Patent Application: US-201414771308-A

Abstract:
the present invention relates generally to the field of generating recombinant chimeric fusion proteins to be used in the cancer therapy , and more specifically , to fusion molecules of anti - egfr1 - tgfβrii , anti - egfr1 - pd1 and anti - ctla4 - pd1 and methods of generating same , wherein the methods reduce production costs and increase homogeneity of the recombinant chimeric fusion proteins .

Description:
in order to facilitate review of the various embodiments of the invention and provide an understanding of the various elements and constituents used in making and using the present invention , the following terms used in the invention description have the following meanings . as used herein , the terms “ polypeptide ,” “ protein ” and “ peptide ” are used interchangeably to denote a sequence polymer of at least two amino acids covalently linked by an amide bond , regardless of length or post - translational modification ( e . g ., glycosylation , phosphorylation , lipidation , myristilation , ubiquitination , etc .). d - and l - amino acids , and mixtures of d - and l - amino acids are also included . chimeric polypeptide refers to an amino acid sequence having two or more parts which generally are not found together in an amino acid sequence in nature . the term “ spacer / linker ” as used herein refers to a molecule that connects two monomeric protein units to form a chimeric molecule and still provides for binding of the parts to the desired receptors . particular examples of spacer / linkers may include an amino acid spacer , wherein thee amino acid sequence can essentially be any length , for example , as few as 5 or as many as 200 or more preferably from about 5 to 30 amino acid residues . the term “ therapeutic ,” as used herein , means a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs . the term “ therapeutically effective amount ,” as used herein means an amount of the chimeric protein that is sufficient to provide a beneficial effect to the subject to which the chimeric protein is administered . another example of a modification is the addition of a heterologous domain that imparts a distinct functionality upon the chimeric polypeptide . a heterologous domain can be any small organic or inorganic molecule or macromolecule , so long as it imparts an additional function . particular examples of heterologous domains that impart a distinct function include an amino acid sequence that imparts targeting ( e . g ., receptor ligand , antibody , etc . ), immunopotentiating function ( e . g ., immunoglobulin , an adjuvant ), enable purification , isolation or detection ( e . g ., myc , t7 tag , polyhistidine , avidin , biotin , lectins , etc .). as exemplified herein , polypeptide sequences may include substitutions , variations , or derivitizations of the amino acid sequence of one or both of the polypeptide sequences that comprise the chimeric polypeptide , so long as the modified chimeric polypeptide has substantially the same activity or function as the unmodified chimeric polypeptide . as used herein , the term “ substantially the same activity or function ,” when used in reference to a chimeric polypeptide so modified , means that the polypeptide retains most , all or more of the activity associated with the unmodified polypeptide , as described herein or known in the art . modified chimeric polypeptides that are “ active ” or “ functional ” included herein can be identified through a routine functional assay . for example , by using antibody binding assays or co - receptor binding assays one can readily determine whether the modified chimeric polypeptide has activity . as the modified chimeric polypeptides will retain activity or function associated with unmodified chimeric polypeptide , modified chimeric polypeptides will generally have an amino acid sequence “ substantially identical ” or “ substantially homologous ” with the amino acid sequence of the unmodified polypeptide . as used herein , the term “ substantially identical ” or “ substantially homologous ,” when used in reference to a polypeptide sequence , means that a sequence of the polypeptide is at least 50 % identical to a reference sequence . modified polypeptides and substantially identical polypeptides will typically have at least 70 %, alternatively 85 %, more likely 90 %, and most likely 95 % homology to a reference polypeptide . as set forth herein , substantially identical or homologous polypeptides include additions , truncations , internal deletions or insertions , conservative and non - conservative substitutions , or other modifications located at positions of the amino acid sequence which do not destroy the function of the chimeric polypeptide ( as determined by functional assays , e . g ., as described herein ). a particular example of a substitution is where one or more amino acids are replaced by another , chemically or biologically similar residue . as used herein , the term “ conservative substitution ” refers to a substitution of one residue with a chemically or biologically similar residue . examples of conservative substitutions include the replacement of a hydrophobic residue , such as isoleucine , valine , leucine , or methionine for another , the replacement of a polar residue for another , such as the substitution of arginine for lysine , glutamic for aspartic acids , or glutamine for asparagine , and the like . those of skill in the art will recognize the numerous amino acids that can be modified or substituted with other chemically similar residues without substantially altering activity . modified polypeptides further include “ chemical derivatives ,” in which one or more of the amino acids therein have a side chain chemically altered or derivatized . such derivatized polypeptides include , for example , amino acids in which free amino groups form amine hydrochlorides , p - toluene sulfonyl groups , carobenzoxy groups ; the free carboxy groups form salts , methyl and ethyl esters ; free hydroxyl groups that form o - acyl or o - alkyl derivatives , as well as naturally occurring amino acid derivatives , for example , 4 - hydroxyproline , for proline , 5 - hydroxylysine for lysine , homoserine for serine , ornithine for lysine , and so forth . also included are d - amino acids and amino acid derivatives that can alter covalent bonding , for example , the disulfide linkage that forms between two cysteine residues that produces a cyclized polypeptide . as used herein , the terms “ isolated ” or “ substantially pure ,” when used as a modifier of invention chimeric polypeptides , sequence fragments thereof , and polynucleotides , means that they are produced by human intervention and are separated from their native in vivo — cellular environment . generally , polypeptides and polynucleotides so separated are substantially free of other proteins , nucleic acids , lipids , carbohydrates or other materials with which they are naturally associated . polypeptides of the present invention may be prepared by standard techniques well known to those skilled in the art . such techniques include , but are not limited to , isolation and purification from tissues known to contain that polypeptide , and expression from cloned dna that encodes such a polypeptide using transformed cells . chimeric polypeptides can be obtained by expression of a polynucleotide encoding the polypeptide in a host cell , such as a bacteria , yeast or mammalian cell , and purifying the expressed chimeric polypeptide by purification using typical biochemical methods ( e . g ., immunoaffinity purification , gel purification , expression screening etc .). other well - known methods are described in deutscher et al ., 1990 . alternatively , the chimeric polypeptide can be chemically synthesized . purity can be measured by any appropriate method , e . g ., polyacrylamide gel electrophoresis , and subsequent staining of the gel ( e . g ., silver stain ) or by hplc analysis . the present invention further provides polynucleotide sequences encoding chimeric polypeptides , fragments thereof , and complementary sequences . as used herein , the terms “ nucleic acid ,” “ polynucleotide ,” “ oligonucleotide ,” and “ primer ” are used interchangeably to refer to deoxyribonucleic acid ( dna ) or ribonucleic ( rna ), either double - or single - stranded , linear or circular . rna can be unspliced or spliced mrna , rrna , trna , or antisense rnai . dna can be complementary dna ( cdna ), genomic dna , or an antisense . specifically included are nucleotide analogues and derivatives , such as those that are resistant to nuclease degradation , which can function to encode an invention chimeric polypeptide . nuclease resistant oligonucleotides and polynucleotides are particularly useful for the present nucleic acid vaccines described herein . an “ isolated ” or “ substantially pure ” polynucleotide means that the nucleic acid is not immediately contiguous with the coding sequences with either the 5 ′ end or the 3 ′ end with which it is immediately contiguous in the naturally occurring genome of the organism from which it is derived . the term therefore includes , for example , a recombinant dna ( e . g ., a cdna or a genomic dna fragment produced by pcr or restriction endonuclease treatment produced during cloning ), as well as a recombinant dna incorporated into a vector , an autonomously replicating plasmid or virus , or a genomic dna of a prokaryote or eukaryote . the polynucleotides sequences of the present invention can be obtained using standard techniques known in the art ( e . g ., molecular cloning , chemical synthesis ) and the purity can be determined by polyacrylamide or agarose gel electrophoresis , sequencing analysis , and the like . polynucleotides also can be isolated using hybridization or computer - based techniques that are well known in the art . such techniques include , but are not limited to : ( 1 ) hybridization of genomic dna or cdna libraries with probes to detect homologous nucleotide sequences ; ( 2 ) antibody screening of polypeptides expressed by dna sequences ( e . g ., using an expression library ); ( 3 ) polymerase chain reaction ( pcr ) of genomic dna or cdna using primers capable of annealing to a nucleic acid sequence of interest ; ( 4 ) computer searches of sequence databases for related sequences ; and ( 5 ) differential screening of a subtracted nucleic acid library . the invention also includes substantially homologous polynucleotides . as used herein , the term “ homologous ,” when used in reference to nucleic acid molecule , refers to similarity