Compositions and methods to manufacture tumor suppressor fusions

Abstract

Provided herein is a fusion protein comprising, or alternatively consisting essentially of, or yet further consisting of an optional signal peptide, a serum albumin, an optional linker, a Phosphatase and Tensin Homolog (PTEN), and an optional purification or detectable marker in any order. Relating polynucleotides, vectors, host cells, pharmaceutical compositions and kits are also disclosed. Further provided are methods for delivering a fusion protein to a subject, treating a cancer or tumor, and/or producing the fusion protein.

1 . A fusion protein comprising consecutive amino acid sequences of serum albumin (SA) and the Phosphatase and Tensin Homolog (PTEN) tumor suppressor. 
     
     
         2 . The fusion protein of  claim 1 ,
 wherein the serum albumin comprises a human serum albumin (HSA), or   wherein the PTEN comprises any one of human PTEN, simian PTEN, rat PTEN, or murine PTEN, or   wherein the serum albumin comprises the HSA and the PTEN comprises any one of: human PTEN, simian PTEN, rat PTEN, or murine PTEN.   
     
     
         3 . The fusion protein of  claim 2 , wherein the serum albumin comprises the polypeptide as set forth in SEQ ID NOs:163 or 13 and the PTEN comprises the polypeptide as set forth in SEQ ID NOs:2 or 3. 
     
     
         4 . The fusion protein of  claim 1 , wherein the fusion protein comprises a polypeptide encoded by the engineered polynucleotide as set forth in any one of SEQ ID NOs: 68, 59, 58, 157, 159, 65, or 136, or a fragment thereof, or a sequence at least 90% identical thereto which encodes the same polypeptide as set forth in any one of SEQ ID NOs: 68, 59, 58, 157, 159, 65, or 136, respectively. 
     
     
         5 . The fusion protein of  claim 1 , comprising the polypeptide as set forth in any one of SEQ ID NOs: 28, 19, 18, 129, 131, 25 or 50, or a fragment, analogue, variant or equivalent of each thereof. 
     
     
         6 . The fusion protein of  claim 1 , comprising any one of the polypeptide as set forth in any one of SEQ ID NOs: 14-17, 20-27, 29-30, 34-37, 49-50, or 109-132, or a fragment, analogue, variant or equivalent of each, or a mutant of each, and optionally further comprising one or more mutations at positions aligned to any one or more of the following: S65, S85, S89, S91, S94, S111, S115, S117, S129, S134, T140, S161, S163, S164 and S168 of SEQ ID NO:1 or S170, N292, S355, S360, S366 and S370 of SEQ ID NO:2, optionally to an aspartate or a glutamate, and optionally wherein the fusion protein is glycosylated, further optionally wherein one or more of the serine (S) or threonine (T) residues of the fusion protein is O-glycosylated, and yet further optionally wherein one or more of asparagine (N) residues of the fusion protein is N-glycosylated. 
     
     
         7 . The fusion protein of  claim 1 , comprising the polypeptide encoded by an engineered polynucleotide as set forth in any one of: SEQ ID NOs: 54-70, 74-77, or 135-160, or a fragment thereof, or a polynucleotide at least 90% identical thereto and which encodes the same polypeptide as set forth in any one of SEQ ID NOs: 54-70, 74-77, or 135-160, respectively. 
     
     
         8 . The fusion protein of  claim 1 , wherein the serum albumin is conjugated directly or indirectly to the N-terminus or C terminus of the PTEN. 
     
