell death-associated receptors. When TRAIL binds to DR4 or DR5, the intracellular death domain of the receptor is activated, thereby transducing apoptotic signals through various signal transduction pathways, leading to apoptotic cell death. TRAIL can also bind to DcR1, DcR2, and osteoprotegerin (OPG), which do not induce apoptosis. There is no significant difference in the expression levels of the cell death-inducing receptors DR4 and DR5 between normal cells and tumor cells. In contrast, the other three receptors that do not induce apoptosis are expressed at high levels in normal cells, but at low levels or not at all in tumor cells. Therefore, in normal cells, TRAIL mainly binds to DcR1, DcR2, and OPG, which do not contain a death domain, and therefore does not induce cell death. In contrast, in cancer cells and over-activated immune cells, apoptosis is induced by the binding of TRAIL to DR4 and DR5, which contain a death domain. This selective apoptosis induction by TRAIL is a particularly attractive feature in medical or pharmaceutical applications.

[0009] TRAIL-mediated apoptosis has been observed in various types of cancer cells including colon cancer, glioma, lung cancer, prostate cancer, brain tumors and multiple myeloma cells. TRAIL has been shown to have very high anti-cancer activity in animals. Good anti-cancer efficacy of TRAIL has been achieved by using TRAIL alone and in combination with other anti-cancer drugs, such as paclitaxel and doxorubicin and radiotherapy. In addition to cancer, TRAIL is also used to treat arthritis (autoimmune disease), alleviating and treating arthritis by inducing the death of overactivated immune cells. In addition to protein therapy, gene therapy has also been attempted by delivering TRAIL genes. TRAIL can also be used to treat T cell-mediated autoimmune diseases, such as lupus, rheumatoid arthritis and type 1 diabetes.

[0010] However, there are some problems with natural TRAIL as a therapeutic agent. The main problem is that the trimer formation rate of natural TRAIL is low. TRAIL monomers do not bind to TRAIL receptors and therefore do not induce cell apoptosis. In this regard, many studies have been conducted with the aim of improving the trimeric structure and trimer formation rate of TRAIL. Zinc ions play a key role in the trimerization of natural TRAIL. TRAIL mutants were developed based on computer analysis results. For the formation of TRAIL trimers, the most useful method seems to be to introduce amino acid sequences that are conducive to trimer folding. Such sequences include leucine zipper (LZ) motifs and isoleucine zipper (iLZ) motifs. Henning Walczak reported the anti-cancer efficacy of trimeric TRAIL derivatives, in which the leucine zipper motif was added to the N-terminus of natural TRAIL (Walczak et al., Nature Medicine, 5:157-163 (1999)). Kim reported a TRAIL derivative containing an isoleucine zipper motif and having good apoptotic activity (Kim et al., BBRC, 321: 930-935 (2004)).

[0011] TRAIL has different half-lives in different species. For example, it is reported that the half-life of TRAIL in rodents is a few minutes, and the half-life in apes is about 30 minutes (Xiang, et al., Drug Metabolism and Disposition, 32:1230-1238 (2004)). Most TRAIL is rapidly excreted through the kidneys. This short half-life is considered to be a shortcoming of TRAIL pharmaceutical use, making it necessary to have TRAIL or its derivatives with extended half-life. Other problems that need to be solved include the low solubility and solution stability of TRAIL. The half-life of recombinant TRAIL is extremely short, unstable and aggregates at high concentrations. Therefore, the use of unmodified TRAIL in in vivo drug development is limited.

[0012] Another problem in the clinical application of TRAIL is its cytotoxicity to normal cells in certain tissues. Most normal cells are resistant to the cytotoxicity caused by the expression of various TRAIL receptors, but some hepatocytes and keratinocytes are sensitive to TRAIL-mediated cytotoxicity (Yagita et al., Cancer Sci., 95:777-783 (2004); Joe et al., Nature Medicine, 6:564-567 (2000); Zheng et al., J. Clin. Invest., 113:58-64 (2004)).

[0013] There remains a need for biologically active, highly pure TRAIL compositions and TRAIL conjugates that retain the biological activity of native TRAIL, have extended serum half-life, increased solubility, are not cytotoxic to healthy cells, and are non-immunogenic in the host.

[0014] Therefore, one object of the present invention is to provide non-immunogenic TRAIL compositions and TRAIL conjugates with prolonged serum half-life, high solubility, high biological activity and low toxicity to healthy cells.

[0015] Another object of the present invention is to provide a method for preparing non-immunogenic TRAIL compositions and TRAIL conjugates.

[0016] It is another object of the present invention to provide methods of using the non-immunogenic TRAIL compositions and TRAIL conjugates. Summary of the Invention

[0017] Fusion polypeptides with modified multimerization domains and TRAIL domains have high expression, solubility, and stability. Compared to other TRAIL polypeptides in the art with unmodified multimerization domains, fusion polypeptides generally have low immunogenicity in the host. Conjugates of fusion polypeptides, polynucleotides encoding fusion polypeptides, and compositions thereof have been developed. Typically, a fusion polypeptide is a recombinant fusion polypeptide comprising a first sequence and a second sequence containing a modified isoleucine zipper (miLZ) domain. The first sequence may comprise any one of SEQ ID NOs: 4-8. The second sequence is typically a TRAIL domain and may comprise any one of SEQ ID NOs: 9-26. The first sequence may be linked to the second sequence via a linker, such as an amino acid linker. The fusion polypeptide may comprise an expressed sequence preceding the first sequence. An exemplary fusion polypeptide has the amino acid sequence set forth in SEQ ID NO: 30.

[0018] The modified isoleucine zipper (miLZ) domain of the fusion polypeptide has an isoleucine residue modified at position 17 and a lysine residue modified at position 26 of the isoleucine zipper (iLZ) known in the art (iLZ containing the sequence set forth in SEQ ID NO: 1 and miLZ containing the sequence set forth in SEQ ID NOs: 4-8). These modifications to iLZ do not affect the multimerization of the fusion polypeptide and can therefore be used to generate stable, soluble, and immunogenic TRAIL trimers with extended in vivo half-life. This is in contrast to the art showing that an isoleucine residue at this position (Ile at position 20 of SEQ ID NO: 2 (corresponding to position 17 of SEQ ID NO: 1) is required to confer stable trimer formation to TRAIL (Harbury et al., Nature, 371: 80-83 (1994); Rozanov et al., Mol Cancer Ther, 8: 1515-1525 (2009), disclosing SEQ ID NO: 3).

[0019] Multimeric forms, such as trimeric fusion polypeptides, are typically highly soluble, with solubility between about 0.2 mg / ml and 100 mg / ml in physiological buffer, physiological pH, and room temperature. Multimeric forms, such as trimeric fusion polypeptides, have a half-maximal inhibitory concentration (IC50) in vitro that is significantly lower than that of fusion polypeptides containing unmodified iLZ domains. The IC50 of a fusion polypeptide can be from about 1 / 10 to about 1 / 2, or less than 1 / 10, that of a fusion polypeptide containing an unmodified iLZ domain. Fusion polypeptides typically have an in vivo half-life greater than 1 hour, e.g., between about 1 hour and about 24 hours, between about 1 hour and about 12 hours, or between about 1 hour and about 6 hours.

[0020] Fusion polypeptides stably conjugated to half-life extension molecules such as polyethylene glycol (PEG) or its derivatives have been developed. Typically, the molecular weight of PEG or its derivatives is between about 5,000 Da (Daltons) and about 100,000 Da. The conjugates are generally highly soluble, having a solubility of up to about 30 mg / ml, for example, about 25 mg / ml, in the presence of physiological concentrations of salt. The conjugates are generally highly stable and do not significantly reduce solubility and activity with repeated freeze-thaw cycles. The conjugates generally have a long in vivo half-life, for example, an in vivo half-life of about 20 hours to about 50 hours in non-human primates. The IC50 of the conjugate can be about 1 / 10 to about 1 / 2, or less than 1 / 10, of that of the conjugate containing the unmodified iLZ domain. Compared to the conjugate containing the unmodified iLZ domain, the immunogenicity of the conjugate in mammalian hosts is generally significantly reduced.

[0021] Polynucleotides encoding recombinant fusion polypeptides have also been developed. The polynucleotides can be in an expression vector. The expression vector typically includes a promoter operably linked to the polynucleotide. The promoter can be any promoter, including constitutively active, inducible, conditional, or tissue-specific promoters. The polynucleotides can be used for expression in yeast (such as Pichia pastoris) or Escherichia coli (E. coli) expression systems.

[0022] The pharmaceutical composition contains an effective amount of fusion polypeptide, polynucleotide or conjugate. These drugs are usually used for injection administration.

[0023] Methods for preparing fusion polypeptides have been developed. Such methods involve transfecting an expression host, such as E. coli cells, with an expression vector containing a polynucleotide encoding the fusion polypeptide and an inducible promoter. The transfected E. coli cells are used to inoculate a culture medium. The culture medium typically includes zinc ions, for example, in the form of zinc chloride.

[0024] Methods for treating proliferative diseases, autoimmune diseases, or fibrotic diseases in individuals in need thereof require administration of a fusion polypeptide, conjugate, polynucleotide, or pharmaceutical composition thereof to the individual. Fusion polypeptides, conjugates, polynucleotides, or pharmaceutical compositions are particularly effective in inducing apoptosis in cancer-related fibroblasts. In some embodiments, the proliferative disease is a cancer with a solid tumor. In some embodiments, the proliferative disease is hepatocellular carcinoma, pancreatic ductal adenocarcinoma, colon cancer, glioma, lung cancer, prostate cancer, or multiple myeloma. The composition is administered in an amount that effectively induces apoptosis in cancer-related fibroblasts and / or reduces tumor size. In some embodiments, the method includes administering a second active agent. In some embodiments, the second active agent is a chemotherapeutic agent, such as a DNA topoisomerase I inhibitor and a DNA topoisomerase II inhibitor. In some embodiments, the second active agent is an immune checkpoint inhibitor, such as a PD-1 antagonist, a PD-1 ligand antagonist, or a CTLA4 antagonist.In some embodiments, the second active agent is doxorubicin, etoposide, camptothecin, irinotecan, cisplatin, oxaliplatin, docetaxel, cyclophosphamide, 5-fluorouracil, carboplatin, mechlorethamine, sorafenib, chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, paclitaxel and its derivatives, topotecan, amsacrine, etoposide phosphate, phosphate), teniposide, epipodophyllotoxins, trastuzumab, cetuximab, rituximab, bevacizumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, pidilizumab, vopratelimab, danvatirsen, cetrelimab, or ipilimumab. In some embodiments, the method further comprises administering adoptive T cell therapy, a cancer vaccine, surgery, and / or radiation therapy to the individual. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figures 1A-1C is a diagram showing the sequences of known fusion polypeptides and modified fusion polypeptides. Figure 1A is a diagram of the sequence of iLZ-TRAIL (His-iLZ-TRAIL) containing a histidine tag according to the prior art (Chae, et al., Molecular cancer therapeutics 9(6):1719-29 (2010)), wherein the recombinant human TRAIL (amino acids 114-281) is preceded by an isoleucine zipper having a low expression sequence and an immunogenic epitope, which is preceded by a histidine (His) tag; Figure 1BmiLZ-TRAIL—recombinant human TRAIL (amino acids 114-281) preceded by an isoleucine zipper with an improved expression sequence, deimmunized epitopes, and no histidine (His) tag ( Figure 1B );as well as Figure 1C Show Figure 1B The sequence in is PEGylated at the N-terminus with 5 kDa PEG. Figure ID shows a diagram of the structural elements of TLY012, with each monomer of the trimer having the amino acid sequence SEQ ID NO: 31.

[0026] Figure 2A Graph showing the melting curves (fluorescence changes within a temperature range (25-98°C)) of prior art His-TRAIL and miLZ-TRAIL clone #5, whose amino acid sequence is shown in FIG. Figure 1C shown. Figure 2B Line graph showing the bioactivity (change (%) in cell viability over a range of protein concentrations (nM)) of prior art His-iLZ-TRAIL and miLZ-TRAIL, clone #5, tested on COLO205 cells.

[0027] Figure 3 Line graph showing the selective cytotoxicity (change (%) in cell viability over a range of protein concentrations (nM)) of TLY012 tested on quiescent HSCs and activated HSCs (myofibroblasts). The IC50 (nM) for two concentrations is shown.

[0028] Figure 4 Shown is a line graph of the cytotoxicity (change in cell viability (%) over a range of protein concentrations (nM)) of PEGylated His-iLZ-TRAIL (His-TRAIL-PEG, containing SEQ ID NO: 3 and SEQ ID NO: 15, as in SEQ ID NO: 32) and PEGylated miIL-TRAIL (comprising SEQ ID NO: 4 and SEQ ID NO: 15, as in SEQ ID NO: 31) tested on COLO-205 colorectal cancer cells.

[0029] Figures 5A-5H miLZ-TRAIL 1876 (123V, K32E) containing SEQ ID NO: 4 is shown ( Figure 5A and Figure 5E ); iLZ-TRAIL 1877 containing SEQ ID NO: 3 ( Figure 5B and 5F ); miLZ-TRAIL 1878 (K32E) containing SEQ ID NO: 6 ( Figure 5C and Figure 5G ); and miLZ-TRAIL 1879(K32Q) containing SEQ ID NO: 7 ( Figure 5D and Figure 5H ) in 30 mM Hepes, 1 M NaCl, 5 mM DTT (pH 8.0) ( Figures 5A-5D ) or in 50 mM MES, 0.5 M NaCl, 5 mM DTT (pH 6.0) ( Figures 5E-5H ) is a graph showing the changes in fluorescence (-R*[T]) within a certain temperature range (25-98°C).

[0030] Figure 6 This table shows the results of an in silico immunogenicity analysis of various miLZ domains using a ProPred-based algorithm. This software queries a broad range of human MHC-II alleles to determine their ability to bind to potential T-cell epitopes (linear nonapeptides) present in the protein sequence. Each amino acid in the potential T-cell epitope is assigned an immunogenicity score. This score is proportional to the likelihood that the peptide will bind to a specific MHC-II allele and trigger a T-cell immune response.

[0031] Figures 7A-7D Intravenous injection (IV, 10 mg / kg, Figure 7A ) or subcutaneous injection (SC, 2 mg / kg or 10 mg / kg, Figure 7B and 7C ) or 50mg / kg( Figure 7D ), a graph showing changes in TLY012 serum concentration over time (hours) at 4 hr.

[0032] Figure 8A Bar graph showing the results of Western blot analysis of tissue extracts for a-SMA, GRP78, DR4, DR5, PDGF-Rb, and C1.PARP-1. Figures 8B-8D Bar graphs showing the mRNA expression levels of fibrosis markers Acta2, TGF-b, and Pdgf-r, respectively.

[0033] Figure 9A Bar graph showing cell viability (%) of ASPC-1 cells grown with or without PSC-CMs and treated with a combination of TLY012 and sorafenib. Figure 9B Bar graph showing quantified data of tumor weight (mg) of ASPC-1 / PSC xenografts obtained from mice treated with sorafenib, TLY012, or a combination of TLY012 and sorafenib.

[0034] Figure 10Bar graph showing caspase 3 / 7 activity (RLU) in pancreatic CAF cells treated with different doses of TLY012 and caspase 3 / 7 activity (RLU) in activated PSCs treated with the same dose of TLY012.

[0035] Figure 11 Bar graph showing the fold increase in caspase 3 / 7 activity (Fold) in pancreatic CAF cells treated with various cytotoxic agents as indicated.

[0036] Figure 12A Shown are histograms of caspase 3 / 7 activity (RLU) in colon CAF cells treated with different doses (ng / ml) of TLY012. Figure 12B Bar graph showing the fold increase in caspase 3 / 7 activity (fold) in colon CAF cells treated with various cytotoxic agents as indicated.

[0037] Figures 13A-13C Bar graph showing the mRNA expression levels of Dr4, DR5, Acta2, TGF-b, Colla2, PD-L1, and PD-L2 in pancreatic and colon CAFs compared to normal (quiescent) PSCs.

[0038] Figure 14 Shown are the tumor volumes (mm) of tumors obtained from orthotopic pancreatic xenografts of KPC cells in mice treated with saline or TLY012. 3 ) changes in the graph.

[0039] Figure 15A is a line graph of bioluminescent activity (total counts) obtained from mouse tumors with orthotopic xenografts of mouse KPC cells and treated with PBS, anti-PD-L1 antibody (B7-H1, anti-mouse PD-L1), TLY012, or a combination of anti-PD-L1 antibody and TLY012 over time (days). Figure 15B is the tumor volume (mm) of tumors obtained from mice with orthotopic xenografts of mouse KPC cells and treated with PBS, anti-PD-L1 antibody, TLY012, or a combination of anti-PD-L1 antibody and TLY012. 3 ) picture. Figure 15C Shown are survival curves showing the percentage survival over days for mice having orthotopic xenografts of mouse KPC cells and treated with PBS, anti-PD-L1 antibody, TLY012, or a combination of anti-PD-L1 antibody and TLY012.

[0040] Figure 16Graph showing Sirius Red-positive area (%) in tissues of mice bearing orthotopic xenografts of mouse KPC cells and treated with PBS, anti-PD-L1 antibody, TLY012, or a combination of anti-PD-L1 antibody and TLY012.

[0041] Figure 17 Bar graph showing the mRNA expression levels of Acta2, Colla2, Col3a1, PDGFR, and CTGF normalized to GAPDH in quiescent HSC cells (qHSC), activated hepatic stellate cells (HSC), and primary liver cancer-associated fibroblasts (liver CAFs).

[0042] Figure 18A and 18B Shown are highly TRAIL-resistant HCC cells (including Huh-7 cells) treated with vehicle (Vehicle) or TLY012 ( Figure 18A ) and Hep-G2 cells ( Figure 18B ) is a bar graph showing the fold increase of caspase 3 / 7 activity (fold). DETAILED DESCRIPTION

[0043] 1. Definition

[0044] As used herein, the term "polypeptide" includes proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. These sequences are written from left to right in the direction from the amino group to the carboxyl terminus. According to standard nomenclature, amino acid residue sequences are named by three letters or single letter codes, as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

[0045] As used herein, the term "variant" refers to a polypeptide or polynucleotide that is different from a reference polypeptide or reference polynucleotide but retains essential properties. A typical variant of a polypeptide differs from another reference polypeptide in amino acid sequence. Typically, the differences are limited so that the sequences of the reference polypeptide and the variant are very similar overall and, in many regions, are identical. The amino acid sequences of the variant and the reference polypeptide may differ by one or more modifications (e.g., substitutions, additions, and / or deletions). The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be an unknown naturally occurring variant.

[0046] Modifications and changes can be made in the structure of a polypeptide and still obtain a molecule with similar characteristics to the polypeptide (e.g., conservative amino acid substitutions). For example, certain amino acids can be substituted for other amino acids in the sequence without a significant loss of activity. Because it is the interaction ability and properties of a polypeptide that determine the biological functional activity of the polypeptide, certain amino acid sequence substitutions can be made in a polypeptide sequence and still obtain a polypeptide with similar properties.

[0047] When making such changes, the hydropathic index of the amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids with similar hydropathic indices or scores and still produce a polypeptide with similar biological activity. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. These indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0048] It is believed that the relative hydrophilicity of amino acids determines the secondary structure of synthetic polypeptides, and the secondary structure in turn determines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies and antigens. It is known in the art that an amino acid can be substituted with another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such variations, it is preferred to substitute amino acids whose hydropathic index is within ±2, particularly those within ±1 and even more particularly those within ±0.5.