between two nucleotide sequences . when a nucleotide position in both of the molecules is occupied by identical nucleotides , then they are homologous at that position . “ substantially homologous ” nucleic acid sequences are at least 50 % homologous , more likely at least 75 % homologous , and most likely 90 % or more homologous . as with substantially homologous invention chimeric polypeptides , polynucleotides substantially homologous to invention polynucleotides encoding chimeric polypeptides encode polypeptides that retain most or all of the activity or function associated with the sequence to which it is homologous . for polynucleotides , the length of comparison between sequences will generally be at least 30 nucleotides , alternatively at least 50 nucleotides , more likely at least 75 nucleotides , and most likely 110 nucleotides or more . algorithms for identifying homologous sequences that account for polynucleotide sequence gaps and mismatched oligonucleotides are known in the art , such as blast ( see altschul , 1990 ). the polynucleotides of the present invention can , if desired : be naked or be in a carrier suitable for passing through a cell membrane ( e . g ., polynucleotide - liposome complex or a colloidal dispersion system ), contained in a vector ( e . g ., retrovirus vector , adenoviral vectors , and the like ), linked to inert beads or other heterologous domains ( e . g ., antibodies , ligands , biotin , streptavidin , lectins , and the like ), or other appropriate compositions disclosed herein or known in the art . thus , viral and non - viral means of polynucleotide delivery can be achieved and are contemplated . the polynucleotides of the present invention can also contain additional nucleic acid sequences linked thereto that encode a polypeptide having a distinct functionality , such as the various heterologous domains set forth herein . the polynucleotides of the present invention can also be modified , for example , to be resistant to nucleases to enhance their stability in a pharmaceutical formulation . the described polynucleotides are useful for encoding chimeric polypeptides of the present invention , especially when such polynucleotides are incorporated into expression systems disclosed herein or known in the art . accordingly , polynucleotides including an expression vector are also included . for propagation or expression in cells , polynucleotides described herein can be inserted into a vector . the term “ vector ” refers to a plasmid , virus , or other vehicle known in the art that can be manipulated by insertion or incorporation of a nucleic acid . such vectors can be used for genetic manipulation ( i . e ., “ cloning vectors ”) or can be used to transcribe or translate the inserted polynucleotide ( i . e ., “ expression vectors ”). a vector generally contains at least an origin of replication for propagation in a cell and a promoter . control elements , including promoters present within an expression vector , are included to facilitate proper transcription and translation ( e . g ., splicing signal for introns , maintenance of the correct reading frame of the gene to permit in - frame translation of mrna and stop codons ). in vivo or in vitro expression of the polynucleotides described herein can be conferred by a promoter operably linked to the nucleic acid . “ promoter ” refers to a minimal nucleic acid sequence sufficient to direct transcription of the nucleic acid to which the promoter is operably linked ( see bitter 1987 ). promoters can constitutively direct transcription , can be tissue - specific , or can render inducible or repressible transcription ; such elements are generally located in the 5 ′ or 3 ′ regions of the gene so regulated . as used herein , the term “ operably linked ” means that a selected polynucleotide ( e . g ., encoding a chimeric polypeptide ) and regulatory sequence ( s ) are connected in such a way as to permit transcription when the appropriate molecules ( e . g ., transcriptional activator proteins ) are bound to the regulatory sequence ( s ). typically , a promoter is located at the 5 ′ end of the polynucleotide and may be in close proximity of the transcription initiation site to allow the promoter to regulate expression of the polynucleotide . when cloning in bacterial systems , constitutive promoters , such as t7 and the like , as well as inducible promoters , such as pl of bacteriophage gamma , plac , ptrp , ptac , may be used . when cloning in mammalian cell systems , constitutive promoters , such as sv40 , rsv and the like , or inducible promoters derived from the genome of mammalian cells ( e . g ., the metallothionein promoter ) or from mammalian viruses ( e . g ., the mouse mammary tumor virus long terminal repeat , the adenovirus late promoter ), may be used . promoters produced by recombinant dna or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention . mammalian expression systems that utilize recombinant viruses or viral elements to direct expression may be engineered . for example , when using adenovirus expression vectors , the nucleic acid sequence may be ligated to an adenovirus transcription / translation control complex , e . g ., the late promoter and tripartite leader sequence . alternatively , the vaccinia virus 7 . 