     
         9 . The fusion protein of  claim 1 , wherein the PTEN comprises any one or more of the following:
 (i) a mature human PTEN-L Unique Domain (LUD), or a fragment, analogue, variant, mutant, or equivalent thereof, optionally wherein the LUD comprises the polypeptide as set forth in SEQ ID NO:5, further optionally wherein the LUD comprises the polypeptide encoded by the engineered polynucleotide as set forth in SEQ ID NO:193, or a polynucleotide at least 90% identical thereto which encodes the same polypeptide as set forth in SEQ ID NO:5, optionally wherein the LUD is glycosylated, further optionally wherein one or more of the serine (S) or threonine (T) residues of the LUD is O-glycosylated;   (ii) a human PTEN-M Unique Domain (MUD), or a fragment, analogue, variant, mutant, or equivalent thereof, optionally wherein the MUD comprises the polypeptide as set forth in SEQ ID NO:9, further optionally wherein the MUD comprises the polypeptide encoded by the engineered polynucleotide as set forth in SEQ ID NO:194, or a polynucleotide at least 90% identical thereto which encodes the same polypeptide as set forth in SEQ ID NO:9, optionally wherein the MUD is glycosylated, further optionally wherein one or more of the serine (S) or threonine (T) residues of the MUD is O-glycosylated;   (iii) a human PTEN-N Unique Domain (NUD), or a fragment, analogue, variant, mutant, or equivalent thereof, optionally wherein the NUD comprises the polypeptide as set forth in SEQ ID NO:10, further optionally wherein the NUD comprises the polypeptide encoded by the engineered polynucleotide as set forth in SEQ ID NO:195, or a polynucleotide at least 90% identical thereto which encodes the same polypeptide as set forth in SEQ ID NO:10, optionally wherein the NUD is glycosylated, further optionally wherein one or more of the serine (S) or threonine (T) residues of the NUD is O-glycosylated;   (iv) a human PTEN-O Unique Domain (OUD), or a fragment, analogue, variant, mutant, or equivalent thereof, optionally wherein the OUD comprises the polypeptide as set forth in SEQ ID NO:11, further optionally wherein the OUD comprises the polypeptide encoded by the engineered polynucleotide as set forth in SEQ ID NO:196, or a polynucleotide at least 90% identical thereto which encodes the same polypeptide as set forth in SEQ ID NO:11, optionally wherein the OUD is glycosylated, further optionally wherein one or more of the serine (S) or threonine (T) residues of the OUD is O-glycosylated;   (v) a minimal cell-penetration domain (MCPD) comprising 6× Arg (SEQ ID NO:39), and optionally wherein the fusion protein comprises a cleavable linker between the PTEN and the albumin;   (vi) a membrane-binding domain (MBD), optionally wherein the MBD comprises the polypeptide as set forth in SEQ ID NO:40;   (vii) a minimal PTEN-S, or a fragment, analogue, variant, mutant, or equivalent thereof, optionally wherein the minimal PTEN-S comprises the polypeptide as set forth in SEQ ID NO:162, further optionally wherein the minimal PTEN-S comprises the polypeptide encoded by the engineered polynucleotide as set forth in SEQ ID NO:197, or a polynucleotide at least 90% identical thereto which encodes the same polypeptide as set forth in SEQ ID NO:162; and   (viii) a PTEN-S, or a fragment, analogue, variant, mutant, or equivalent thereof, optionally wherein the PTEN-S comprises the polypeptide as set forth in SEQ ID NO:2, further optionally wherein the PTEN-S comprises the polypeptide encoded by the engineered polynucleotide as set forth in SEQ ID NO:172, or a polynucleotide at least 90% identical thereto which encodes the same polypeptide as set forth in SEQ ID NO:2.   
     
     
         10 . The fusion protein of  claim 1 , further comprising one or more of the following: a signal peptide (and optionally wherein the signal peptide comprises a human serum albumin signal peptide), a first linker between the albumin and the PTEN, a second linker at the C terminus of the signal peptide and a third linker at the N terminus of the fusion protein, and optionally wherein one or more of the linkers are cleavable, a detectable marker, or a purification marker. 
     
     
         11 . A vector comprising a polynucleotide encoding the fusion protein of  claim 1 , further optionally wherein the vector is a viral vector, optionally wherein the vector is selected from the group of: rabies virus, flavivirus, lentivirus, baculovirus, retroviral vector, adenovirus vector (AdV), adeno-associated virus vector, herpes simplex virus vector (HSV), alphavirus vector, Seneca Valley virus, poliovirus, vaccinia virus, reovirus, Coxsackievirus, parvovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, or infectious tobacco mosaic virus (TMV), and further optionally wherein the vector is suitable for an anti-cancer therapy or a gene therapy or both. 
     
     
         12 . A method of inhibiting the growth of, or metastasis of a cancer or tumor cell, comprising contacting the cell with an effective amount of the fusion protein of  claim 1  or a vector comprising a polynucleotide encoding the fusion protein, and optionally further comprising contacting the cell with an effective amount of a combined therapy. 
     