[0049] Substitution of similar amino acids can also be made based on hydrophilicity. The hydrophilicity values ​​of amino acid residues are as follows: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 ± 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine ​​(1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (2.3); phenylalanine (-2.5); tryptophan (-3.4). It should be understood that an amino acid can be substituted with another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, particularly immunoequivalent, polypeptide. In such changes, it is preferred to substitute amino acids whose hydropathic indices are within ±2, particularly preferred those within ±1 and even more particularly preferred those within ±0.5.

[0050] As mentioned above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge and size. In particular, embodiments of the polypeptide may include variants having about 50%, 60%, 70%, 80%, 90% and 95% sequence identity with the polypeptide of interest.

[0051] As used herein, the term "identity," as known in the art, refers to the relationship between two or more polypeptide sequences as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptides as determined by the match between these sequence strings. "Identity" can also refer to the degree of sequence relatedness between a polypeptide and a reference polypeptide over its entire length. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to (Computational Molecular Biology, Lesk, AM, Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Described in Math., 48:1073 (1988).

[0052] Various programs and alignment algorithms are described in: Smith & Waterman, Adv Appl Math 2, 482 (1981); Needleman & Wunsch, J Mol Biol 48, 443 (1970); Pearson & Lipman, Proc Natl Acad Sci USA 85, 2444 (1988); Higgins & Sharp, Gene 73, 237-244 (1988); Higgins & Sharp, CABIOS 5, 151-153 (1989); Corpet et al, Nuc Acids Res 16, 10881-10890 (1988); Huang et al, Computer App Biosci 8, 155-165 (1992); and Pearson et al, Meth Mol Bio 24, 307-331 (1994). Additionally, Altschul et al, J Mol Biol 215, 403-410 (1990), consider sequence alignment methods and homology calculations in detail.

[0053] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al, (1990) supra) is available from a number of sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and the internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. For more information, please visit the NCBI website. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, the specified output file will display these homology regions as aligned sequences. If the two compared sequences do not share homology, the specified output file will not display the aligned sequences.

[0054] The preferred method for determining identity is designed to provide maximum matching between the sequences tested. Methods for determining identity and similarity are incorporated into publicly available computer programs. The percent identity between two sequences can be determined using analysis software (i.e., Sequence Analysis Software Package of Genetics Computer Group, Madison Wis.) comprising the Needelman and Wunsch (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST and XBLAST). Default parameters are used to determine the identity of a polypeptide.

[0055] For example, a polypeptide sequence can be identical to a reference sequence, i.e., 100% identical, or the polypeptide sequence can include up to a certain integer number of amino acid changes compared to the reference sequence such that the percent identity is less than 100%. Such changes are selected from at least one amino acid deletion, substitution (including conservative and non-conservative substitutions), or insertion, wherein the changes may occur at the amino or carboxyl terminal positions of the reference polypeptide sequence or at any position between these terminal positions, interspersed individually between amino acids in the reference sequence or in one or more adjacent groups within the reference sequence. The number of amino acid changes for a given percent identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percentage of the corresponding percent identity (divided by 100) and then subtracting this product from the total number of amino acids in the reference polypeptide.

[0056] The term "percent (%) sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a reference nucleic acid or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. Alignment for determining percent sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment can be determined by known methods, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0057] As used herein, the term "recombinant polynucleotide" generally refers to a polynucleotide obtained by genetic engineering techniques.

[0058] As used herein, the term "recombinant polynucleotide" generally refers to a polypeptide obtained from a recombinant polynucleotide. A recombinant nucleic acid or polypeptide is a nucleic acid or polypeptide having a sequence that does not occur naturally or that is formed by the artificial combination of two or more otherwise isolated sequence fragments. This artificial combination is typically achieved through chemical synthesis or, more commonly, through the artificial manipulation of isolated nucleic acid fragments, such as through genetic engineering techniques. A recombinant polypeptide may also refer to a polypeptide prepared using a recombinant nucleic acid, including a recombinant nucleic acid transferred to a host organism that is not the natural source of the polypeptide.

[0059] As used herein, the term "purified" refers to a molecule or compound in a form that is substantially free (at least 60% free, preferably 75% free, and most preferably 90% free) from other components normally associated with the molecule or compound in its natural environment.

[0060] As used herein, the term "fusion polypeptide" refers to two or more polypeptides joined by a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide, or a polypeptide formed by linking one polypeptide to another by a reaction between amino acid linkers or amino acid side chains (e.g., a disulfide bond between cysteine ​​residues on each polypeptide). A fusion protein can be formed by chemical coupling of the constituent polypeptides, or can be expressed as a single polypeptide from a nucleic acid sequence encoding a single continuous fusion protein. Fusion proteins can be prepared using conventional techniques in molecular biology to link two genes in frame into a single nucleic acid sequence, and then expressing the nucleic acid in an appropriate host cell under conditions that express the fusion protein.

[0061] As used herein, the term "monomer" refers to a single fusion polypeptide molecule. As used herein, the terms "dimer," "trimer," "tetramer," or "multimer" refer to two, three, four, or more monomers forming a polypeptide molecule, respectively. A dimer, trimer, tetramer, or multimer may be a homodimer, homotrimer, homotetramer, or homomultimer, wherein each monomer in the monomers forming the dimer, trimer, tetramer, or multimer contains the same amino acid sequence. A dimer, trimer, tetramer, or multimer may be a heterodimer, heterotrimer, heterotetramer, or heteromultimer, wherein each monomer in the monomers forming the dimer, trimer, tetramer, or multimer contains a different amino acid sequence.

[0062] As used herein, the term "solubility" refers to the maximum concentration of a polypeptide or conjugate in a physiological buffer at physiological pH and room temperature at which the polypeptide or conjugate does not substantially aggregate. Solubility and / or aggregation can be detected by chromatography, gel electrophoresis, or melting / aggregation analysis.

[0063] As used herein, the term "in vivo half-life" generally refers to half of the maximum time that a polypeptide or conjugate remains in the in vivo circulation. In vivo half-life can be measured by testing the activity of the polypeptide or conjugate after obtaining plasma samples at different time intervals from an individual to whom the polypeptide or conjugate is administered. In vivo half-life can be measured by a detection assay that detects the presence of the polypeptide or conjugate in plasma samples obtained at different time intervals from an individual to whom the polypeptide or conjugate is administered.

[0064] As used herein, the term "nucleic acid" refers to any natural or synthetic linear continuous array of nucleotides and nucleosides, such as DNA, including complementary DNA (cDNA), replicating RNA (repRNA) and messenger RNA (mRNA). The term "nucleic acid" further includes modified or derivatized nucleotides and nucleosides, such as, but not limited to, halogenated nucleotides, such as 5-bromouracil, and derivatized nucleotides, such as biotin-labeled nucleotides.

[0065] As used herein, the term "polynucleotide" refers to a single molecule comprising more than one nucleic acid molecule, wherein each nucleic acid molecule encodes a different protein and / or performs a different function in an expression vector. The polynucleotide can be in an expression vector, such as a plasmid, cosmid, or viral vector.

[0066] As used herein, the term "expression vector" refers to a recombinant genetic molecule containing one or more isolated polynucleotide sequences. Expression vectors for polynucleotide expression in a host organism include a promoter sequence in the 5'-3' direction; a sequence encoding the polynucleotide of interest; and a termination sequence. The vector may also include a selectable marker gene and other regulatory elements for expression. The vector may be suitable for expression in prokaryotic cells such as Escherichia coli, or in eukaryotic cells such as yeast cells or mammalian cells.

[0067] As used herein, the term "host," "subject," or "patient" refers to any individual to whom administration is directed.

[0068] As used herein, the term "immunogenic" in the context of a polypeptide, conjugate, or composition refers to a polypeptide, conjugate, or composition that is capable of inducing an immune response and is therefore antigenic. An "immune response" refers to any reaction of the immune system. These reactions include alterations in the activity of an organism's immune system in response to a polypeptide, polynucleotide, conjugate, or composition thereof, and may involve, for example, antibody production, induction of cell-mediated immunity, complement activation, or the development of immune tolerance.

[0069] As used herein, the phrase "significantly less immunogenicity" provides that the immunogenicity score is reduced or inhibited by greater than at least about 25%, at least about 20%, at least about 15%, at least about 12.5%, at least about 10%, or at least about 5% relative to the immunogenicity of the corresponding polypeptide or the corresponding conjugate in a host or the immunogenicity of the corresponding polypeptide or the corresponding conjugate in ex vivo immune cells as predicted by computer simulation.

[0070] As used herein, the term "corresponding polypeptide" refers to a polypeptide that is generally formed by a similar first sequence and / or a similar second sequence as a reference polypeptide, but does not have modifications of the reference polypeptide.

[0071] As used herein, the term "corresponding conjugate" refers to a conjugate that is generally formed from the same or similar first sequence and / or the same or similar second sequence as a reference conjugate, but does not have the modifications of the reference conjugate.

[0072] As used herein, the term "treating" includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.

[0073] The terms "reduce," "inhibit," "alleviate," or "reduce" are used relative to a control. One skilled in the art will readily identify the appropriate control for each experiment. For example, the response in an individual or cell treated with a polypeptide is reduced compared to a control, such as the response of an individual or cell not treated with the polypeptide or the corresponding polypeptide or conjugate. Reduction can be complete inhibition or reduction of activity, expression, or symptoms, or partial inhibition or reduction. Inhibition can be a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, %, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0074] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to treat, suppress or alleviate one or more symptoms of the disease state being treated or otherwise provide a desired pharmacological and / or physiological effect. The exact dosage will vary according to a variety of factors, such as individual-related variables (e.g., age, immune system health status, etc.), the disease or condition, and the ongoing treatment. The effect of an effective amount can be relative to a control. Such controls are known in the art and discussed herein, and can be, for example, the condition of an individual before or without administration of a drug or drug combination, or, in the case of a drug combination, the effect of the combination can be compared to the effect of administering only one drug.

[0075] As used herein, the term "combination therapy" refers to a method of treating a disease or its symptoms, or achieving a desired physiological change, by administering effective amounts of two or more agents or components to treat the disease or condition or produce a physiological change, wherein the agents or components are administered together, e.g., as part of the same composition, or separately and independently at the same time or different times (i.e., the administration of each agent or component is separated from each other by a limited period of time).

[0076] As used herein, the term "dosage regime" refers to the administration regimen, administration route, dosage, administration interval, and treatment duration of a polypeptide, polynucleotide, conjugate, or composition thereof.

[0077] As used herein, the term "substantially" refers to a comparative measurement of at least about 25%, at least about 20%, at least about 15%, at least about 12.5%, at least about 10%, or at least about 5% relative to a control.

[0078] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0079] Use of the term "about" is intended to describe a value that is within a range of about + / - 10 above or below the stated value.

[0080] 2. Compositions

[0081] A. Modified fusion polypeptides

[0082] Fusion polypeptides comprise a modified multimerization domain as a first sequence and a protein of interest as a second sequence. Conjugates of fusion polypeptides are also described. Conjugates typically include a fusion polypeptide and a half-life extending molecule. The modified multimerization domain provides the fusion polypeptide with high expression, solubility, stability, and low immunogenicity.

[0083] The TRAIL compositions with modified multimerization domains show improved physicochemical and biological properties compared to TRAIL compositions with state of the art multimerization domains. The TRAIL compositions are also less immunogenic compared to TRAIL compositions with state of the art multimerization domains.

[0084] Also described are polynucleotides encoding fusion polypeptides. The polynucleotides can be contained in expression vectors. The fusion polypeptides produced by expressing the vectors in prokaryotic or eukaryotic cells can be frozen and stored in the cells, or purified from the cells for further use.

[0085] Fusion polypeptides generally include a first sequence and a second sequence. The first sequence is the amino acid sequence of a modified multimerization domain, such as a modified isoleucine zipper (miLZ) domain. The second sequence is the amino acid sequence of a protein or peptide with a dimer, trimer, tetramer or multimer tertiary structure. An exemplary protein or peptide is a TRAIL protein monomer, which is a homotrimer in its tertiary structure.

[0086] 1. First sequence

[0087] Typically, the first amino acid sequence includes a modified multimerization sequence of a yeast GCN4 pII leucine zipper (LZ). It is recognized in the art that GCN4-pII (Ile (I)) modified at certain amino acid positions to replace leucine (Leu (L)) with isoleucine (Ile (I)) (Harbury et al, Nature, 371: 80-83 (1994); Rozanov et al., Mol Cancer Ther, 8: 1515-1525 (2009)) to form an isoleucine zipper (iLZ) sequence. It is also recognized in the art that the use of these modifications and these zipper sequences is immunogenic in humans (US 2016 / 0280761).

[0088] In the fusion polypeptides described herein, the first sequence typically includes a modified isoleucine zipper (miLZ) sequence that has reduced immunogenicity in mammalian hosts. The modified isoleucine zipper sequence is shown below, and in this section, the modified amino acid is shown at the position relative to the following sequence:

[0089] 1) core sequence (SEQ ID NO: 1),

[0090] 2) the prior art iLZ sequence (SEQ ID NO: 2; described in Rozanov et al., Mol Cancer Ther, 8: 1515-1525 (2009)); and

[0091] 3) Second prior art iLZ sequence (SEQ ID NO: 3).

[0092] Prior art LZ and iLZ sequences are expressed as:

[0093] KQIEDKIEEILSKIYHIENEIARIKKLIGE (SEQ ID NO: 1, a fragment from a modified yeast GCN4-pII leucine zipper (LZ) motif containing isoleucine substitutions at positions 6, 10, 13, 17, 20, 24, 27, and 31 (GRMKQIEDKIEEILSKIYHIENEIARIKKLIGER, SEQ ID NO: 2, iLZ); Harbury et al, Nature, 371: 80-83 (1994); Rozanov et al, Mol Cancer Ther, 8: 1515-1525 (2009)), or

[0094] PGMCGGKQIEDKIEEILSKIYHIENEIARIKKLIGEDGV (SEQ ID NO: 3), iLZ).

[0095] For consistency in the present invention, the first sequence is represented by its SEQ ID NO and the modified amino acids are represented by their positions relative to SEQ ID NO: 1.

[0096] The first sequence comprising miLZ typically includes any one of SEQ ID NOs: 4-8 as part of miLZ:

[0097] KQIEDKIEEILSKIYHVENEIARIKELIGE (SEQ ID NO: 4; I17V, K26E; or relative to SEQ ID NO: 2, I20V, K29E, or relative to SEQ ID NO: 3, I23V, K32E)

[0098] KQIEDKIEEILSKIYHVENEIARIKKLIGE (SEQ ID NO: 5; I17V, or relative to SEQ ID NO: 2, I20V, or relative to SEQ ID NO: 3, I23V)

[0099] KQIEDKIEEILSKIYHIENEIARIKELIGE (SEQ ID NO: 6; K26E, or relative to SEQ ID NO: 2, K29E, or relative to SEQ ID NO: 3, K32E)

[0100] KQIEDKIEEILSKIYHIENEIARIKQLIGE (SEQ ID NO: 7; K26Q; relative to SEQ ID NO: 2, K29Q, or relative to SEQ ID NO: 3, K32Q)

[0101] KQIEDKIEEILSKVYHIENEIARIKELIGE (SEQ ID NO: 8; I14V, K26E, or relative to SEQ ID NO: 2, I17V; K29E, or relative to SEQ ID NO: 3, I17V, K32E).

[0102] The miLZ domain of the fusion polypeptide has the following modifications in the iLZ known in the art: an isoleucine residue at position 17 and a lysine residue at position 26 (iLZ containing the sequence shown in SEQ ID NO: 1, miLZ containing the sequences shown in SEQ ID NOs: 4-8). These modifications of the prior art iLZ do not affect the multimerization of the fusion polypeptide and produce stable, soluble, TRAIL trimers with increased in vivo half-life and reduced immunogenicity. This is in contrast to the prior art, which suggests that isoleucine residues at these positions (Ile at position 20 of SEQ ID NO: 2 (corresponding to position 17 in SEQ ID NO: 1) are required and confer stable trimer formation to TRAIL (Harbury et al., Nature, 371: 80-83 (1994); Rozanov et al., Mol Cancer Ther, 8: 1515-1525 (2009), disclosing SEQ ID NO: 3).

[0103] 2.Second sequence

[0104] The second sequence can be the amino acid sequence of a protein or polypeptide that typically functions as a homodimer or heterodimer, homotrimer or heterotrimer, homotetramer or heterotetramer, or homomultimer or heteromultimer. An exemplary second sequence is the complete sequence or a fragment, variant, or homolog of a human TRAIL polypeptide.

[0105] TRAIL / Apo2L (TNFSF10) was initially identified in an EST database search for genes homologous to known TNF superfamily ligands (Benedict et al., J. Exp. Med., 209(11):1903-1906(2012)). In humans, TRAIL binds to two pro-apoptotic death receptors (DRs), TRAIL-R1 and TRAIL-R2 (TNFRSF10A and TNFRSF10B), as well as two other membrane receptors that do not induce death but instead act as decoys for death signals. Binding of TRAIL to its cognate DRs induces the formation of a death-inducing signaling complex, ultimately leading to caspase activation and the initiation of apoptosis (Benedict et al., J. Exp. Med., 209(11):1903-1906(2012)).

[0106] In some embodiments, the second sequence comprises the amino acid sequence of a TRAIL peptide monomer.

[0107] The nucleic acid and amino acid sequences of human TRAIL are known in the art. For example, the amino acid sequence of human TRAIL is MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG (SEQ ID NO: 9, (UniProtKB database accession number P50591 (TNF10_HUMAN)). In some embodiments, the second sequence comprises a TRAIL peptide comprising or having the amino acid sequence of SEQ ID NO: 10.

[0108] A TRAIL homolog or variant may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 9. A conservative amino acid substitution is a substitution of one amino acid with a structurally similar amino acid.

[0109] Preferably, TRAIL is soluble TRAIL. Endogenous full-length TRAIL includes a cytoplasmic domain, a transmembrane domain, and an extracellular domain. Typically, soluble TRAIL is a fragment of full-length TRAIL that does not contain the cytoplasmic domain and the transmembrane domain. Therefore, soluble TRAIL can be the extracellular domain of TRAIL (e.g., the extracellular domain of SEQ ID NO: 9) or a functional fragment thereof. The common extracellular domain of TRAIL of SEQ ID NO: 9 is amino acids 39-281 of SEQ ID NO: 9 (SEQ ID NO: 10). Thus, in some embodiments, the second sequence comprises a TRAIL peptide or a functional fragment or variant thereof, wherein the TRAIL peptide comprises or has amino acids 39-281 (SEQ ID NO: 10), 41-281 (SEQ ID NO: 11), 91-281 (SEQ ID NO: 12), 92-281 (SEQ ID NO: 13), 95-281 (SEQ ID NO: 14) and 114-281 (SEQ ID NO: 15) of SEQ ID NO: 9.

[0110] In some embodiments, the second sequence comprises a functional fragment or variant of SEQ ID NO: 7, which can inhibit signaling through TRAIL-R1 and / or TRAIL-R2. The fragment or variant of SEQ ID NO: 9 can have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 9.

[0111] Preferably, the functional fragment or variant thereof comprises the extracellular domain of SEQ ID NO: 9 or a functional fragment thereof. It is believed that the C-terminal 150 amino acids of TRAIL comprise the receptor binding domain. Thus, in some embodiments, the functional fragment comprises amino acids 132-281 of SEQ ID NO: 9 (SEQ ID NO: 16). In other specific embodiments, the fragment is amino acids 95-281 of SEQ ID NO: 9 (SEQ ID NO: 14) or amino acids 114-281 of SEQ ID NO: 9 (SEQ ID NO: 15).

[0112] The variant may have one or more substitutions, deletions, or additions relative to SEQ ID NOs: 9-15, or any combination thereof. In some embodiments, the variant is a naturally occurring alternative sequence, splice variant, or substitution, addition, or deletion variant, or an extracellular domain or a functional fragment thereof, or an alternative sequence, splice variant, or substitution, addition, or deletion variant. Naturally occurring alternative sequences and variants are disclosed in the UniProtKB database under accession number P50591 (TNF10_HUMAN), version 140 (last modified on January 22, 2014).

[0113] a. TRAIL analogs

[0114] TRAIL can interact with its receptor as a trimer. In some embodiments, the second sequence can form a multimer, preferably a trimer. The trimer can be a homotrimer or a heterotrimer.

[0115] All TRAIL proteins described herein can be prepared using standard techniques for isolating native or recombinant proteins and chemically modified as described herein.

[0116] The second sequence can include a TRAIL analog or an agonistic TRAIL receptor binding fragment or variant thereof. TRAIL analogs are known in the art. In preferred embodiments, the analog has increased affinity or specificity for one or more agonistic TRAIL receptors (e.g., TRAIL-R1 (DR4) and / or TRAIL-R2 (DR5)) and decreased affinity or specificity for one or more antagonistic or decoy TRAIL receptors (e.g., receptors DcR1 and DcR2) or a combination thereof, compared to wild-type or endogenous TRAIL.

[0117] In some embodiments, the analog is a DR4-selective mutant of wild-type TRAIL. DR-4-selective mutants are known in the art and described, for example, in Tur, J. Biological Chemistry, 283(29):20560-8 (2008). In certain embodiments, the analog is a variant of SEQ ID NO: 9 having a D218H (SEQ ID NO: 17) or D218Y (SEQ ID NO: 18) substitution, or a functional fragment thereof (e.g., an extracellular domain).

[0118] In some embodiments, the analog is a DR5 selective mutant of wild-type TRAIL. Specific DR-5 selective mutants include variants of SEQ ID NO: 9 with D269H (SEQ ID NO: 19), D269H / E195R (SEQ ID NO: 20), or D269H / T214R (SEQ ID NO: 21), and functional fragments thereof (e.g., extracellular domains). These variants are described in van der Sloot, Proc. Nat. Acad. Sci. USA 103(23):8634-9 (2006).

[0119] b. Other TRAIL analogs

[0120] In yet further examples, the second sequence comprises one or more stabilizing mutations to the native TRAIL sequence SEQ ID NO: 9, such as S133P (SEQ ID NO: 22), S156C (SEQ ID NO: 23), L196C (SEQ ID NO: 24), T127C (SEQ ID NO: 25), or H270C (SEQ ID NO: 26). Examples of sequences having such mutations include SEQ ID NOs: 11-26.

[0121] Typically, the second amino acid sequence comprises the sequence of any one of SEQ ID NOs: 9-26.

[0122] 3. Connectors

[0123] The first sequence can be fused to the second sequence with or without a linker.

[0124] The linker can be an amino acid linker, for example, an amino acid linker of 1 to 4 or 1 to 6 amino acids in length. Exemplary amino acid linkers include D, G, DG, KGSG (SEQ ID NO: 27), GSG, SG, and RGSG (SEQ ID NO: 28).

[0125] 4. Expression sequence

[0126] The fusion polypeptide may include an expressed sequence preceding the first amino acid sequence. The expressed sequence may be 2 to 6 or 2 to 8 amino acids in length. Exemplary expressed sequences include RM, GRM, CGG, PGMCGG (SEQ ID NO: 29).

[0127] 5. Exemplary Fusion Polypeptides

[0128] An exemplary fusion polypeptide is presented as SEQ ID NO:30 and comprises SEQ ID NO:4 as the first amino acid sequence, SEQ ID NO:15 as the second amino acid sequence, linked together by a DG linker (underlined), and preceded by a GRM expression sequence (underlined):

[0129] GRM KQIEDKIEEILSKIYHVENEIARIKELIGE DG VRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYYYSQTYFRFQEEKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG (SEQ ID NO: 30, used as clone #5 in the Examples).

[0130] Another exemplary sequence is SEQ ID NO: 31:

[0131] It shows the miLZ sequence having more favorable properties than the iLZ sequence in the prior art. It comprises SEQ ID NO: 4 (bold) and SEQ ID NO: 15 (italic).

[0132] An exemplary prior art construct is SEQ ID NO: 32:

[0133] It contains SEQ ID NO: 30 (bold) and SEQ ID NO: 15 (italic) as the iLZ and TRAIL sequences, respectively.

[0134] Other exemplary fusion polypeptides include polypeptides comprising any one of the sequences of SEQ ID NOs: 4-8 in combination with any one of SEQ ID NOs: 9-26.

[0135] Fusion polypeptides can be dimerized, trimerized, or multimerized. Dimerization, trimerization, or multimerization can occur between two or more fusion proteins via a dimerization domain, trimerization domain, or multimerization domain. Alternatively, dimerization, trimerization, or multimerization of the fusion protein can occur via chemical cross-linking. The dimers, trimers, or multimers formed can be homodimers / homomultimers or heterodimers / heteromultimers.

[0136] 6. Properties of Fusion Peptides

[0137] The fusion polypeptides exhibit improved physicochemical and biological properties relative to TRAIL compositions with multimerization domains known in the art. The fusion polypeptides also have lower immunogenicity compared to TRAIL compositions with multimerization domains known in the art.

[0138] a. Solubility

[0139] Typically, the solubility of the fusion polypeptide is between about 0.2 mg / ml and 30 mg / ml (in physiological buffer, physiological pH and room temperature). Typically, no substantial or detectable aggregation is observed in solutions containing the fusion polypeptide at a concentration between 0.2 mg / ml and 30 mg / ml. No substantial or detectable aggregation of the polypeptide is shown in solutions containing the fusion polypeptide at a concentration between about 0.2 mg / ml and 30 mg / ml, between about 0.5 mg / ml and 25 mg / ml, between about 0.5 mg / ml and 20 mg / ml, between about 0.5 mg / ml and 15 mg / ml, between about 0.5 mg / ml and 10 mg / ml, or between about 0.5 mg / ml and 5 mg / ml.

[0140] b. Stability and half-life

[0141] Typically, the fusion polypeptide has improved stability when stored at 4°C or frozen. The fusion polypeptide also has improved stability when stored frozen in cells expressing the fusion polypeptide (as frozen cell blocks (cell mass)). The expressing cells can produce the fusion polypeptide at a production level of about 1g of fusion polypeptide per milliliter of cell culture. The expressing cells with the fusion polypeptide can be stored for more than one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve months without a large loss of soluble fusion polypeptide when thawed. Typically, the fusion polypeptide remains stable in the expressing cells when repeated freeze-thaw cycles.

[0142] As shown in the following examples, compared with the fusion polypeptides with iLZ motif and His tag purification sequence in the art (SEQ ID NO: 3 and SEQ ID NO: 15; Figure 2A), the fusion polypeptide comprising SEQ ID NO: 4 as the first sequence and SEQ ID NO: 15 as the second sequence had statistically higher thermodynamic stability in a thermal shift assay.

[0143] The half-life of the fusion polypeptide in vivo is generally between about 1 hour and 36 hours.

[0144] c. Biological activity

[0145] Typically, the fusion polypeptide has improved biological activity compared to fusion polypeptides having an iLZ motif known in the art. Typically, when tested on COLO 205 cells, the fusion polypeptide has an in vitro half-maximal inhibitory concentration (IC50) of between about 0.001 nM and about 0.1 nM, preferably between about 0.001 nM and about 0.05 nM.

[0146] Typically, the fusion polypeptide has a biological activity (e.g., IC50) that is at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 times greater when compared to fusion polypeptides having an iLZ motif known in the art.

[0147] As shown in the following examples, a fusion polypeptide comprising SEQ ID NO: 4 as a first sequence and SEQ ID NO: 15 as a second sequence has about 5-fold higher biological activity in a cell viability assay compared to a fusion polypeptide comprising an iLZ motif and a His-tag purification sequence (SEQ ID NO: 3 and SEQ ID NO: 15; Figure 2B ).

[0148] d. Low immunogenicity

[0149] Typically, the modified fusion polypeptide carrying miLZ has lower immunogenicity in a host compared to the immunogenicity of the unmodified polypeptide carrying iLZ in the same host. Typically, the host is a mammal, preferably a human.

[0150] The lower immunogenicity of the modified fusion polypeptide carrying miLZ may be a lower immunogenicity score compared to the unmodified polypeptide carrying iLZ.

[0151] For therapeutic applications, the "immune response" of human individuals is of particular concern because it may cause toxicity, alter or counteract the therapeutic effect of a treatment modality (e.g., a polypeptide). To predict the immunogenicity of iLZ-TRAIL fusion polypeptides, in silico analysis of the primary amino acid sequence has been used. This method is based on the well-established PROPRED model for predicting MHC class II binding regions in antigenic protein sequences (Singh & Raghava, Bioinformatics, v17, N 12, p 1236; and Brison et al., Biodrugs, v.24, N 1, p 1, 2010). As a next step in comparing the immunogenicity of fusion polypeptides with modified multimerization domains and TRAIL compositions with multimerization domains in the art, in situ testing is being performed using the EPISCREENT MDC:T cell assay provided by ABZENA (San Diego, CA). The test uses dendritic cells (DCs) derived from peripheral blood mononuclear cells (PBMCs), typically from 50 individual donors whose HLA-DR allotype distribution (coverage and frequency) represents the population of interest. The DCs are differentiated into an immature DC phenotype and loaded with the test protein. Once mature, the DCs are incubated with autologous CD4+ T cells and T cell activation markers are measured.

[0152] Typically, the fusion polypeptide carrying the miLZ modification produces minimal or lower levels of T cell activation markers in a host compared to the levels of T cell activation markers produced in the same host by the unmodified polypeptide carrying iLZ as detected by the EPISCREENT MDC: T cell assay or an equivalent assay.

[0153] Typically, the fusion polypeptide has an overall immunogenicity score that is reduced by at least about 15%, at least about 30%, at least about 50% when compared to a fusion polypeptide having an iLZ motif (eg, SEQ ID NO: 3) known in the art.

[0154] For example, Abzena used the prediction algorithm iTope AI to analyze two protein sequences of interest to identify peptides that bind to human MHC class II and / or have homology to known T cell epitopes. From this analysis, SEQ ID NO: 32 (containing SEQ ID NO: 3 and SEQ ID NO: 15) and SEQ ID NO: 31 (containing SEQ ID NO: 4 and SEQ ID NO: 15) were determined to have total scores of 27 and 5, respectively, where neither of the peptide sequences had any homology to the peptides from TCED. TM (Abzena database) T cell epitope matching.

[0155] As shown below, SEQ ID NO: 31 contains a miLZ sequence with more favorable properties than the iLZ sequence in SEQ ID NO: 32, and has a significantly better overall score (5 compared to 27), which matches the best score for a monoclonal human antibody.

[0156] The low immunogenicity of the miLZ-TRAIL conjugate is generally comparable to the immunogenicity of therapeutic human proteins (e.g., monoclonal human antibodies). Thus, the total MHCII binding score of miLZ-TRAIL (SEQ ID 4 and SEQ ID 15) in computer simulations is comparable to or better than the immunogenicity of a typical human monoclonal antibody. Compared to iLZ TRAIL, miLZ-TRAIL has a lower immunogenicity, with the total MHCII binding score in computer simulations being several-fold lower (5 compared to 27). Typically, the immunogenicity of the modified fusion polypeptide is reduced by at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times, preferably between about 2 and 10 times.

[0157] B. Fusion Peptide Conjugates

[0158] The fusion polypeptide may be coupled to more than one half-life extending molecule. Typically, the conjugate is a stable conjugate that does not release the half-life extending molecule in vivo.

[0159] 1. Half-life extension molecules

[0160] Hydrophilic polymers such as polyalkylene oxides or copolymers thereof such as those sold by BASF It can be covalently bound to fusion polypeptides to improve their pharmacokinetic and pharmacodynamic properties (Kim, et al., Bioconjugate Chem., 22(8), pp 1631–1637 (2011)).

[0161] In other embodiments, biopolymers or polypeptides can be used by reported methods such as, but not limited to, chemically conjugated hyaluronic acid (Yang et al., Biomaterials, 32(33):8722-8729 (2011), depot forming polypeptides (Amiram et al., Proc Natl Acad Sci USA, 110(8):2792-2792 (2013), U.S. Published Application No. US 2013 / 0178416 A1), and fusion polypeptides linked to extended recombinant polypeptides (U.S. Published Application No. US 2010 / 0239554 A1), fusion polypeptides can be derived into long-acting polypeptides with extended half-lives.

[0162] 2. Fusion polypeptide-PEG conjugate

[0163] Fusion polypeptides can be stably conjugated to more than one PEG molecule.

[0164] Studies have shown that TRAIL analogs derivatized with PEG maintain anticancer activity (such as inducing apoptosis of cancer cells) while exhibiting higher metabolic stability, prolonged pharmacokinetic profile, and longer circulating half-life in plasma (Chae, et al., Molecular cancer therapeutics 9(6):1719-29 (2010); Kim, et al., Bioconjugate chemistry, 22(8):1631-7 (2011a); Kim, et al., Journal of pharmaceutical sciences 100(2):482-91 (2011b); Kim, et al., Journal of controlled release: official journal of the Controlled Release Society 150(1):639 (2011c)).

[0165] Thus, in some embodiments, the fusion polypeptide is derivatized with one or more ethylene glycol (EG) units, more preferably two or more EG units (i.e., polyethylene glycol (PEG)) or a derivative thereof. Derivatives of PEG include, but are not limited to, methoxypolyethylene glycol succinimidyl propionate, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol aldehyde, methoxypolyethylene glycol maleimide, and multi-branched polyethylene glycol.

[0166] The precise number of EG or derivative units depends on the desired activity, plasma stability, and pharmacokinetic profile. For example, Kim et al. (Kim et al., 2011a) reported that 2K, 5K, 10K, 20K, and 30K-PEG-TRAIL resulted in higher circulation half-lives in mice, being 3.9 hours, 5.3 hours, 6.2 hours, 12.3 hours, and 17.7 hours, respectively, compared to a circulation half-life of 1.1 hours for TRAIL. In some embodiments, the molecular weight of PEG is between about 1 and 100 kDa. For example, PEG can have a molecular weight of "N" kDa, where N is any integer between 1 and 100. PEG can have a molecular weight of "N" Da, where N is any integer between 1,000 and 1,000,000. In a particular embodiment, the molecular weight of PEG is "N" Da, where "N" is between 5,000 and 50,000, preferably 5,000.

[0167] Fusion polypeptides can be conjugated to linear or branched PEG. Some studies have shown that proteins derivatized with branched PEG have prolonged in vivo circulation half-life compared to linear PEG proteins, which is believed to be partly due to the larger hydrodynamic volume of branched PEG proteins. Fee, et al., Biotechnol Bioeng., 98(4):725-3 (2007).

[0168] The peptide ligands may be derivatized at the C-terminus, or preferably at the N-terminus, using methods known in the art.

[0169] The conjugate can be described by the following formula:

[0170] XL-(PEG) n ,

[0171] in

[0172] X represents TRAIL protein or TRAIL fusion polypeptide,

[0173] L represents a connector,

[0174] PEG represents a linear or branched poly(ethylene glycol) chain, and

[0175] n is an integer selected from 2, 3, 4, 5, 6, 7 or 8.

[0176] In certain embodiments, n is 2.

[0177] The polyalkylene oxide is bound to the protein via a linker. The linker may be a polyalkylene oxide, and preferably two polyalkylene oxide polymers are connected to the protein.

[0178] In certain embodiments, the fusion polypeptide conjugate is a PEG conjugate comprising a TRAIL domain comprising a truncated form of human TRAIL (e.g., Arginine-114 to Glycine-281 of the full-length form of human TRAIL (1-281)) and PEG having a molecular weight of between 1,000 and 100,000 Daltons, preferably between 5,000 and 100,000 Daltons, e.g., between 5,000 and 80,000 Daltons, between 5,000 and 60,000 Daltons, or between 5,000 and 40,000 Daltons.

[0179] N-terminally modified PEG-TRAIL conjugates can be obtained by reacting the N-terminal amine of the TRAIL domain with the aldehyde group of PEG in the presence of a reducing agent. PEG and TRAIL can be reacted in a molar ratio (PEG / TRAIL) of 2 to 50, or preferably 5 to 7.5. Linear or branched PEG molecules can be used.

[0180] The PEG chains preferably, but not necessarily, have equal molecular weights. An exemplary molecular weight range for each PEG chain is between about 10 kDa and 60 kDa, preferably between about 20 kDa and 40 kDa. PEG40 is a branched PEG moiety having a molecular weight of 40 kDa: 20 + 20 kDa (per PEG chain).

[0181] The trimeric PEG moiety may consist of a branched PEG chain attached to a linker arm.A visual depiction of the trimeric PEG moiety is as follows.

[0182]

[0183] Branched PEG PEG linker arm

[0184] Total molecular weight 10-60kDa Total molecular weight 1-30kDa

[0185] Preferred 20-40 kDa Preferred 2-20 kDa

[0186] The following trimeric PEGs were synthesized: YPEG42, YPEG43.5, YPEG45, YPEG50, and YPEG60.

[0187] YPEG42 is a trimeric PEG moiety with a molecular weight of 42 kDa: (20 + 20 kDa) (branched PEG) + 2 kDa (linker arm).

[0188] YPEG43.5 is a trimeric PEG moiety with a molecular weight of 43.5 kDa: (20 + 20 kDa) (branched PEG) + 3.5 kDa (linker arm).

[0189] YPEG45 is a trimeric PEG moiety with a molecular weight of 45 kDa: (20 + 20 kDa) (branched PEG) + 5 kDa (linker arm).

[0190] YPEG50 is a trimeric PEG moiety with a molecular weight of 50 kDa: (20 + 20 kDa) (branched PEG) + 10 kDa (linker arm).

[0191] YPEG60 is a trimeric PEG moiety with a molecular weight of 60 kDa: (20 + 20 kDa) (branched PEG) + 20 kDa (linker arm).

[0192] a. Connector

[0193] Fusion polypeptide can be covalently linked to the PEG portion via a linker. The linker is a polymer, typically having an atomic length of at least 800 angstroms. Typically, the atomic length of the linker is from about 800 to about 2,000 angstroms, from about 800 to 1,500 angstroms, from about 800 to about 1,000 angstroms, or from about 900 to about 1,000 angstroms. The atomic distance refers to a fully extended polymer, and when in solid state or solution, the linker can fold or curl so that the actual distance between the branched PEG and the protein or peptide is less than the atomic length.

[0194] In certain embodiments, the linker is a poly(ethylene glycol) derivative having a molecular weight between about 1 kDa and 30 kDa, preferably about 2 kDa to 20 kDa. The linker can also be at least 80 units of natural or unnatural amino acids in length.

[0195] Alternatives to PEG for linkers may include synthetic or natural water-soluble biocompatible polymers such as polyethylene oxide, polyvinyl alcohol, polyacrylamide, proteins such as hyaluronic acid and chondroitin sulfate, celluloses such as hydroxymethylcellulose, polyvinyl alcohol, and polyhydroxyalkyl (meth)acrylates.