     
         13 . The method of  claim 12 , wherein the cancer or tumor cell has low expression or lacks expression of a PTEN, or wherein the subject has a high expression level of an miRNA targeting the 3′ of the PTEN tumor suppressor mRNA compared to a control, optionally wherein the control is a biological sample isolated from a subject who is free of any cancer or tumor, optionally wherein the control has any other cancer or tumor, optionally wherein the miRNA is an onco-miR, optionally wherein the onco-miR is one or more of miR-106a, miR-130a, miR-181b-1, miR-21, miR-214, miR-26a-5p, miR-301a, miR-486-5p, mir-1273g-3p, miR-200b, miR-26a, miR-103, miR-106b, miR-106b-93, miR-107, miR-10b, miR-1297, miR-130, mir-130b, miR-142-5p, miR-144, miR-146b, miR-153, miR-155, miR-17, miR-17-5p, miR-181, miR-181a, miR-181c, miR-18a, miR-19, miR-197-3p, miR-19a, miR-19b, miR-200, miR-200a, miR-205, miR-205-5p, miR-20b, miR-218, miR-22, miR-221, miR-222, miR-224, miR-23a, miR-23b-3p, miR-25, mir-29c, miR-301a-3p, miR-32-5p, miR-335, miR-338-3p, miR-374a, miR-410-3p, miR-4299, miR-454, miR-494, miR-543, miR-548, miR-616-3p, miR-7, miR-718, miR-9, miR-92a, miR-92b, miR-93, miR-93-5p, miR-940 or miR-let 7b, optionally wherein the level of the miRNA is detected in a liquid biological sample of a subject having the cancer or tumor cell, and further optionally wherein the liquid biological sample is selected from the group of blood, serum or plasma. 
     
     
         14 . The method of  claim 12 , wherein the cancer or tumor cell is any one or more of: a primary or metastatic cancer or tumor cell; a solid tumor cell; a gastric cancer cell, a colorectal cancer cell, a prostate cancer cell, a breast cancer cell, a triple negative breast cancer cell, an ovarian carcinoma cell, a renal cell carcinoma cell, an Ewing sarcoma cell, a melanoma cell, a mesothelioma cell, a lung cancer cell, a non-small cell lung cancer cell, a stage IV lung cancer cell, a brain cancer cell, a glioblastoma cell, a lymphoma cell, a leukemia cell, a multiple myeloma (MM) cell; a cell of a solid tumor or a cancer affecting the blood or bone marrow or both; or a cell of a cancer or tumor in a tissue which normally lacks expression (or has a low expression level) of a PTEN protein, optionally wherein the cancer or tumor having an H score of about 20 or lower, wherein the H score is calculated as the product of the percentage of cells staining at an established intensity (0=no staining, 1+=intermediate/decreased, 2+=full) relative to the internal positive control (optionally wherein the internal positive control comprises endothelium or tumor stroma having an intensity set at 2+) giving a product ranging from 0 to 200, optionally wherein the staining intensity of the IHC sample is evaluated blindly by at least two independent pathologists or is computer assisted. 
     
     
         15 . A method of producing the fusion protein of  claim 1 , comprising
 (i) culturing a host cell capable of expressing the fusion protein under conditions suitable for expression of the fusion protein, and optionally wherein the host cell lacks (or has a low level of) expression of a protease which is able to cleave the fusion protein; and   (ii) purifying or isolating the fusion protein optionally from the cell or cell culture medium, optionally comprising an albumin affinity chromatography or a heparin affinity chromatography or both.   
     
     
         16 . (canceled) 
     
     
         17 . The vector of  claim 11 , wherein the vector is an oncolytic HSV comprising the polynucleotide encoding the fusion protein. 
     
     
         18 . The vector of  claim 11 , further comprising an additional polynucleotide encoding an monoclonal antibody (mAb) or an equivalent thereof recognizing and binding to an immune checkpoint. 
     
     
         19 . The vector of  claim 18 , wherein the immune checkpoint comprises CTLA4 and optionally wherein the mAb or equivalent thereof comprises ipilimumab. 
     
     
         20 . The vector of  claim 18 , wherein the immune checkpoint comprises PD-1 and optionally wherein the mAb or equivalent thereof comprises pembrolizumab or nivolumab. 
     
     
         21 . The vector of  claim 18 , wherein the immune checkpoint comprises PD-L1 and optionally wherein the mAb or equivalent thereof comprises atezolizumab.