[0196] Conventional chemical methods can be used to covalently bind fusion polypeptides to linkers. For example, primary amine groups present at the N-terminus or in lysine residues will react with aldehydes and their equivalents under reducing conditions to form amines. (Molineux, Current Pharmaceutical Design, 10(11):1235-1244(2004)). For example, sulfhydryl (-SH) groups present in cysteine ​​residues can be conjugated to various Michael acceptors, including acrylic and methacrylic acid derivatives and maleimides (Gong et al., British Journal of Pharmacology, 163(2):399-412(2011)). Other suitable nucleophilic groups present in peptides and proteins include disulfide bonds in fusion polypeptides (Brocchini, et al., Nature protocols, 1:2241-2252 (2006)) and histidine residues (Cong, et al., Bioconjugate Chemistry, 23(2):248-263 (2012)).

[0197] The linker can be covalently attached to the protein or peptide using conventional chemical methods. For example, the linker polymer can be derivatized at one end with an electrophilic group such as an aldehyde, an epoxide, a halogen (chlorine, bromine, iodine), a sulfonate (toluene sulfonate, mesylate), a Michael acceptor or an activated carboxylate, and then reacted with a nucleophilic amine or sulfhydryl group in the protein or peptide. Suitable Michael acceptors include acrylic and methacrylic acid derivatives, such as acrylamide, methacrylamide, acrylate and methacrylate, and maleimide. Suitable activated carboxylates include nitrophenyl carbonate and NHS (N-hydroxysuccinic acid) ester. In other embodiments, peptides and proteins containing arginine residues can be covalently attached to a linker containing a reactive 1,3 diketone functional group.

[0198] The conjugate can be prepared by first attaching the linker to the fusion polypeptide and then attaching the linker to the branched poly(ethylene glycol), or first attaching the linker to the branched poly(ethylene glycol) and then attaching the linker to the fusion polypeptide. The optimal order of bond formation is determined by the specific chemical transformations involved.

[0199] 3. Characteristics of the conjugate

[0200] Compared to prior art conjugates with iLZ domains, long-acting fusion polypeptide-PEG conjugates have lower immunogenicity. Typically, the long-acting conjugates have higher solubility, lower immunogenicity, and significantly improved potency than prior art long-acting conjugates.

[0201] a. Solubility

[0202] Typically, the solubility of the conjugate is between about 0.2 mg / ml and 50 mg / ml (in physiological buffer, physiological pH and room temperature). Typically, no substantial or detectable aggregation is observed in solutions containing the conjugate at a concentration between 0.2 mg / ml and 50 mg / ml. Solutions containing the conjugate at a concentration between about 0.2 mg / ml and 50 mg / ml, between about 0.2 mg / ml and 40 mg / ml, between about 0.2 mg / ml and 30 mg / ml, or between about 0.2 mg / ml and 25 mg / ml generally do not show substantial or detectable aggregation of the conjugate.

[0203] b. Stability and half-life

[0204] Typically, the stability of the conjugate is enhanced when stored at 4°C in the presence of physiological concentrations of salt or when frozen. Typically, the conjugate is generally stable to repeated freeze-thaw cycles without substantial loss of biological activity.

[0205] Stability can be assessed by measuring the ratio of soluble to insoluble material that can be separated by centrifugation. The intact structure of the stored material can also be confirmed by IEX-HPLC (SP-NPR) and RP-HPLC (C4).

[0206] As shown in Example 5 below, an exemplary conjugate comprising SEQ ID NO: 4 as a first sequence and SEQ ID NO: 15 as a second sequence was resistant to multiple freeze-thaw cycles at a concentration of 21-22 mg / ml, both in terms of biological activity and performance in HPLC analysis. This example demonstrates that the exemplary fusion polypeptide-PEG conjugate is highly soluble and stable to repeated freeze-thaw cycles.

[0207] Typically, the in vivo half-life of the fusion polypeptide is generally between about 20 hours and about 50 hours in non-human primates, between about 20 hours and about 40 hours, or between about 25 hours and about 40 hours in non-human primates.

[0208] The half-life (t 1 / 2 ) was 0.9 hours, while after PEGylation, t 1 / 2 The PEGylated iLZ-TRAIL variant has a circulating t 1 / 2 Exceeding the t of non-PEGylated TRAIL 1 / 2 The following examples show that the t 1 / 2 (about 20 hours, about 30 hours, about 40 hours, such as 15 hours to 50 hours) than the t of PEGylated iLZ TRAIL (8.6 hours). 1 / 2 Several times larger.

[0209] c. Biological activity

[0210] Typically, the conjugates have improved biological activity compared to conjugates having an iLZ motif in the art. Typically, the in vitro IC50 of the conjugates when tested on COLO 205 cells is between about 0.001 nM and about 0.1 nM, preferably between about 0.001 nM and about 0.05 nM ( Figure 4 When tested on activated hepatic stellate cell (HSC) Colo-205 cells, the in vitro IC50 of the miLZ conjugate was between about 0.01 nM and about 1 nM ( Figure 2B ).

[0211] Typically, the conjugate has at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 times greater biological activity (e.g., IC50) than a conjugate having an iLZ motif. Biological activity is often expressed, for example, as IC50 (half maximal inhibitory concentration), which decreases as biological activity increases.

[0212] As shown in the following examples, the TLY012 conjugate containing SEQ ID NO: 4 as the first sequence and SEQ ID NO: 15 as the second sequence was cytotoxic to activated HSCs at an IC50 of 0.142 nM and had no effect on quiescent HSCs at a high concentration (100 nM) ( Figure 3 ).

[0213] d. Low immunogenicity

[0214] Typically, the low immunogenicity of the PEGylated modified fusion polypeptide is similar to the low immunogenicity of the modified fusion polypeptide.

[0215] Typically, the modified fusion polypeptide carrying miLZ has lower immunogenicity in a host compared to the immunogenicity of the unmodified polypeptide carrying iLZ in the same host. Typically, the host is a mammal, preferably a human.

[0216] The lower immunogenicity of the modified fusion polypeptide carrying miLZ may be a lower immunogenicity score compared to the unmodified polypeptide carrying iLZ, as described above.

[0217] The low immunogenicity of the miLZ-TRAIL conjugate is generally comparable to that of therapeutic human proteins, such as monoclonal human antibodies. Thus, the total MHC II binding scores of the in silico miLZ-TRAIL (SEQ ID 4 and SEQ ID 15) were comparable to or better than the immunogenicity of typical human monoclonal antibodies. The lower immunogenicity of miLZ-TRAIL, compared to iLZ-TRAIL, was defined by a several-fold reduction in the total in silico MHC II binding score (5 compared to 27).

[0218] For example, Abzena used the prediction algorithm iTope-AI to analyze two protein sequences of interest to identify peptides that bind to human MHC class II and / or have homology to known T cell epitopes. From this analysis, SEQ ID NO: 32 (containing SEQ ID NO: 3 and SEQ ID NO: 15) and SEQ ID NO: 31 (containing SEQ ID NO: 4 and SEQ ID NO: 15) were determined to have total scores of 27 and 5, respectively, with none of the peptide sequences matching those from TCED. TM(Abzena database) T cell epitope matching.

[0219] SEQ ID NO: 31 comprises a miLZ sequence with more favorable properties than the iLZ sequence in SEQ ID NO: 32, as shown below, and has a significantly better overall score (5 compared to 27), which matches the best score for a monoclonal human antibody.

[0220] The low immunogenicity of the miLZ-TRAIL conjugate is generally comparable to the immunogenicity of therapeutic human proteins (e.g., monoclonal human antibodies). Thus, the total MHCII binding scores of miLZ-TRAIL (SEQ ID 4 and SEQ ID 15) simulated in silico are comparable to or better than the immunogenicity of typical human monoclonal antibodies. The lower immunogenicity of miLZ-TRAIL compared to iLZ-TRAIL is demonstrated by a several-fold reduction in the total score of computer-simulated MHCII binding (5 compared to 27). Typically, the immunogenicity of the modified fusion polypeptide is reduced by at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, preferably about 2 to 10-fold.

[0221] 4. Exemplary Fusion Polypeptide-PEG Conjugates

[0222] An exemplary conjugate of a PEG moiety and a TRAIL fusion polypeptide is the TLY012 molecule. This is a PEGylated form of recombinant human TRAIL (rhTRAIL) genetically fused at the N-terminus to a humanized coiled-coil isoleucine "zipper" (iLZ) that favors trimer formation ( Figure 1D The TRAIL portion of the construct is an extracellular domain beginning with V36, as shown in SEQ ID NO: 31. The miLZ "zipper" domain is derived from a yeast protein called GCN41. The miLZ "zipper" contains isoleucine substitutions at positions 6, 10, 13, 17, 24, 27, and 31 to enhance trimer formation and two amino acid substitutions, as well as I20V and K29E (relative to the substitutions shown in SEQ ID NO: 2) to reduce the probability of immunogenicity based on computer simulation analysis. Residues can be added to facilitate expression of miLZ-TRAIL in E. coli, such as an initial G and / or an amino acid linker D or DG.

[0223] Prior to PEGylation, miLZ rhTRAIL is a zinc-complexed homotrimer composed of three identical single polypeptide chains with an estimated molecular weight of 71.2 kDa (71,175 Da). TLY012 is a mixture of several miLZ-rhTRAIL forms PEGylated at various positions to varying degrees, with the 5 kDa PEG-aldehyde predominating in mono- and di-PEGylated forms. All PEGylated forms of miLZ-TRAIL present in TLY012 are functionally active in cell-based bioassays ( Figure 3 The amino acid sequence of TLY012 is shown below, and the N-terminal methionine was processed after miLZ-TRAIL was expressed in E. coli:

[0224] GRMKQIEDKIEEILSKIYH V ENEIARIK E LIGEDGVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG (SEQ ID NO: 30).

[0225] 5. Complexes

[0226] The fusion polypeptide can be complexed with a negatively charged moiety. In some embodiments, the negatively charged moiety can help load the fusion polypeptide into the nanoparticle for extended delivery, sustained delivery, or slow-release delivery. In some embodiments, the negatively charged moiety itself mediates the extended delivery, sustained delivery, or slow-release delivery of the fusion polypeptide. Preferably, the negatively charged moiety does not substantially reduce the ability of the fusion polypeptide to induce or enhance apoptosis.

[0227] The formation of a complex (CS / TRAIL) between positively charged TRAIL and negatively charged chondroitin sulfate (CS) has been shown to facilitate the loading of TRAIL into poly(lactide-co-glycolide) (PLGA) microspheres (MS) without compromising the activity of TRAIL (Kim, et al., Journal of Pharmacy and Pharmacology, 65(1):11–21(2013)). At pH 5.0, a nanocomplex of approximately 200 nm was formed with a weight ratio of 2TRAIL to CS (TC2). The loading efficiency of this complex in PLGA MS prepared by a multiple emulsion method was >95% higher than that of native TRAIL. Therefore, in some embodiments, the fusion polypeptide or conjugate is complexed with chondroitin sulfate and optionally loaded into microparticles or nanoparticles, such as PLGA-based particles.

[0228] In other embodiments, the fusion polypeptide or its conjugate is compounded with hyaluronic acid (HA). Nanocomplexes of PEG-TRAIL and HA prepared by mixing positively charged PEG-TRAIL and negatively charged HA have shown sustained delivery in vivo with negligible loss of bioactivity compared to PEG-TRAIL (Kim, et al., Biomaterials, 31(34):9057-64(2010)). Delivery is further enhanced by administering nanoparticles in a solution containing 1% HA.

[0229] C. Polynucleotide encoding fusion polypeptide

[0230] Disclosed are polynucleotides encoding recombinant fusion polypeptides. Such polynucleotides can be provided in an expression vector. Typically, the expression vector includes a promoter operably linked to the polynucleotide. The promoter can be any promoter, including constitutively active, inducible, conditional, or tissue-specific promoters. The polynucleotides can be suitable for expression in yeast (e.g., Pichia pastoris) or Escherichia coli expression systems.

[0231] Methods for expressing these polynucleotides are known in the art. These methods include transfecting E. coli cells with an expression vector, provided that the expression vector includes an inducible promoter. The transfected E. coli cells are used to inoculate a culture medium. Typically, the culture medium includes zinc ions, for example, in the form of zinc chloride.

[0232] The nucleotide sequence encoding the fusion polypeptide is listed below, with codons of interest indicated in boxes:

[0233]

[0234]

[0235]

[0236] (SEQ ID NO: 35);

[0237]

[0238]

[0239]

[0240]

[0241]

[0242] D. Pharmaceutical Compositions

[0243] The pharmaceutical composition comprises an effective amount of the fusion polypeptide (or polynucleotide) or conjugate, and optionally a pharmaceutically acceptable carrier. These pharmaceutical compositions can be used to treat proliferative, autoimmune or fibrotic diseases.

[0244] A pharmaceutical composition can include a fusion polypeptide (or polynucleotide) or conjugate as a first therapeutic, prophylactic, or diagnostic agent and a second, or more therapeutic, prophylactic, or diagnostic agents.

[0245] Typically, typical excipients include sterile water, sterile saline or sterile buffered water or sterile buffered saline, which are commonly used in the formulation of parenteral administration.

[0246] 3. Preparation method

[0247] A. Preparation method of fusion polypeptide

[0248] Fusion polypeptide can be made using conventional techniques known in the art. Isolation polypeptide can be obtained by, for example, chemical synthesis or recombinant production in a host cell. For recombinant production of polypeptide, nucleic acid containing the nucleotide sequence encoding the fusion protein can be used for transformation, transduction or transfection of bacteria or eukaryotic host cells (e.g., insect, yeast or mammalian cells). Usually, nucleic acid constructs include regulatory sequences operably connected to the nucleotide sequence encoding the polypeptide. Typically, regulatory sequences (also referred to herein as expression control sequences) do not encode gene products, but affect the expression of the nucleotide sequences operably connected to them.

[0249] Useful prokaryotic and eukaryotic systems for expression and production of polypeptides are well known in the art and include, for example, E. coli strains such as BL-21 and cultured mammalian cells such as CHO cells.

[0250] Expression of glycoproteins in mammalian cells generally results in mammalian-type glycosylation. For human proteins, this is ideal, however some cell lines add the non-human Galα1-3Gal epitope and N-glycylneuraminic acid (NGNA). Insect expression systems add shorter N-glycans with little to no sialylation. Typically, plant cells include glycans containing additional trehalose and xylose residues. Yeast expression systems have a very different glycosylation pattern than mammalian cells, with only mannose-containing glycans. In general, iLZ-TRAIL and miLZ do not rely on glycosylation, so bacterial and mammalian cell lines can be used to express the constructs.

[0251] In eukaryotic host cells, a number of viral-based expression systems can be used to express polypeptides. Viral-based expression systems are well known in the art and include, but are not limited to, baculovirus, SV40, retrovirus, or vaccinia virus-based viral vectors.

[0252] Mammalian cell lines stably expressing polypeptides can be produced using expression vectors with appropriate control elements and selective markers. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023 (B) (see Wong et al. (1985) Science 228: 810-815) are suitable for expressing variant polypeptides in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Other suitable expression systems include the GS gene expression system available from Lonza Group, Inc. TM (GS Gene Expression SystemTM ).

[0253] Stable cell lines can be selected after introduction of the expression vector by electroporation, lipofection, calcium phosphate or calcium chloride coprecipitation, DEAE-dextran, or other suitable transfection methods (e.g., by metabolic selection or antibiotic resistance to G418, kanamycin, or hygromycin). The transfected cells can be cultured to express the polypeptide of interest, and the polypeptide can be recovered from, for example, the cell culture supernatant or lysed cells.

[0254] Polypeptides can be separated using chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. In some embodiments, polypeptides can be engineered to contain additional domain-containing amino acid sequences that allow the polypeptide to be captured on an affinity matrix. For example, Fc-fusion polypeptides in cell culture supernatants or cytoplasmic extracts can be separated using a protein A column. Tags such as c-myc, hemagglutinin, polyhistidine, or FLAG™ (Kodak) can be used to aid in polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including at the carboxyl or amino termini. Other useful fusions include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography can also be used to purify polypeptides. In addition, polypeptides can be engineered to contain a secretion signal (if no secretion signal is already present) that causes the polypeptide to be secreted by the cells producing the polypeptide. The secreted polypeptide can then be conveniently isolated from the cell culture medium.

[0255] 1. Exemplary Methods

[0256] A fusion polypeptide comprising SEQ ID NO: 4 as a first sequence and SEQ ID NO: 15 as a second sequence can be prepared according to the following method.

[0257] The polynucleotides encoding SEQ ID NO: 4 and SEQ ID NO: 15 in the expression cassette were produced in E. coli using the pET23 expression vector. After amplification in E. coli, expression of the fusion polypeptide from an inducible promoter was induced using isopropyl-L-thio-BD-galactopyranoside (0.02-1 mmol / L, 27°C for 7 hours). The cells were then harvested, lysed, and the soluble fusion polypeptide was purified by affinity chromatography (Zn-NTA) and HIC (C4) chromatography.

[0258] B. Preparation Method of Conjugate

[0259] The PEG-fusion polypeptide conjugate can be obtained by reacting the N-terminal amine of the first or second sequence of the fusion polypeptide with the aldehyde group of PEG in the presence of a reducing agent. PEG and the fusion polypeptide can be reacted at a molar ratio (PEG / miLZ-TRAIL) of 2 to 50, or preferably 5 to 7.5.

[0260] Typically, the method for preparing a PEG-fusion polypeptide conjugate comprises reacting the fusion polypeptide with an aldehyde group of PEG or a derivative thereof. Exemplary derivatives of PEG include methoxypolyethylene glycol succinimidyl propionate, methoxypolyethylene glycol N-hydroxysuccinimide, methoxypolyethylene glycol aldehyde, methoxypolyethylene glycol maleimide, and multi-branched polyethylene glycol. Typically, the reaction occurs in the presence of a reducing agent. Typically, PEG or a derivative thereof has a molecular weight between 1,000 and 100,000 as determined by SDS-PAGE and MALDI-TOF.

[0261] In an exemplary method, PEG and the fusion polypeptide are reacted at a molar ratio (PEG / fusion polypeptide) of 2 to 10, preferably 5 to 7.5. Typically, the reducing agent is NaCNBH3.

[0262] 4. Usage

[0263] Typically, the formulation is used in a method of preventing or treating one or more symptoms of a disease. The method generally comprises administering a solid or liquid composition of the fusion polypeptide, polynucleotide or conjugate to an individual in need thereof.

[0264] A fusion polypeptide, polynucleotide, conjugate or pharmaceutical composition containing an effective amount of a fusion polypeptide, polynucleotide or conjugate to treat or alleviate one or more symptoms of a disease or disorder, and an optional pharmaceutically acceptable carrier, is typically administered parenterally, such as by injection, topically (e.g., during surgery) or applied to a mucosal surface (rectum, vagina, oral cavity or lung). These drugs can be administered in the form of a solution, implant or gel, or administered in the form of a dry powder or after redissolution or resuspension.

[0265] Typically, the method involves using the fusion polypeptide, polynucleotide, or conjugate, alone or in combination, to treat a proliferative disease, such as cancer, an autoimmune disease, such as rheumatoid arthritis, or a fibrotic disease.

[0266] After administration, the individual and the treating physician will typically monitor the state, symptoms, or clinical manifestations of the disease. In a preferred embodiment, the administration of the formulation partially or completely reduces the severity of one or more symptoms or clinical manifestations of the disease compared to the severity of the symptoms before administration. This reduction in symptom severity alleviates, improves, alleviates the same or other symptoms or specific diseases, disorders, and / or conditions in the individual, delays the onset of the individual, inhibits the progression of the same or other symptoms or specific diseases, disorders, and / or conditions, reduces the severity of the same or other symptoms or specific diseases, disorders, and / or conditions, and / or reduces the incidence of the same or other symptoms or specific diseases, disorders, and / or conditions.

[0267] The fusion polypeptide, polynucleotide or conjugate or a composition thereof can be formulated into various formulations for parenteral administration in clinical applications. The formulation is administered to an individual to deliver a therapeutically effective amount of the fusion polypeptide, polynucleotide or conjugate or a composition thereof to cells and tissues.

[0268] The desired dose can be delivered once a day or multiple times a day. For example, the desired dose can be delivered three times a day, twice a day, once a day, twice a week, once a week, or once a week. In certain embodiments, the desired dose can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

[0269] The dosage for a particular patient will be adjusted based on the patient's weight, age, sex, health status and diet, duration of administration, route of administration, excretion rate, and severity of the disease. Typically, an effective dose may be administered once every one to two weeks. Alternatively, the dose may be taken as a single dose or in divided doses within the daily effective dose.

[0270] Typically, the fusion polypeptide, polynucleotide or conjugate, or a composition thereof, is formulated in dosage unit form for ease of administration and uniformity of dosage. However, it will be understood that the total daily dosage of the composition will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular individual will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific active agent being used; the specific components being used; the age, weight, general health, sex, and diet of the individual; the time of administration, route of administration, and excretion rate of the specific active agent being used; the duration of treatment; drugs used in combination with or concurrently with the specific active agent being used; and similar factors known in the medical art.

[0271] In certain embodiments, a dosage unit contains an amount of the fusion polypeptide, polynucleotide or conjugate, or composition thereof, in an amount of about 0.001 mg / kg of subject body weight to about 100 mg / kg of subject body weight, about 0.01 mg / kg of subject body weight to about 50 mg / kg of subject body weight, about 0.1 mg / kg of subject body weight to about 40 mg / kg of subject body weight, about 0.5 mg / kg of subject body weight to about 30 mg / kg of subject body weight, about 0.01 mg / kg of subject body weight to about 10 mg / kg of subject body weight, about 0.1 mg / kg to about 10 mg / kg of subject body weight, or about 1 mg / kg to about 25 mg / kg of subject body weight per day, one or more times per day to achieve the desired therapeutic effect. The desired dose can be delivered three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once every three weeks, or once every four weeks. In certain embodiments, multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more administrations) can be used to deliver the desired dose.

[0272] A. Diseases that require treatment

[0273] 1. Proliferative diseases, such as cancer

[0274] The fusion polypeptides, polynucleotides, conjugates, or compositions thereof can be used to treat individuals with benign or malignant tumors or other hyperproliferative diseases by delaying or inhibiting the growth of a tumor in the individual, reducing the growth or size of a tumor, inhibiting or reducing metastasis of a tumor, and / or inhibiting or reducing symptoms associated with tumor development or growth.

[0275] Treatable malignant tumors can be classified according to the embryonic origin of the tissue from which they originate. Cancers are tumors that arise from endoderm or ectoderm tissues, such as the skin or the epithelial layer of internal organs and glands. Sarcomas occur less frequently and arise from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemias and lymphomas are malignant tumors of the blood-forming cells of the bone marrow. Leukemias multiply as single cells, while lymphomas grow as tumors. Malignant tumors can arise in many organs or tissues of the body, forming cancers.

[0276] Proliferative diseases can be cancers, wherein the primary tumor originates in the liver, pancreas, colon, brain, breast, prostate, lung, head and neck, stomach, lymph nodes and skin. The tumor can be a primary tumor or a metastatic tumor. The tumor can be a solid tumor.

[0277] a. Targeting cancer-associated fibroblasts

[0278] Immunotherapy, or more specifically, the use of monoclonal antibodies to block inhibitory immune checkpoint molecules to enhance the immune response to tumors, has shown clinical promise in advanced solid tumors. Such therapies include PD-1, PD-L1, CTLA-4, and IDO pathway blockers. Significant progress has been made in tumor treatment, including the development of cancer immunotherapies based on checkpoint inhibitors. However, immunotherapy has only been beneficial for patients with selected cancer types such as melanoma and lung cancer, while the development of effective immunotherapies for solid cancers, including hepatocellular carcinoma (HCC), pancreatic ductal adenocarcinoma (PDAC), or colon cancer (CRC), has been relatively slow. It is now widely believed that the immunosuppressive tumor microenvironment (TME) is one of the main components of primary resistance to immunotherapy. The establishment of the tumor microenvironment (TME) not only allows tumor development, but also allows it to recruit components of the host immune system. In addition to promoting tumor growth, the TME also primarily acts as a cellular barrier to prevent any infiltration of anti-tumor immune cells (Weiner, Clinical Advances in Hematology & Oncology.; 13(5): 299-306 (2015), Chiriva-Internati and Bot. International Reviews of Immunology; 34(2): 101–103 (2015)). The formation of a stromal layer surrounding the cancerous mass establishes a physical barrier characterized by several features known to promote cancer growth, including hypoxic conditions and the formation of abnormal tumor neovascularization (Klener et al., Current Pharmaceutical Biotechnology; 16(9): 771–781 (2015)). The TME is a heterogeneous cell population composed of tumor cells and fibrotic stromal cells. The most important sources of stromal cells are α-smooth muscle actin (α-SMA)+ myofibroblasts (MFBs, activated fibroblasts) and cancer-associated fibroblasts (CAFs), which are known to contribute to excessive extracellular matrix (ECM) deposition, tumor initiation and progression, and potent immunosuppressive properties in solid cancers.

[0279] When used for cancer treatment, the fusion polypeptides and fusion polypeptide conjugates target cancer-associated fibroblasts and induce apoptosis in cancer-associated fibroblasts.

[0280] Here we demonstrate that the fusion peptide conjugate TLY012 possesses anti-fibrotic function and targets fibrotic tumors with cancer-associated fibroblasts, which, in the absence of treatment, are resistant to immunotherapy due to the fibrotic cell barrier.

[0281] 2. Autoimmune diseases

[0282] The fusion polypeptide, polynucleotide, conjugate, or a composition thereof can be used to prevent or treat one or more symptoms of an autoimmune disease. Representative autoimmune diseases include lupus, rheumatoid arthritis, and type I diabetes.

[0283] Systemic lupus erythematosus (SLE) is an autoimmune disease with a wide range of clinical and immunological abnormalities. The presence of autoantibodies, particularly those against double-stranded DNA, is a hallmark of the disease. SLE can affect various organ systems, including the skin, joints, central and peripheral nervous systems, kidneys, and liver. The cause of the disease is still unclear. However, increasing evidence suggests that the presence and accumulation of apoptotic cells play a role in autoimmunity (Hooge et al., Ann Rheum Dis, 64:854–858 (2005)).

[0284] Elevated serum soluble TRAIL concentrations have been observed in patients with SLE and have been found to be disease-specific, with levels generally higher in patients with inactive disease than in those with active disease.

[0285] Rheumatoid arthritis (RA) is a type of arthritis that causes joint pain, swelling, stiffness, and loss of function. It can affect any joint but is most common in the wrists and fingers. More women than men develop RA. The disease may only last a short time, or symptoms may come and go. Severe forms can last a lifetime.

[0286] Rheumatoid arthritis is different from osteoarthritis (OA), a common form of arthritis that often develops with aging. RA can affect parts of the body other than joints, such as the eyes, mouth, and lungs. RA is an autoimmune disease, meaning the arthritis is caused by the immune system attacking the body's own tissues.

[0287] Compared with osteoarthritis (OA) patients, rheumatoid arthritis (RA) patients have increased TRAIL expression. The inflammatory environment in the arthritic joints of RA patients appears to promote TRAIL expression in fibroblast-like synoviocytes (FLS) (Audo et al., Arthritis and Rheumatism, 63(4):904–913 (2011)).

[0288] Diabetes (diabetes mellitus) occurs when the pancreas doesn't produce enough insulin, or when the body's cells don't respond properly to the insulin that's produced. There are three main types of diabetes:

[0289] Type 1 diabetes is caused by the pancreas not producing enough insulin. This type was previously called "insulin-dependent diabetes mellitus" (IDDM) or "juvenile diabetes."

[0290] Type 2 diabetes begins with insulin resistance, a condition in which cells fail to respond appropriately to insulin. As the disease progresses, insulin deficiency may also develop. This type was previously known as "non-insulin-dependent diabetes mellitus" (NIDDM) or "adult-onset diabetes."

[0291] Gestational diabetes, the third major form, occurs when pregnant women with no history of diabetes develop elevated blood sugar levels.

[0292] Type 1 diabetes requires insulin therapy for survival. Type 1 diabetes is an autoimmune inflammatory disease of the pancreatic islets. In both human and rodent models of type 1 diabetes, insulin-producing pancreatic β-cells are selectively destroyed by infiltrating inflammatory cells.

[0293] The role of TRAIL in the body may include suppressing autoimmune inflammation of the pancreatic islets. TRAIL can inhibit insulitis and suppress autoimmune diabetes.

[0294] Both T cells and macrophages are involved in mediating the β-cell damage in this disease. TRAIL may modulate diabetes by acting on one or both of these cells. TRAIL can also block DNA synthesis and cell cycle progression in T cells activated by anti-CD3 antibodies. TRAIL may mediate negative selection of thymocytes. Thus, TRAIL may inhibit diabetic inflammation and autoreactive T cell activation (Lamhamedi-Cherradi et al., Diabetes 52:2274–2278 (2003)).

[0295] The following examples illustrate that activated cells, such as hepatic stellate cells, can be specifically targeted and killed by the PEGylated fusion polypeptide conjugate TLY012, without affecting quiescent hepatic stellate cells. This treatment results in TRAIL-induced apoptosis, thereby preventing or reversing fibrosis. Importantly, by eliminating such activated stellate cells, highly upregulated fibrosis-related molecules can be simultaneously downregulated. This suggests that the compounds can be used to treat pathological conditions in which activated fibroblasts, myofibroblasts, myofibroblasts, and activated endothelial and epithelial cells produce or induce excessive extracellular matrix, leading to unwanted fibrosis or scarring. Scarring or fibrosis can occur in the liver, pancreas, lungs, heart, kidneys, intestines, skin, or arteries.

[0296] B. Combination therapy

[0297] One or more of the recombinant fusion polypeptide, polynucleotide or fusion polypeptide conjugate can be administered to an individual in need alone or in combination with one or more additional active agents. In these embodiments, the recombinant fusion polypeptide, polynucleotide or fusion polypeptide conjugate is a first active agent, and the additional active agent is a second active agent. In some embodiments, the second active agent is an agent known in the art for treating proliferative diseases, autoimmune diseases or fibrotic diseases. In some embodiments, the second active agent is an agent for regulating host cells, for example, regulating cancer cell proliferation, reducing stellate cell activation or activity, increasing cancer cells or stellate cell apoptosis, reducing deposition of extracellular matrix or its components (particularly collagen), increasing extracellular matrix and its components, particularly collagen or any combination thereof. In some embodiments, the first active agent increases efficacy, enhances effect or otherwise improves the performance or sensitivity of cells to the second active agent.

[0298] 1. Second active agent

[0299] The second active agent that can be used with the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate can be a chemotherapeutic agent or an anti-inflammatory agent.

[0300] a. Chemotherapeutic agents

[0301] The use of recombinant fusion polypeptides, recombinant polynucleotides, or recombinant fusion polypeptide conjugates in the treatment of cancer has been studied, either alone or in combination with conventional cancer treatments such as chemotherapeutic agents. Some reports suggest that chemotherapeutic drugs can sensitize cells to TRAIL-induced apoptosis, and some results suggest that the combination of two drugs is more effective than the sum of the effects of the drugs when used alone (Cuello, et al., Gynecol Oncol., 81(3):380-90(2001); Wu, et al., Vitam Horm., 67:365-83(2004)). Therefore, in some embodiments, the disclosed individuals and diseases are treated with a combination of one or more recombinant fusion polypeptides or one or more recombinant fusion polypeptide conjugates and a chemotherapeutic agent. In some embodiments, the individual suffers from cancer. In some embodiments, the individual suffers from cancer with a solid tumor.

[0302] Exemplary chemotherapeutic drugs include, but are not limited to, doxorubicin, etoposide, camptothecin, irinotecan, cisplatin, oxaliplatin, docetaxel, cyclophosphamide, 5-fluorouracil, carboplatin, mechlorethamine, sorafenib, chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, paclitaxel and its derivatives, topotecan, amsacrine, etoposide phosphate, teniposide, epipodophyllotoxin, trastuzumab Cetuximab and rituximab ( or ), bevacizumab and combinations thereof.

[0303] i. Immune checkpoint inhibitors

[0304] The second active agent may include one or more immune checkpoint inhibitors (ICIs). Typically, ICIs include small molecules, antibodies, or antibody fragments that bind to programmed cell death protein 1 (PD-1), small molecules, antibodies, or antibody fragments that target PD-1 ligand 1 (PD-L1), and small molecules, antibodies, or antibody fragments that target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).

[0305] Typically, the second active agent comprises between about 0.1 mg / kg and about 100 mg / kg of the patient's body weight of ICI in one injection. Suitable amounts of ICI in the vaccine include between about 0.1 mg / kg and about 500 mg / kg, between about 0.1 mg / kg and about 250 mg / kg, between about 0.1 mg / kg and about 100 mg / kg, between about 0.1 mg / kg and about 80 mg / kg, and between about 0.1 mg / kg and about 60 mg / kg, for example, between about 0.5 mg / kg and about 20 mg / kg, or between about 1 mg / kg and about 10 mg / kg. Specific concentrations of ICI include 0.1mg / kg, 0.2mg / kg, 0.3mg / kg, 0.4μM, 0.5mg / kg, 0.6mg / kg, 0.7mg / kg, 0.8mg / kg, 0.9mg / kg, 1mg / kg, 2mg / kg, 3mg / kg, 4mg / kg, 5mg / kg, 6mg / kg, 7mg / kg, 8mg / kg, 9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg, 36mg / kg, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 41mg / kg, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, 49mg / kg and 50mg / kg.

[0306] PD-1 antagonists

[0307] The second active agent may be a PD-1 and / or PD-L1 / PD-L2 antagonist.

[0308] T cell activation typically depends on antigen-specific signals following contact between the T cell receptor (TCR) and antigenic peptides presented via the major histocompatibility complex (MHC), and the extent of this response is controlled by antigen-independent positive and negative signals from a variety of co-stimulatory molecules. The latter are typically members of the CD28 / B7 family. In contrast, programmed death-1 (PD-1), a member of the CD28 receptor family, delivers a negative immune response when induced on T cells. Contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces T cell proliferation and / or the intensity and / or duration of the T cell response. Suitable PD-1 antagonists are described in U.S. Patent Nos. 8,114,845, 8,609,089, and 8,709,416, and include compounds or agents that bind to and block PD-1 ligands to interfere with or inhibit the binding of the ligand to the PD-1 receptor, or compounds or agents that directly bind to and block the PD-1 receptor without inducing inhibitory signal transduction through the PD-1 receptor.

[0309] In some embodiments, a PD-1 receptor antagonist directly binds to the PD-1 receptor without triggering inhibitory signal transduction, and binds to a PD-1 receptor ligand to reduce or inhibit the ligand from triggering signal transduction through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and trigger inhibitory signal transduction, fewer cells are attenuated by the negative signals delivered by PD-1 signaling, and a more robust immune response can be achieved.

[0310] PD-1 signaling is thought to be driven by tight binding of PD-1 ligands, such as B7-H1 or B7-DC, to peptide antigens presented by the major histocompatibility complex (MHC) (e.g., see Freeman, Proc. Natl. Acad. Sci. USA, 105:10275-10276 (2008)).

[0311] Therefore, proteins, antibodies, or small molecules that prevent the co-ligation of PD-1 and TCR on the T cell membrane are also useful PD-1 antagonists.

[0312] Other PD-1 antagonists include antibodies and other antibodies that bind to PD-1 or PD-1 ligands.

[0313] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following publications: PCT / IL03 / 00425 (Hardy et al., WO / 2003 / 099196), PCT / JP2006 / 309606 (Korman et al., WO / 2006 / 121168), PCT / US2008 / 008925 (Li et al., WO / 2009 / 014708), PCT / JP03 / 08420 (Honjo et al., WO / 2004 / 004771), PCT / JP04 / 00549 (Honjo et al., WO / 2004 / 00549 ...3 / 00425 (Hardy et al., WO / 2003 / 099196), PCT / JP04 / 00549 (Korman et al., WO / 2006 / 121168), , WO / 2004 / 072286), PCT / IB2003 / 006304 (Collins et al., WO / 2004 / 056875), PCT / US2007 / 088851 (Ahmed et al., WO / 2008 / 083174), PCT / US2006 / 026046 (Korman et al., WO / 2007 / 005874), PCT / US2008 / 084923 (Terrett et al., WO / 2009 / 073533) and Berger et al., Clin. Cancer Res., 14(10):3044-51 (2008).

[0314] A specific example of an anti-PD-1 antibody is MDX-1106 (nivolumab, see clinical trial number NCT00441337 and Kosak, US 20070166281 (published July 19, 2007), paragraph 42), which is a human anti-PD-1 antibody, preferably administered at a dose of 3 mg / kg.

[0315] Exemplary anti-B7-H1 antibodies include, but are not limited to, those described in the following publications: PCT / US06 / 022423 (WO / 2006 / 133396, published December 14, 2006), PCT / US07 / 088851 (WO / 2008 / 083174, published July 10, 2008), US2006 / 0110383 (published May 25, 2006)

[0316] A specific example of an anti-B7-H1 antibody is MDX-1105 (WO / 2007 / 005874, published January 11, 2007), which is a human anti-B7-H1 antibody. See 7,411,051, 7,052,694, 7,390,888, and U.S. Published Application No. 2006 / 0099203 for more information on anti-B7-DC antibodies.

[0317] The antibody can be a bispecific antibody comprising an antibody that binds to the PD-1 receptor bridged with an antibody that binds to a ligand of PD-1 (e.g., B7-H1). In some embodiments, the PD-1 binding moiety reduces or inhibits signal transduction through the PD-1 receptor.

[0318] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologs and variants of these polypeptides, and active fragments of any of the above, as well as proteins comprising any of these. In a preferred embodiment, the protein comprises a soluble portion of B7-DC coupled to the Fc portion of an antibody (e.g., human IgG), and does not comprise all or part of the transmembrane portion of human B7-DC.

[0319] The PD-1 antagonist may also be a fragment of mammalian B7-H1, preferably from mouse or primate, preferably human, wherein the fragment binds to and blocks PD-1 but does not cause inhibitory signal transduction through PD-1. The fragment may also be part of a fusion protein, such as an Ig fusion protein.

[0320] Other useful polypeptide PD-1 antagonists include those that bind to the ligands of PD-1 receptors. These antagonists include PD-1 receptor proteins or soluble fragments thereof that can bind to PD-1 ligands, such as B7-H1 or B7-DC, and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signal transduction. B7-H1 has also been shown to bind to protein B7.1 ((Butte et al, Immunity, Vol. 27, pp. 1 11-122, (2007)). These fragments also include the soluble ECD portion of PD-1 protein, which includes mutations such as A99L mutations that increase binding to natural ligands (Molnar et al, PNAS, 105: 10483-10488 (2008)). B7-1 or its soluble fragments, which can bind to B7-H1 ligands and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signal transduction, are also useful.

[0321] PD-1 and B7-H1 antisense nucleic acids, DNA and RNA and siRNA molecules can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells and the production of T cell ligands such as B7-H1, PD-L1 and / or PD-L2. For example, a complex of siRNA (e.g., about 21 nucleotides in length, specific for genes encoding PD-1 or encoding PD-1 ligands, whose oligonucleotides can be easily purchased commercially) and a carrier such as polyethyleneimine (see Cubillos-Ruiz et al, J. Clin. Invest. 119 (8): 2231-2244 (2009)) is easily taken up by cells expressing PD-1 and PD-1 ligands, and reduces the expression of these receptors and ligands to achieve a reduction in inhibitory signal transduction in T cells, thereby activating T cells.

[0322] CTLA-4 antagonists

[0323] Other molecules for use in combination therapy include CTLA-4 antagonists. For example, in some embodiments, the molecule is an agent that binds to CTLA4.

[0324] Dosages for anti-PD-1, anti-B7-H1, and anti-CTLA4 antibodies are known in the art and are in the range of 0.1 mg / kg to 100 mg / kg, preferably in the narrower range of 1 mg / kg to 50 mg / kg, and more preferably in the range of 10 mg / kg to 20 mg / kg. Suitable doses for human subjects are between 5 mg / kg and 15 mg / kg, with 10 mg / kg of antibody (e.g., human anti-PD-1 antibody, such as MDX-1106).

[0325] Specific examples of anti-CTLA4 antibodies useful as described herein are ipilimumab, also known as MDX-010 or MDX-101, a human anti-CTLA4 antibody, preferably administered at a dose of about 10 mg / kg, and tremelimumab, a human anti-CTLA4 antibody, preferably administered at a dose of about 15 mg / kg. See also Sammartino, et al, Clinical Kidney Journal, 3(2):135-137 (2010), published online December 2009.

[0326] In other embodiments, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al, J. Biol. Chem., 277: 7363-7368 (2002)). Such small organic compounds can be administered alone or in combination with anti-CTLA4 antibodies to reduce inhibitory signal transduction of T cells.

[0327] 2. Exemplary ICI

[0328] One or more recombinant fusion polypeptides or recombinant fusion polypeptides, recombinant polynucleotides or recombinant fusion polypeptide conjugates can be used in combination with one or more ICIs. Exemplary ICIs include nivolumab (targeting PD-1), pembrolizumab (targeting PD-1), atezolizumab (targeting PD-L1), avelumab (targeting PD-L1), durvalumab (blocking the interaction of PD-L1 with PD-1 (CD279)), cemiplizumab (targeting PD-L1), pidilizumab (targeting Delta-like 1 (DLL1) as the primary binding target and PD-1 as the secondary binding target), wopilizumab (targeting the inducible co-stimulatory molecule (ICOS) of T cells to generate anti-tumor immune responses), danvatirsen, cetuximab and ipilimumab (targeting CTLA-4).

[0329] b. Anti-inflammatory agents

[0330] The second active agent used in combination with the recombinant fusion polypeptide, recombinant polynucleotide or recombinant fusion polypeptide conjugate can be a protein, peptide, carbohydrate, nucleic acid, lipid, small molecule or a combination thereof for treating inflammatory or autoimmune diseases. In some embodiments, the second active agent is an agent known in the art for treating inflammatory or autoimmune diseases. Exemplary agents include analgesics, nonsteroidal anti-inflammatory drugs, and biological disease-modifying anti-rheumatic drugs (DMARDs) that target components of the immune response. Some such therapies target the release of excessive inflammatory mediators by infiltrating lymphocytes responsible for tissue destruction. Some people believe that TNF-α antagonists are the most effective conventional biological agents for treating inflammation (Audo, et al., Cytokine, 63(2):81-90(2013)). Other targets in the TNF family include receptor activator of nuclear factor kappa-beta ligand ligand (RANKL) and its receptor RANK and osteoprotegerin (OPG) (Lamhamedi-Cherradi, et al., Nature immunology 4(3):255-60(2003)).

[0331] In short, the second active agent can be an active agent that regulates immune cells. The active agent can reduce or inhibit the proliferation or activity of proinflammatory immune cells, induce or increase the proliferation or activity of anti-inflammatory immune cells (such as regulatory T cells) or any combination thereof. In some embodiments, the second active agent reduces one or more proinflammatory molecules, including but not limited to TNF-α, IL-1α, IFN-γ, IL-2, IL-6, IL-8, IL-1β, TGF-β, IL-17, IL-6, IL-23, IL-22, IL-21, prostaglandins and matrix metalloproteinases (MMP) expression or circulation.

[0332] 2. Dosage and treatment regimen of combined therapy

[0333] Therapeutic methods generally include treating a disease or its symptoms, or achieving a method for a desired physiological change, including administering an effective amount of a recombinant fusion polypeptide, a recombinant polynucleotide or a recombinant fusion polypeptide conjugate to an animal, such as a mammal, especially a human being, to treat a proliferative disease, an autoimmune disease or a fibrotic disease and / or its symptoms. In some embodiments, a recombinant fusion polypeptide, a recombinant polynucleotide or a recombinant fusion polypeptide conjugate is combined with another active agent. The recombinant fusion polypeptide, a recombinant polynucleotide or a recombinant fusion polypeptide conjugate and another active agent can be administered together, such as as part of the same composition, or separately and independently at the same time or at different times (i.e., the recombinant fusion polypeptide, the recombinant polynucleotide or the recombinant fusion polypeptide conjugate and the second active agent are administered at a limited time interval from each other). Therefore, the term "combination" or "combined" is used to refer to the concomitant, simultaneous or sequential administration of a recombinant fusion polypeptide, a recombinant polynucleotide or a recombinant fusion polypeptide conjugate and a second active agent. The combination can be administered concomitantly (e.g., as a mixture), separately but simultaneously (e.g., through separate intravenous lines; one agent is administered orally and the other is administered by infusion or injection, etc.), or sequentially (e.g., one agent is administered first and the second agent is administered secondarily).

[0334] In preferred embodiments, the results achieved by administering the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate in combination with the second active agent are greater than those achieved by administering the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate and the second active agent alone or separately (i.e., the results achieved by the combination are greater than the sum of the results achieved by each component alone). In some embodiments, the effective amount of one or both agents used in combination is less than the effective amount of each agent when administered alone. In some embodiments, when used in combination therapy, the amount of one or both agents is less than the therapeutic amount when used alone.

[0335] A combination therapy regimen may include one or more administrations.

[0336] In some embodiments, the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate is administered prior to the first administration of the second active agent. In other embodiments, the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate is administered concurrently with the first administration of the second active agent. In other embodiments, the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate is administered after the first administration of the second active agent.

[0337] The recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate can be administered at least 1, 2, 3, 5, 10, 15, 20, 24, or 30 hours, or at least 1, 2, 3, 5, 10, 15, 20, 24, or 30 days before or after administration of the second active agent.

[0338] The dosage regimens or cycles of the agents may overlap completely or partially, or may be sequential. For example, in some embodiments, all such administrations of the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate occur before or after the administration of the second active agent. Alternatively, the administration of one or more doses of the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate may be temporally staggered with the administration of the second therapeutic agent to form an entirely identical course of treatment or a different course of treatment, wherein one or more doses of the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate are administered, followed by one or more doses of the second active agent, followed by one or more doses of the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate; or one or more doses of the second active agent are administered, followed by one or more doses of the recombinant fusion polypeptide, recombinant polynucleotide, or recombinant fusion polypeptide conjugate, followed by one or more doses of the second active agent; and so on, all of which are selected according to any schedule selected or desired by the researcher or clinician administering the treatment.

[0339] The effective amount of each agent can be administered as a single unit dose (e.g., as a dosage unit) or as a low therapeutic dose administered at limited time intervals. Such unit doses can be administered daily for a limited period of time, e.g., up to 3 days, up to 5 days, up to 7 days, up to 10 days, up to 15 days, up to 20 days, or up to 25 days.

[0340] Typically, for every kind of reagent, the first activating agent and the second activating agent are used with effective dose.The effective dose of every kind of reagent can be between about 0.1mg / kg patient weight and about 100mg / kg patient weight for a dose of injection.The suitable amount of every kind of reagent is included between about 0.1mg / kg and about 500mg / kg, between about 0.1mg / kg and about 250mg / kg, between about 0.1mg / kg and about 100mg / kg, between about 0.1mg / kg and about 80mg / kg, between about 0.1mg / kg and about 60mg / kg, for example, between about 0.5mg / kg and about 20mg / kg or between about 1mg / kg and about 10mg / kg. Specific dosages of each agent include about 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 μM, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg , 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg , 36mg / kg, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 41mg / kg, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, 49mg / kg and 50mg / kg.

[0341] 5. Test Kit

[0342] Also disclosed are dosage kits. The dosage kit may include, for example, a dosage supply of a recombinant fusion polypeptide, a recombinant polynucleotide, or a recombinant fusion polypeptide, alone or in combination with a second therapeutic agent. When combined with a second therapeutic agent, the active agent may be supplied alone (e.g., lyophilized) or in the form of a pharmaceutical composition (e.g., a mixture). The active agent may be a unit dose or a stock to be diluted before administration. In some embodiments, the kit includes a supply of a pharmaceutically acceptable carrier. The kit may also include a device for administering the active agent or composition, such as a syringe. The kit may include printed instructions for administering the compound in the above-mentioned uses.

[0343] The present invention will be further understood by reference to the following non-limiting examples.

[0344] Example

[0345] Example 1. miLZ-TRAIL variants with high expression levels and low immunogenicity.

[0346] Materials and methods

[0347] Computer simulation analysis was used to identify structural elements that were incompatible with clinical application ( Figure 1A , boxed area). Thirty expression constructs were made and tested for expression levels.

[0348] The construct was cloned into the pSX2 plasmid (Scarab Genomics). Transformed E. coli cells (strain SG6620; Scarab Genomics) were grown in TB medium at 28°C and protein expression was induced with 0.02-1 mM IPTG at a cell density of approximately 2 absorbance units (AU) (A600). Cells were grown for different time periods, collected by centrifugation, and used Lysis was performed and TRAIL content was analyzed by SDG-PAGE.

[0349] Two leader sequences were selected based on the improvement of soluble protein expression levels. Both sequences were designed without the HisTag at the N-terminus and amino acid substitutions were made in the iLZ domain to destroy the immunogenic epitope ( Figure 1B and Figure 1C , indicated by arrows). A total of 400 mg of protein was prepared for further PEGylation experiments.

[0350] The protein was purified using NTA agarose and tested for purity, thermodynamic stability, and biological activity. Cells containing iLZ-TRAIL were lysed and a supernatant containing high salt (1M NaCl) was prepared by centrifugation. Nucleic acids were digested using benzonase and the resulting cell lysate was incubated with Zn-NTA resin (batch or column) in the presence of low imidazole (usually 10mM) until the binding capacity of the resin was saturated with iLZ-TRAIL. In the presence of low imidazole, the resin as the batch or column contents was washed with a high salt buffer and the iLZ-TRAIL was eluted with 125-250mM imidazole. The eluted protein was dialyzed against the buffer required for the specific assay.

[0351] result

[0352] The data showed that the purity of the protein preparation was high (over 95%). Compared with the HisTag-iLZ-TRAIL protein prototype, significantly higher thermodynamic stability was observed in the thermal shift assay ( Figure 2A HisTag-iLZ-TRAIL produced two peaks: the first peak was in the range of 55-75°C, and the second peak was in the range of 75-98°C. miLZ-TRAIL had only a small shoulder instead of the first peak, and its second peak was larger than the peak observed for His-tag-iLZ-TRAIL. Both lead candidates showed about 5-fold higher bioactivity in the cell viability bioassay. Figure 2B Table 1 shows the IC50 (nM) of the tested polypeptides.

[0353] miLZ-TRAIL can be repeatedly frozen and thawed (tested for up to 5 cycles) without significant loss of its functional activity (cell assay) or noticeable changes in its IEX-HPLC profile.

[0354] Table 1. Figure 2B IC50 (nM) of the peptides tested.

[0355] protein IC50, nM His-iLZ-TRAIL 0.07670 miLZ-TRAIL#5-06 0.01558 miLZ-TRAIL#5-05 0.01787

[0356] Example 2. In vitro biological activity of PEGylated lead compounds (such as TLY012 lead) used in vivo.

[0357] Materials and methods

[0358] In addition to standard physicochemical properties, the lead's on-target efficacy and non-target toxicity were also tested. Initially, the selective apoptotic activity of various TLY012 leads was tested in normal (non-target) and myofibroblasts (target cells) by utilizing resting and activated primary human hepatic stellate cells (HSCs), respectively. Briefly, resting and culture-activated HSCs were cultured with TLY012 leads and cell viability was determined by cytotoxicity assays.

[0359] TLY012 is a PEGylated form of rhTRAIL genetically fused to a humanized coiled-coil isoleucine "zipper" (miLZ) at its N-terminus. It is a DR4 / DR5 receptor agonist and is therefore known to induce apoptosis in human colon cancer cells COLO 205, thereby inhibiting cell proliferation. Luminescence was measured in a conventional luciferase assay system using CELL TITER-GOL™ (Promega, WI). This assay is a homogeneous method based on the quantitative determination of the number of metabolically active cells in the presence of ATP. A single reagent ( Reagent) added directly to cells cultured in serum-supplemented medium results in cell lysis and the generation of a luminescent signal that is proportional to the amount of ATP present, which is proportional to the number of cells present in culture.

[0360] Test procedure. TLY012 samples were tested three times in a serial dilution range from 1000 ng / mL to 0.05 ng / mL. Initially, a 2x concentration of TLY012 was prepared by diluting TLY012 10x twice. All other concentrations were prepared using one 1 / 10 and eight 1 / 3 dilutions, respectively. Dilutions were performed using a sterile 12-channel reservoir. According to the plate map, 50 μl of diluted reference standard sample and test sample were transferred to a 96-well plate. The 96-well plate was placed in a monolayer cell culture incubator at 37±1.0°C; 5±0.5% CO2; ≥85% relative humidity for 1±0.5 hours until the cells were ready. 20,000 cells in 50 μl of growth medium and the previously added TLY012 dilution were placed in a 96-well plate and the experiment was performed for 22-24 hours. The DR5 receptor agonist TLY012 binds to the receptor and causes a cytotoxic effect. The ATP level present in the culture indicates the metabolic activity of COLO 205 cells. TM The inhibition of cell proliferation is measured by lysing the cells to release ATP, which reacts with the Cell Titer Glow solution to produce a stable "glow-type" luminescent signal.

[0361] The growth medium was RPMI1640 containing 10% FBS, glutamine, and penicillin / streptomycin solution. Using a 12-channel pipette, the cell suspension (50 μL / well - 2 × 10 4 Cells) were transferred to the inner 60 wells of a 96-well assay plate. Additionally, well BCD12 was loaded with the cell suspension. Wells EFG12 and B1-G1 were loaded with culture medium alone. The remaining wells contained 100 μL of assay medium. The 96-well plate was placed in a monolayer cell culture incubator at 37 ± 1.0°C, 5 ± 0.5% CO2, and ≥ 85% relative humidity for 22-24 hours. Following incubation, the CELL TITER-GLOTM Luciferase Assay System solution was added to the cells. The cell suspension was transferred to a white 96-well plate and luminescence was measured using a BIOTEK plate reader.

[0362] Data analysis. Data were analyzed using BioTek Gen5 Security Software. Dose-response curves were generated using a four-parameter logistic model, and IC50 values ​​were calculated for the reference standard and test samples. The potency of the test sample was expressed as the ratio of the IC50 values ​​of the test sample to the reference standard.

[0363] Statistical analysis. For statistical analysis, a four-parameter logistic model was used to model the measurements of ATP release as a function of TLY012 concentration. The statistical model applied to this assay was given by the following nonlinear regression equation:

[0364] Y=(A–D) / (1+(X / C) B )+D

[0365] In this equation, Y (relative luminescence units) is a function of X (concentration), and the four parameters A, B, C (IC50), and D have specific interpretations. Asymptote A refers to the upper limit of the response, asymptote D refers to the lower limit of the response, IC50 refers to the inhibitory concentration that inhibits 50% of the maximum signal, and B is a scale parameter related to the shape and steepness of the curve itself. By fitting this model to a reference standard sample and a test sample analyzed in the same experiment, when two concentrations produce the same effect, the relative potency (RP) is calculated as follows:

[0366] RP=IC 50 (reference) / IC50(test).

[0367] All relative potency values ​​as well as EC50 values ​​and four parameters were calculated using BIOTEK software Gen5 secure.

[0368] result

[0369] The TLY012 lead showed no off-target toxicity in quiescent astrocytes but exhibited robust efficacy only in activated astrocytes ( Figure 3 Importantly, the TLY012 lead did not show any off-target toxicity at concentrations up to 100 nM in primary human hepatocytes, while exhibiting subnanomolar potency in cell viability bioassays (0.142 nM in activated HSCs and 0.01545 nM in colorectal cancer cells COLO-205). Figure 3 and 4 The IC50 values ​​of the PEGylated peptides are shown in Table 2.

[0370] Table 2. Figure 4 IC50 (nM) of the PEGylated peptides tested in .

[0371] protein IC50, nM PEG-TRAIL 0.01545 His-TRAIL-PEG 0.1414

[0372] Example 3. miLZ-TRAIL has significantly improved production in E. coli compared to a composition with histidine-tag-iLZ-TRAIL

[0373] Materials and methods

[0374] The construct was cloned into the pSX2 plasmid (Scarab Genomics). Transformed E. coli cells (strain SG6620; Scarab Genomics) were grown in TB medium at 28°C and induced for protein expression with IPTG at a cell density of approximately 2 AU (A600). Cells were cultured overnight in TB medium, harvested by centrifugation, and lysed using a BUGBUSTER. TRAIL content was analyzed by SDG-PAGE.

[0375] The following fusion peptides were used in this study:

[0376] The original His-tag-TRAIL comprising the sequences SEQ ID NO: 3 (bold) and SEQ ID NO: 15 (italic)

[0377]

[0378] miLZ-TRAIL (clone #5) comprising SEQ ID NO: 4 (bold) and SEQ ID NO: 15 (italics)

[0379]

[0380] result

[0381] The production efficiency of histidine-tag-iLZ-TRAIL (Chae, et al., Molecular cancer therapeutics 9(6):1719-29 (2010)) in rich media (TB) does not exceed 100 mg / L. Histidine-tag-iLZ-TRAIL produced in minimal media is unstable within cells and cannot be stored as frozen cell blocks.

[0382] The production efficiency of miLZ-TRAIL is approximately 1 g / ml, and the protein can be stored in frozen cells for several months.

[0383] Example 4. miLZ-TRAIL has significantly increased solubility

[0384] Materials and methods

[0385] The fusion polypeptides used in this study were the same as those in Example 3.

[0386] result

[0387] His-tag-iLZ-TRAIL has very limited solubility. The maximum concentration of His-iLZ-TRAIL used for PEGylation in a pH 5.0 solution did not exceed 100 μg / ml (Chae et al. Mol Cancer Ther. 9(6):1719–1729 (2010)). PEGylation experiments at pH 5.0 were performed at a starting concentration of 15 mg / ml of miLZ-TRAIL. No solubility issues were observed, and the final product met all quality control ("QC") requirements.

[0388] Example 5. PEGylated miLZ-TRAIL shows significantly increased solubility and stability compared to prior PEGylated TRAIL

[0389] Materials and methods

[0390] The fusion polypeptides used in this study were the same as those in Example 4.

[0391] result

[0392] The PEGylated miLZ-TRAIL formulation was stable in the presence of physiological concentrations of salt at concentrations up to 25 mg / ml. When analyzed by IEX-HPLC, the formulation retained the same protein concentration, showed no detectable precipitation, and exhibited the same protein pattern. Stability studies demonstrated that the formulated PEGylated miLZ-TRAIL (21-22 mg / ml) was resistant to multiple freeze-thaw cycles in terms of both biological activity and performance in HPLC analysis.

[0393] When the proteins were heated in the presence of a fluorescent dye sensitive to protein aggregation / denaturation using the ENZO PROTEOSTAT aggregation kit, melting / aggregation curves with three easily discernible phases were observed for all iLZ-TRAIL and miLZ-TRAIL variants.

[0394] Phase 1 (25-55°C) reflects weak protein-dye interactions and is more pronounced at pH 8.0 than at pH 6.0. This may be due to initial differences in the exposure of hydrophobic regions or charge-mediated binding context.

[0395] The temperature peaks in phase 2 (60-80°C at pH 8.0 and 55-75°C at pH 6.0) and phase 3 (80-98°C at pH 8.0 and 75-98°C at pH 6.0) were similar for all variants melting in the same buffer.

[0396] Phase 2 at pH 6.0 showed the highest signal several degrees lower than that at pH 8.0.

[0397] The variant with the lowest predicted immunogenicity, miLZ-TRAIL (1876 (K32E, I23V), corresponding to the miLZ sequence of SEQ ID NO: 4), had a less pronounced first phase at pH 6.0 compared to all other variants.

[0398] The results are as follows Figures 5A-5H shown.

[0399] Example 6. PEGylated miLZ-TRAIL shows significantly increased potency (IC50, in vitro) compared to PEGylated TRAIL with a HIS-tag

[0400] Materials and methods

[0401] The fusion polypeptides used in this study were the same as those in Example 4.

[0402] result

[0403] When tested for cytotoxicity using a COLO 205 cell assay, the final PEGylated TRAIL was 9-fold higher in bioactivity than the prototype ( Figure 4 and Table 2 ).

[0404] Example 7. Pharmacokinetic (PK) parameters of PEGylated miLZ-TRAIL in primates

[0405] Materials and methods

[0406] PK parameters were studied in cynomolgus monkeys. This was a single-dose, parallel-group, dose-escalation study in five normal male and five female cynomolgus monkeys aged 34 to 44 months. At study entry, body weight ranged from 2.9 kg to 3.9 kg for males and 2.9 kg to 3.2 kg for females. The study design is summarized in Table 3. Blood samples were collected at the time points specified in Table 3, and serum samples were prepared for analysis of drug levels.

[0407] Table 3: Test drug allocation and blood sample collection schedule for monkeys.

[0408]

[0409] Sample analysis. A sandwich enzyme-linked immunosorbent assay (ELISA) for human TRAIL was developed to measure the concentration of TLY012 in monkey serum. Briefly, serum samples were diluted in 0.5% normal monkey serum and incubated with an anti-TRAIL capture antibody immobilized on an ELISA plate. Test samples were analyzed at dilutions ranging from 1:200 to 1:168,000 (depending on the predicted concentration) to fit the range of the standard curve (97 pg / mL to 12.5 ng / mL). All data within the experimental range were reported and provided for pharmacokinetic analysis.

[0410] PK parameter analysis. The pharmacokinetic analysis of the dose and serum concentration-time data was performed independently of the compartment model using Phoenix 64 WinNonlin software. The parameters discussed included: maximum serum concentration (C max ), reach C max Time (T max ), reaching the final quantifiable concentration (C last ) time (T last ), elimination half-life (t 1 / 2 ). From Zero to C last The area under the curve (AUC last), the total area under the curve (AUC) from zero to infinity. The total systemic clearance (CL) and steady-state distribution volume (V) of group IV were calculated. ss The clearance (CL / F), volume of distribution (Vz / F), and bioavailability (F) of the SC group were calculated. In addition, the dose-exposure relationship was evaluated by linear regression analysis.

[0411] result

[0412] Following intravenous (IV) administration of a 10 mg / kg dose, TLY012 serum concentrations declined log-linearly, with first order terminal elimination half-lives (Table 4) of 30.9 hours in females and 38.0 hours in males. Serum concentrations appeared to decline in a log-linear fashion, with a slight deviation from the barrel shape, suggesting a nonlinear elimination process ( Figures 7A-7D ).

[0413] Table 4. Pharmacokinetic parameters of TLY012 after intravenous (IV) administration in cynomolgus monkeys

[0414]

[0415] Following SC doses (2, 10, or 50 mg / kg) of TLY012 (Groups 2-4), the time to maximum (Tmax) serum concentrations ranged from 6 to 72 hours after administration. Serum concentrations at 2 mg / kg decreased in a clear log-linear fashion, with a first-order terminal elimination half-life of 34.7 hours in males and 42.7 hours in females. The half-life values ​​for the 10 mg / kg group were 20.7 hours in males and 30.4 hours in females, and the half-life values ​​for the 50 mg / kg group were 43.9 hours in males and 46.7 hours in females. Following SC administration of TLY012 to monkeys, the absolute bioavailability of TLY012 appeared to increase with increasing dose. Absolute bioavailability (F%) was 37.4%, 75.4%, and 77.8% in males after administration of 2 mg / kg, 10 mg / kg, and 50 mg / kg of TYL012, respectively. After administration of 2 mg / kg, 10 mg / kg, and 50 mg / kg TYL012 to females, F was 53.7%, 79.6%, and 114%, respectively.

[0416] Table 5: Pharmacokinetic parameters of TLY012 after SC administration to cynomolgus monkeys

[0417]

[0418] In the dose range studied (2-50 mg / kg), the maximum serum (Cmax) concentration increased proportionally with dose, and the total area under the curve (AUC) and exposure (AUC) increased with dose in a disproportionately large manner. The dose increased 25-fold, while the exposure increased 40-fold to 70-fold (Table 5).

[0419] The elimination half-life was 20.7 hours to 46.7 hours. In contrast, the t 1 / 2 was only 0.9 h, while the t 1 / 2 is 8.6 hours (Hepatology; 64(1):209–223(2016)).

[0420] Example 8. TLY012 induces apoptosis and reduces fibrosis in in vivo analysis of PSC / ASPC-1 xenografts

[0421] Materials and methods

[0422] Six-week-old athymic nude mice were anesthetized for all procedures and 5 × 10 6 ASPC-1 cells (a human pancreatic cancer ascites metastatic cell line derived from nude mouse xenografts initiated with cells from ascites of a 62-year-old female Caucasian patient with pancreatic cancer) were inoculated with 5 × 10 4 PSC cells were mixed and injected subcutaneously into athymic nude mice. Before injection, cells were trypsinized, counted, washed twice in 1× PBS, and resuspended in 100 μl The cells were fixed with a 400 μg / ml PBS solution (Corning) and injected between the shoulder blades. Two weeks after tumor implantation, mice were injected with TLY012 for 2 weeks via intravenous (IV) injection (10 mg / kg every other day), while control mice received PBS. Tissue extracts were Western blotted with antibodies against GAPDH (Santa Cruz Biotechnology, sc-1694), α-SMA (Sigma, A2547), GRP78 (Santa Cruz Biotechnology, sc-13968), DR4 (Abcam, Cambridge, MA, #13890), DR5 (Abcam, #47179), PDGF-Rb (Santa Cruz Biotechnology, sc-432), and C1.PARP-1 (Cell Signaling Technology, #5625).

[0423] Comparative quantitative RT-PCR (qPCR) was performed in tumor samples to detect fibrosis markers. qPCR was performed in triplicate for each sample using the Rapid SYBR Green Master Mix (Thermo Fisher Scientific) and the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Target gene expression levels were normalized to 18s expression and calculated based on the comparative cycle threshold Ct method (2-ΔΔCt).

[0424] Collagen deposition was examined using Masson's Trichrome staining and microscopy.

[0425] result

[0426] Western blot analysis showed that the levels of PDGF Rb, DR5, DR4, and GRP78 were significantly decreased compared with the PBS-treated control group. The level of PARP-1 was increased ( Figure 8A ).

[0427] The anti-tumor / fibrogenic efficacy of TLY012 was studied in PSC / AsPC-1 (human primary pancreatic stellate cell / pancreatic adenocarcinoma cell line) tumor-bearing mice. Tissue samples treated with TLY012 showed high levels of cleaved PARP-1, a typical apoptosis marker. In addition, PDGF-Rb, DR5, and DR4 protein levels were significantly reduced in tumors as evaluated by Western blotting. Acta2, Pdgf-r, and Tgf-β mRNA levels were reduced during TLY012 treatment, and collagen-positive signals were highly increased in PSC-AsPC-1 mixed tumor tissue samples, but TLY012 treatment reduced collagen levels by Masson's trichrome staining.

[0428] When normalized to GAPDH, the expression levels of Acta2 and Tgf-β were significantly decreased in the TLY012-treated group compared with the control group ( Figures 8B-8D ).

[0429] Images and digital quantification of collagen-positive areas stained with Masson's trichrome showed a significant reduction in collagen deposition after TLY012 treatment.

[0430] Example 9. Combination treatment with TLY012 and sorafenib reduces tumor size in PSC / ASPC-1 xenografts.

[0431] Materials and methods

[0432] ASPC-1 cells or ASPC-1 cells with PSC-conditioned medium (PSC-CM) were cultured in vitro in culture wells for 24 hours and then treated with control (DMSO) or 100 ng / mL TLY012 and 1 μM sorafenib for 8 hours. Microscope images were acquired from the culture wells after 8 hours of culture.

[0433] Mouse xenografts were prepared as described in Example 8. Two weeks after tumor implantation, mice were injected intravenously (IV) with TLY012 (10 mg / kg every other day) and / or administered with sorafenib (20 mg / kg orally daily) for 2 weeks. When the tumor volume reached 1500 mm 3 , mice were euthanized, images of excised xenografts were recorded, and tumor weights were measured.

[0434] result

[0435] Images of ASPC-1 cells or ASPC-1 cells cultured with PSC CM for 24 hours followed by 8 hours of TLY012 and sorafenib treatment show that ASPC-11 cells treated with TLY012 and sorafenib for 8 hours showed a decrease in cell number compared to control-treated ASPC-1. The decrease in ASPC-1 cell number was significantly greater when ASPC-1 cells were treated with PSC CM and treated with TLY012 and sorafenib. This significant decrease was statistically significant relative to the number of ASPC-1 cells receiving control-treated PSC CM or relative to ASPC-1 cells treated with TLY012 and sorafenib for 8 hours. Figure 9A shown.

[0436] Figure 9B The tumor weights (mg) of control and treated mice are shown. The combined effect was synergistic. Table 6 summarizes Figure 9B data.

[0437] Table 6. Quantification of tumor weight (mg) of ASPC-1 / PSC xenografts obtained from mice treated with Sorafenib, TLY012, or the combination of TLY012 and Sorafenib.

[0438] Group mean SEM comparison 1454 79.71449 Sorafenib 967 89.83207 TLY012 1109.5 180.3952 Sorafenib / TLY012 567.6667 76.09059

[0439] This demonstrates that sensitization of pancreatic cells to sorafenib (a multikinase inhibitor) significantly increased TLY012-induced apoptosis in AsPC-1 cells after pretreatment with conditioned medium from activated PSCs.6 AsPC-1 cells were mixed with 5×10 4 PSC cell mixture 100 μl The antitumor effect of TLY012 in AsPC-1 cells bearing PSCs was investigated by subcutaneous injection of xenograft athymic mice with stromal cells (Corning). The data in Figure 9 show that sorafenib sensitized the antitumor effect in TLY012-induced fibroblast tumors.

[0440] Example 10. DR5 agonist, TLY012 induces apoptosis in pancreatic CAFs and aPSCs.

[0441] α-SMA+MFB is one of the main sources of cancer-associated fibroblasts (CAFs) and connective tissue generation (desmoplasia), which is known to have strong immunosuppressive properties in solid tumors and contribute to the occurrence and development of tumors. TRAIL-based therapy has attracted much attention due to its anti-tumor effect rather than anti-fibrotic function. The potential anti-fibrotic effect of TRAIL signaling in the fibrosis-associated tumor microenvironment (TME) and immune cells of CAFs or α-SMA+MFB has not been reported before. The main reasons for the challenges of immunotherapy in cancer treatment are insufficient effector T cell response and lack of immunogenicity. Cancer-associated fibroblasts (CAFs) are the main components of TME and are involved in regulating the innate immune system by interfering with the function of cytotoxic T cells. Stromal cells, including stellate cells, endothelial cells and a variety of immune cells, are known to be the source of TME and can be used for cancer survival and progression, with strong immunosuppressive properties.

[0442] Materials and methods

[0443] Human primary pancreatic stellate cells (PSCs) and stellate cell culture medium (SteCM) were obtained from ScienCell Research Laboratories (Carlsbad, CA). PSCs were cultured on poly-L-lysine coated plates in SteCM medium supplemented with 2% FBS, 1% stellate cell growth supplement, and 1% penicillin / streptomycin solution according to the manufacturer's instructions. Pancreatic cancer-associated fibroblasts (CAF08) and maintenance medium (PC00B5) were obtained from Vitro Biopharma. 100 PSCs were cultured and activated for 7 days to obtain a confluent activated cell population, or 2 × 10 4CAF08 cells were cultured in 96-well microplates (Corning, CLS3917) for 24 hours and incubated with TLY012 (at 30 ng / ml or 100 ng / ml) for 3 hours. After incubation, 50 μL of caspase 3 / 7 substrate / lysis buffer was added to each well after equilibration for 30 minutes to room temperature. Luminescence of each sample was measured on a plate reader (Bio-Tek Instruments Inc) with a 1-minute delay time and a 0.5-second / well read time (n=3).

[0444] For immunoblotting, 100 PSCs were cultured and activated in 6-well plates for 7 days, or 2 × 10 5 CAF08 cells were seeded in 6-well plates and then treated with control or TLY012 (30 ng / ml or 100 ng / ml) for 3 hours. Cell lysates were tested for the presence of C1, PARP-1, and SMA using Western blotting.

[0445] result

[0446] Luminescence of each well is reported as relative luminescence units (RLU). The data show that caspase 3 / 7 is activated in both PSC and CAF08 cells after TLY012 treatment, but not in control-treated cells ( Figure 10 ).

[0447] Western blot analysis detected Cl.PARP-1 in TLY012-treated PSCs and CAF08 cells. Cl.PARP-1 was not detected in control-treated PSCs or CAF08 cells. SMA levels in CAF08 cells were significantly higher than in PSCs treated with either treatment.

[0448] This confirmed that TLY012 induced apoptosis in primary cancer-associated fibroblasts derived from pancreatic cancer patients, compared with activated primary human pancreatic stellate cells, as assessed by caspase 3 / 7 activity assay and Western blot for cleaved PARP-1. The study also found that pancreatic CAFs expressed high levels of α-SMA protein compared with activated stellate cells.

[0449] Example 11. TLY012 is superior to other cancer therapies in inducing apoptosis in human pancreatic cancer-associated fibroblasts

[0450] Materials and methods

[0451] Pancreatic cancer-associated fibroblasts (CAF08) and maintenance medium (PC00B5) were obtained from Vitro Biopharma and maintained in pancreatic astrocyte CAF maintenance medium (Vitro Biopharma, Cat. No. PC00B5). Approximately 2×10 4 Cells were cultured in 96-well microplates (Corning, CLS3917) for 24 hours and incubated with 30 ng / ml TLY012 for 3 hours, or with the cytotoxic agents gemcitabine (50 μM) for 24 hours, doxorubicin (10 μM) for 24 hours, cisplatin (50 μM) for 24 hours, 5-fluorouracil (5-FU) (25 μg / ml) for 24 hours, or irinotecan (10 μM) for 24 hours in a dose-dependent manner.

[0452] After incubation, after a 30-minute equilibration period to room temperature, 50 μL of caspase 3 / 7 substrate / lysis buffer from the Caspase 3 / 7 Glo Assay Kit (Promega, Madison USA) was added to each well and incubated for 30 minutes. Luminescence of each sample was measured on a plate reader (Bio-Tek Instruments Inc) with a 1-minute delay time and a 0.5-second / well read time (n=3).

[0453] For Western blotting, 2 × 10 5 Cells were plated in 6-well plates and incubated with the same cytotoxic agents as indicated, and then Western blot analysis of Cl.PARP-1, PD-L1, DR5, DR4, α-SMA, and β-actin was performed.

[0454] result

[0455] Figure 11 Luminescence results reported as fold increase in luminescence (Fold) under different treatment conditions are shown.TLY012 outperforms other cancer therapies in activating caspase 3 / 7 in human pancreatic cancer-associated fibroblasts.

[0456] The data showed that among the cytotoxic agents tested, including gemcitabine, doxorubicin, cisplatin, 5-FU, and irinotecan, only TLY012 induced apoptosis ( Figure 11 Importantly, the protein level of α-SMA in pancreatic CAFs was significantly reduced by TLY012 treatment, as assessed by Western blotting.

[0457] Example 12. TLY012 induces apoptosis in human colon cancer-associated fibroblasts and is superior to other cancer treatments in inducing apoptosis.

[0458] Materials and methods

[0459] Human colon cancer fibroblasts HC-6231 were obtained from Cell Biologics (Cat. No. HC-6231) and maintained in fibroblast culture medium (Cell Biologics, Cat. No. M2267). 4 The cells were cultured in 96-well microplates (Corning, CLS3917) for 24 hours, and

[0460] (a) incubating with TLY012 at a dose of 0 ng / ml, 10 ng / ml, 30 ng / ml, 100 ng / ml or 1000 ng / ml in a dose-dependent manner for 3 hours; or

[0461] b) Incubation with TLY012 or cytotoxic agents as indicated: 30 ng / ml TLY012 for 3 hours, 50 μM gemcitabine for 24 hours, 10 μM doxorubicin for 24 hours, 50 μM cisplatin for 24 hours, 25 μg / ml 5-FU for 24 hours, or 10 μM irinotecan for 24 hours.

[0462] Then, after a 30-minute equilibration period to room temperature, 50 μL of caspase 3 / 7 substrate / lysis buffer was added to each well and incubated for 30 minutes. Luminescence of each sample was measured on a plate reader (Bio-Tek Instruments Inc) with a 1-minute delay time and a 0.5-second / well read time (n=3).

[0463] result

[0464] Figure 12A Shown are the luminescence results of relative luminescence units (RLU) of activated caspase 3 / 7 in colon cancer fibroblasts treated with different doses of TLY012 for 3 hours. Figure 12B Shown are the fold increases (Fold) in caspase 3 / 7 activation in colon cancer fibroblasts treated with different cytotoxic agents at the indicated doses and times.

[0465] This study demonstrated that TLY012 induced apoptosis in primary cancer-associated fibroblasts derived from colon cancer patients, as assessed by caspase 3 / 7 activity assay ( Figure 12A TLY012 induced significantly more apoptosis among the cytotoxic agents tested, including gemcitabine, doxorubicin, cisplatin, 5-FU, and irinotecan ( Figure 12B ).

[0466] Example 13. CAFs highly express fibroblast markers and PD-L1 / 2.

[0467] Programmed cell death protein 1 (PD-1) on T cells and PD-1 ligands PD-L1 or PD-L2 on tumor cells are important factors in tumor cell evasion of host immunity. The PD-1 / PD-L1 pathway controls the induction and maintenance of immune tolerance within the tumor microenvironment. The activity of PD-1 and its ligands PD-L1 or PD-L2 is responsible for T cell activation, proliferation, and cytotoxicity secretion in cancer to degenerate antitumor immune responses (Han et al., Am J Cancer Res; 10(3):727-742(2020)). By eliminating CAFs, TLY012 opens up the tumor microenvironment to host antitumor immune responses. Systemic DR5 agonists (TLY012) combined with established emerging immunotherapies (e.g., checkpoint inhibitors) can enable drugs and immune cells to enter tumors while restoring immune responses in the tumor microenvironment, thereby showing synergistic effects in hepatocellular carcinoma (HCC), colorectal cancer, or pancreatic ductal adenocarcinoma (PDAC).

[0468] Materials and methods

[0469] According to the manufacturer's instructions, Purelink RNA kit (Thermo Fisher Scientific, Waltham, MA) was used to extract total RNA from cultured cells. RNA concentration was measured using NanoDrop2000 (Thermo Fisher Scientific) spectrophotometry. 1-2 μg of total RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription system (Reverse Transcription System, Thermo Fisher Scientific). Three comparative quantitative RT-PCR (qPCR) analyses were performed on each sample using fast SYBR Green MasterMix (Thermo Fisher Scientific) and StepOnePlus real-time PCR systems (StepOnePlus Real-Time PCR System, Thermo Fisher Scientific). The expression level of the target gene was standardized to 18s expression and calculated based on the comparative cycle threshold Ct method (2-ΔΔCt). The sequences of the primers are shown in Table 7.

[0470] Table 7. Primer sequences used for qPCR.

[0471]

[0472] result

[0473] Figures 13A-13C DR4 or DR5 ( Figure 13A ), PD-L1 or PD-L2 ( Figure 13B ) or Acta2, TGF-β or Col1a2 ( Figure 13C )'s relative expression of mRNA. Figure 13B and 13C It was shown that CAFs highly expressed fibroblast markers and PD-L1 / 2.

[0474] This study showed that pancreatic CAFs and colon CAFs expressed Dr4, Dr5, Acta2, Tgf-b, Col1a2, PD-L1, and PD-L2 mRNA levels compared to normal cells, quiescent PSCs, or Conlon fibroblasts. Both pancreatic CAFs and colon CAFs showed high levels of PD-L1 or PD-L2 mRNA. Compared to normal cells, colon CAFs showed high levels of Dr4 / 5, and pancreatic CAFs showed significantly higher levels of Acta2, Tgf-b, and Col1a2 mRNA fibroblast markers. Taken together, these data suggest that removing fibrosis is an effective strategy for treating cancer.

[0475] Example 14. TLY012 reduces fibrosis levels and tumor volume in orthotopic xenografts of KPC cells in mice.

[0476] Materials and methods

[0477] Method for inoculating KPC into the pancreas of B6.129 mice

[0478] To generate solid tumors, 10 7 Mouse pancreatic ductal adenocarcinoma UN-KPC-960-luc2 cells suspended in 100 μl PBS were inoculated into the flanks of syngeneic B6.129 mice. Once the tumors reached 8-10 mm in diameter, 3 , KPC tumors of donor mice were removed under sterile conditions. The donor tumors were cut into 1-1.5 mm pieces using a surgical blade. 3 of blocks.

[0479] For orthotopic xenograft tumors in the pancreas, laparotomy was performed through a left flank incision to expose the pancreatic head, and the donor tumor piece was inserted into the pancreas of recipient B6.129 mice (n = 7 / group), and the incision was closed in two layers with 5–0 absorbable sutures. Two weeks after tumor transplantation, mice were injected with TLY012 and / or anti-PD-L1 (B7-H1, anti-mouse PD-L1, 2.5 mg / kg, twice a week) via the intraperitoneal route (10 mg / kg, every other day) for 2 weeks. Transplanted animals were imaged once a week for 5 weeks. Bioluminescence images were acquired using IVIS 10-15 minutes after intraperitoneal administration of D-luciferin (150 mg / kg) (GOLDBIO, St. Louis, MO, USA).

[0480] For tumor volume studies, orthotopic xenograft tumors were generated in the pancreas. Laparotomy was performed through a left flank incision to expose the pancreatic head, and 5 × 10 6 UN-KPC-960 cells were transplanted into the pancreas of recipient B6.129 mice (n=5 / group), and then the two layers were sutured with 5-0 absorbable sutures. Four weeks after transplantation, mice were injected with TLY012 or saline via the intraperitoneal route (10 mg / kg, every other day) for 2 weeks. Then, images captured by a) and 1 / 2ab 2 a) Tumor volume after TLY012 treatment, and b) collagen and α-SMA positive areas were measured using Sirius Red and anti-α-SMA staining to study the resected tumors.

[0481] Double immunofluorescence staining

[0482] Paraffin-embedded KPC tumor specimens were immunofluorescently stained (IF) with α-SMA and CD4 or CD8. Tissues were deparaffinized, hydrated, and heated in a microwave in citrate buffer (Thermo Fisher Scientific) for antigen retrieval. Slides were washed, treated with blocking solution (3% normal horse serum, Vectastain ABC System, Vector Labs, Burlingame, CA), and mouse anti-α-SMA antibodies were applied, along with primary antibodies: rabbit anti-CD4 antibodies (Abcam, ab183685) or rabbit anti-CD8 antibodies (Abcam, ab217344). Tissues were incubated overnight at 4°C, washed, and anti-rabbit IgG conjugated to Alexa fluor 488 and mouse IgG conjugated to Alexa fluor 633 (Thermo Fisher Scientific) were applied and incubated at room temperature. After washing the slides, the samples were mounted with Fluorescence Mounting Medium with DAPI and captured under an Axiovert microscope (Carl Zeiss Microscopy, LLC. Thornwood, NY).

[0483] Histological studies and immunohistochemistry

[0484] The resected tumors were immediately fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 4 μm thickness. The sections were then stained with Masson's trichrome or Sirius red to determine collagen deposition. The stained tissues were imaged under a light microscope (Nikon Eclipse E600 connected to a Nikon DS-Fi2 camera, Nikon USA). Ten randomly selected stained pancreatic tissue images from a single mouse were used to quantify the positive staining area using Image J software (NIH, Bethesda, MD).

[0485] result

[0486] TLY012 reduced fibrosis and tumor volume in orthotopic KPC cell xenografts in mice ( Figure 14 The data showed that only TLY012 reduced tumor volume compared to the saline-treated group. Tissue samples of xenograft tumors showed high levels of collagen deposition and α-SMA-positive areas, but TLY012 treatment reduced collagen levels assessed by immunohistochemistry.

[0487] Treatment with TLY012 promoted the migration of cytotoxic T cells to the tumor site in orthotopic xenografts of mouse KPC cells. CD4+ and CD8+ cells were detected in the tumor tissue. The data showed that cytotoxic T cells (including CD8 and CD4 cells) in the tumor tissue TLY012 samples co-localized with α-SMA cells compared to saline-treated samples. This strategy reduced the fibrosis of PDAC tumors and destroyed the stromal barrier. Finally, TLY012 enhanced the infiltration of cytotoxic T cells into the tumor site.

[0488] Combination therapy of TLY012 and anti-PD-L1 reduced luciferase activity in tumors, decreased tumor volume, and improved survival in mice bearing orthotopic xenografts of mouse KPC cells ( Figures 15A-15C ). The results are also shown in Tables 8-10.

[0489] Table 8. Bioluminescent activity (total counts) over time (days) obtained from mouse tumors bearing orthotopic xenografts of mouse KPC cells, treated with PBS, anti-PD-L1 antibody, TLY012, or a combination of anti-PD-L1 antibody and TLY012.

[0490]

[0491] Table 9. Mean tumor volume (mm) of tumors obtained from mice bearing orthotopic xenografts of mouse KPC cells and treated with PBS, anti-PD-L1 antibody, TLY012, or a combination of anti-PD-L1 antibody and TLY012. 3 ).

[0492]

[0493] Table 10. Survival rate over days of mice bearing orthotopic xenografts of mouse KPC cells and treated with PBS, anti-PD-L1 antibody, TLY012, or a combination of anti-PD-L1 antibody and TLY012.

[0494] sky PBS Anti-PD-L1 TLY012 joint 0 100 100 100 100 14 90 21 80 88.889 28 70 35 70 88.899 100 100

[0495] pass Spectrum in vivo imaging system evaluation showed that the bioluminescence intensity of tumors in samples treated with TLY012 or PD-L1 combination therapy was significantly reduced ( Figure 15A This confirmed that the PD-L1 and TLY012 combination therapy showed the most effective efficacy in the treatment group ( Figure 15B ). In addition, it was found that the combined treatment increased the survival rate of the animals ( Figure 15C ).

[0496] The combination of TLY012 and anti-PD-L1 reduced the level of collagen deposition, as determined from captured images of Sirius red staining of collagen-positive areas ( Figure 16 This confirmed that tissue samples of xenograft tumors showed high levels of collagen deposition in control mice, but the combined treatment of PD-L1 and TLY012 reduced the collagen-positive area (Sirius Red) compared to the other groups ( Figure 16 ).

[0497] Example 15. Liver CAFs highly express fibroblast markers

[0498] Materials and methods

[0499] Liver CAF (BioIVT, DT01046P1) cells were cultured in DMEM. Total RNA from the cultured cells was extracted using the PURELINK RNA kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. RNA concentration was measured spectrophotometrically using a NanoDrop 2000 (Thermo Fisher Scientific). 1-2 μg of total RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription system (Thermo Fisher Scientific). Three comparative quantitative RT-PCR (qPCR) analyses were performed for each sample using a fast SYBR Green Master Mix (Thermo Fisher Scientific) and a Quantstudio 5 Real-Time PCR System (Thermo Fisher Scientific). The expression levels of the target genes were normalized to the expression of GAPDH and calculated based on the comparative cycle threshold Ct method (2-ΔΔCt).

[0500] result

[0501] It has been demonstrated that mRNA levels of Acta2, Col1a2, Col3a1, PDGFR, and CTGF are significantly increased in activated hepatic stellate cells and primary HCC-associated fibroblasts compared with quiescent HSCs ( Figure 17 These data suggest that targeting aHSCs or hepatic CAFs to eliminate fibrosis is one of the therapeutic strategies for HCC by safely eliminating primitive cells in the tumor microenvironment.

[0502] Example 16. Increased apoptosis levels in HCC cells treated with TLY012 using liver CAF conditioned medium (CM)

[0503] Materials and methods

[0504] 2×10 4 HCC cells (Huh-7 or Hep-G2) were cultured for 24 hours on a 96-well microplate (Corning, CLS3917) and cultured for 3 hours with conditioned medium from liver CAFs at 100 ng / mL TLY012. After a 30-minute equilibration period to room temperature, 50 μL of caspase 3 / 7 substrate / lysis buffer was added to each well and cultured for 30 minutes. Luminescence of each sample was measured on a plate reader (Bio-Tek Instruments Inc) with a 1-minute delay and a 0.5-second / well read time (n=3).

[0505] result

[0506] It has been demonstrated that TLY012 induces apoptosis in highly TRAIL-resistant HCC cells of Huh-7 and Hep-G2 treated with conditioned medium (CM) from primary cancer-associated fibroblasts, as assessed by caspase 3 / 7 activity assay ( Figure 18A and Figure 18B ).

[0507] Di Modugno et al., Journal of Experimental and Clinical Cancer Research, 38(117)(2019) discussed the current knowledge about the tumor microenvironment, focusing on T cells, cancer-associated fibroblasts, and the extracellular matrix. TRAIL signaling, which interacts with CAFs and immune cells, plays a potential anti-fibrotic role in the tumor microenvironment associated with fibrosis.

[0508] Activated PSCs are the main source of cancer-associated fibroblasts (CAFs) and connective tissue production, which are known to contribute to tumor initiation and progression and have potent immunosuppressive properties. Importantly, PDAC has an excessive tumor microenvironment and is highly resistant to conventional anticancer drugs because drug delivery becomes difficult due to penetration.

[0509] The combination of TLY012 and anti-PD-L1 immunotherapy showed the best results and may be the mainstay of treatment, including PD-1, PD-L1, CTLA-4, and IDO. Blocking immune checkpoints is the most promising approach to date for activating therapeutic anti-tumor immunotherapy. Programmed cell death-1 (PD-1) is a key immune checkpoint on T lymphocytes. However, PD-L1 from tumor cells binds to PD-1 and inhibits the anti-tumor ability of T lymphocytes by reducing T cell activity and proliferation, which is known as the "don't eat me signal." One of the main reasons for poor prognosis with immunotherapy is desmoplasia, which is the excessive proliferation of fibrotic tissue. Desmoplastic responses induce physiological and biological signals that promote tumor cell proliferation and drug resistance.

[0510] Here, we show that TLY012 has anti-fibrotic function and targets fibrotic tumors with cancer-associated fibroblasts that, if left untreated, are resistant to immunotherapy due to the fibrotic cell barrier.

[0511] 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 the disclosed invention belongs.Publications cited herein and the materials they cite are specifically incorporated by reference.

[0512] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A recombinant fusion polypeptide consisting of the amino acid sequence GRMKQIEDKIEEILSKIYHVENE IARIKELIGEDGVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG. 2 . The recombinant fusion polypeptide according to claim 1 , having a solubility between 0.2 mg / ml and 100 mg / ml in a physiological buffer and at a physiological pH.

3. The recombinant fusion polypeptide of claim 1, wherein the polypeptide is an inhibitory polypeptide having an in vitro half-maximal inhibitory concentration (IC50) between 0.001 nM and 0.1 nM when tested on COLO 205 cells. The recombinant fusion polypeptide according to claim 1 , which has an in vivo half-life of between 1 hour and 24 hours.

5. The recombinant fusion polypeptide according to claim 1, which has significantly lower immunogenicity in a human host than the immunogenicity of a corresponding polypeptide in which the amino acid at position 14, 17 or 26 of SEQ ID NO: 4 is not selected from valine (V), glutamic acid (E) and glutamine (Q).

6. A recombinant fusion polypeptide comprising two or more polypeptides according to claim 1 in the form of dimers, trimers, tetramers or multimers.

7. A conjugate comprising the recombinant fusion polypeptide according to any one of claims 1 to 6 and a half-life extending molecule.

8. The conjugate of claim 7, wherein the half-life extending molecule comprises polyethylene glycol or a derivative thereof.

9. The conjugate according to claim 7 or 8, wherein the half-life extending molecule comprises polyethylene glycol or a derivative thereof having a molecular weight between 5000 Da and 100000 Da.

10. The conjugate of claim 7, which has a solubility of up to 50 mg / ml in the presence of physiological concentrations of salt. The conjugate according to claim 7 , which has an in vivo half-life of 20 to 50 hours in non-human primates.

12. The conjugate of claim 7, having an in vitro IC50 between 0.01 nM and 1 nM when tested on activated hepatic stellate cells (HSC).

13. The conjugate of claim 7, wherein two or more freeze-thaw cycles of the recombinant fusion polypeptide do not reduce the solubility and activity of the conjugate.

14. The conjugate of claim 7, which has significantly lower immunogenicity in a human host relative to the immunogenicity of the corresponding polypeptides wherein the amino acid at position 14, 17 or 26 of SEQ ID NOs: 4-8 is not selected from valine (V), glutamic acid (E) and glutamine (Q).

15. The conjugate according to claim 7, comprising (i) a recombinant fusion polypeptide consisting of the amino acid sequence GRMKQIEDKIEEILSKIYHVENEIARIKELIGEDG VRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG; and (ii) polyethylene glycol with a molecular weight between 5000 Da and 100000 Da, wherein the polyethylene glycol is conjugated to the polypeptide. The conjugate according to claim 15 , which is in the form of a dimer, trimer, tetramer or multimer.

17. A recombinant polynucleotide comprising a nucleic acid sequence encoding the fusion polypeptide of any one of claims 1-6.

18. The recombinant polynucleotide according to claim 17, which is in an expression vector for expression in prokaryotic or eukaryotic cells.

19. The recombinant polynucleotide of claim 17 or 18, constructed for expression in a prokaryotic cell to produce between 0.2 g / ml and 2 g / ml of the polypeptide.

20. A prokaryotic or eukaryotic cell comprising the recombinant polynucleotide of claim 17.

21. A method for preparing the fusion polypeptide of any one of claims 1-6, comprising expressing the recombinant polynucleotide of any one of claims 17-19 in a prokaryotic cell or a eukaryotic cell, and purifying the fusion polypeptide to obtain a purified fusion polypeptide.

22. The method according to claim 21, wherein The purified fusion polypeptide is in a buffer comprising at least 90% of the purified fusion polypeptide.

23. The method of claim 21, further comprising conjugating the purified fusion polypeptide to a half-life extending molecule.

24. Use of the recombinant fusion polypeptide of any one of claims 1-6, the recombinant polynucleotide of claim 17 or 18, or the conjugate of any one of claims 7-16 in the preparation of a medicament for treating a proliferative disease in an individual, wherein the proliferative disease is selected from pancreatic ductal adenocarcinoma and colon cancer.

25. The use according to claim 24, wherein the effective amount of the fusion polypeptide or the effective amount of the conjugate is between 0.001 mg / kg and 50 mg / kg.

26. The use according to claim 24 or 25, wherein the fusion polypeptide or the conjugate is used in combination with a second active agent.

27. The use according to claim 24, wherein the medicament is used to induce apoptosis in cancer-associated fibroblasts, wherein the cancer is selected from pancreatic ductal adenocarcinoma and colon cancer.

28. The use according to claim 24, wherein the medicament is used to reduce the size of a tumor in the proliferative disease.

29. The use according to claim 24, wherein the recombinant fusion polypeptide, the recombinant polynucleotide or the conjugate is used in combination with a second active agent, wherein the second active agent is a chemotherapeutic agent.

30. The use according to claim 24, wherein the recombinant fusion polypeptide, the recombinant polynucleotide or the conjugate is used in combination with a second active agent, wherein the second active agent is a chemotherapeutic agent that is a DNA topoisomerase I inhibitor, a DNA topoisomerase II inhibitor and / or an immune checkpoint inhibitor (ICI).

31. The use according to claim 24, wherein the recombinant fusion polypeptide, the recombinant polynucleotide or the conjugate is used in combination with a second active agent, wherein the second active agent is one or more immune checkpoint regulators selected from PD-1 antagonists, PD-1 ligand antagonists and CTLA4 antagonists.

32. The use according to claim 24, wherein the recombinant fusion polypeptide, the recombinant polynucleotide or the conjugate is used in combination with a second active agent, wherein the second active agent is selected from the group consisting of doxorubicin, etoposide, camptothecin, irinotecan, cisplatin, oxaliplatin, docetaxel, cyclophosphamide, 5-fluorouracil, carboplatin, mechlorethamine, sorafenib, chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, paclitaxel and its derivatives, topotecan, amsacrine, teniposide, epipodophyllotoxin, trastuzumab, cetuximab, rituximab, bevacizumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplizumab, pidilizumab, wolpidemab, danwasser, cetuximab and ipilimumab.

33. The use according to claim 24, wherein the recombinant fusion polypeptide, the recombinant polynucleotide or the conjugate is used in combination with a second active agent, wherein the second active agent is etoposide phosphate. 34 . The use according to claim 24 , wherein the recombinant fusion polypeptide, the recombinant polynucleotide or the conjugate is used in combination with adoptive T cell therapy and / or cancer vaccine.

35. The use according to claim 24, wherein the recombinant fusion polypeptide, the recombinant polynucleotide or the conjugate is used in combination with surgery or radiotherapy.

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