5k promoter may be used ( see mackett 1982 ; mackett 1984 ; panicali 1982 ). for yeast expression , a number of vectors containing constitutive or inducible promoters may be used ( see ausubel 1988 ; grant 1987 ; glover 1986 ; bitter 1987 ; and strathem 1982 ). the polynucleotides may be inserted into an expression vector for expression in vitro ( e . g ., using in vitro transcription / translation kits , which are available commercially ), or may be inserted into an expression vector that contains a promoter sequence that facilitates expression in either prokaryotes or eukaryotes by transfer of an appropriate nucleic acid into a suitable cell , organ , tissue , or organism in vivo . as used herein , a “ transgene ” is any piece of a polynucleotide inserted by artifice into a host cell , and becomes part of the organism that develops from that cell . a transgene can include one or more promoters and any other dna , such as introns , necessary for expression of the selected dna , all operably linked to the selected dna , and may include an enhancer sequence . a transgene may include a polynucleotide that is partly or entirely heterologous ( i . e ., foreign ) to the transgenic organism , or may represent a gene homologous to an endogenous gene of the organism . transgenes may integrate into the host cell &# 39 ; s genome or be maintained as a self - replicating plasmid . as used herein , a “ host cell ” is a cell into which a polynucleotide is introduced that can be propagated , transcribed , or encoded polypeptide expressed . the term also includes any progeny of the subject host cell . it is understood that all progeny may not be identical to the parental cell , since there may be mutations that occur during replication . host cells include but are not limited to bacteria , yeast , insect , and mammalian cells . for example , bacteria transformed with recombinant bacteriophage polynucleotide , plasmid nucleic acid , or cosmid nucleic acid expression vectors ; yeast transformed with recombinant yeast expression vectors ; plant cell systems infected with recombinant virus expression vectors ( e . g ., cauliflower mosaic virus , camv ; tobacco mosaic virus , tmv ), or transformed with recombinant plasmid expression vectors ( e . g ., ti plasmid ), insect cell systems infected with recombinant virus expression vectors ( e . g ., baculovirus ), or animal cell systems infected with recombinant virus expression vectors ( e . g ., retroviruses , adenovirus , vaccinia virus ), or transformed animal cell systems engineered for stable expression . as used herein , the term “ transformation ” means a genetic change in a cell following incorporation of a polynucleotide ( e . g ., a transgene ) exogenous to the cell . thus , a “ transformed cell ” is a cell into which , or a progeny of which , a polynucleotide has been introduced by means of recombinant techniques . transformation of a host cell may be carried out by conventional techniques known to those skilled in the art . when the host cell is a eukaryote , methods of dna transformation include , for example , calcium phosphate , microinjection , electroporation , liposomes , and viral vectors . eukaryotic cells also can be co - transformed with invention polynucleotide sequences or fragments thereof , and a second dna molecule encoding a selectable marker , as described herein or otherwise known in the art . another method is to use a eukaryotic viral vector , such as simian virus 40 ( sv40 ) or bovine papilloma virus , to transiently infect or transform eukaryotic cells , and express the protein ( see gluzman 1982 ). when the host is prokaryotic ( e . g ., e . coli ), competent cells that are capable of dna uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the cacl 2 method using procedures well - known in the art . transformation of prokaryotes also can be performed by protoplast fusion of the host cell . chimeric polypeptides , polynucleotides , and expression vectors containing same of the present invention can be encapsulated within liposomes using standard techniques and introduced into cells or whole organisms . cationic liposomes are preferred for delivery of polynucleotides . the use of liposomes for introducing various compositions in vitro or in vivo , including proteins and polynucleotides , is known to those of skill in the art . liposomes can be targeted to a cell type or tissue of interest by the addition to the liposome preparation of a ligand , such as a polypeptide , for which a corresponding cellular receptor has been identified . monoclonal antibodies can also be used for targeting ; many such antibodies specific for a wide variety of cell surface proteins are known to those skilled in the art and are available . the selected ligand is covalently conjugated to a lipid anchor in either preformed liposomes or are incorporated during liposome preparation ( see lee 1994 and lee 1995 ). as the chimeric polypeptides or polynucleotides of the present invention will be administered to humans , the present invention also provides pharmaceutical formulations comprising the disclosed chimeric polypeptides or polynucleotides . the compositions administered to a subject will therefore be in a “ pharmaceutically acceptable ” or “ physiologically acceptable ” formulation . as used herein , the terms “ pharmaceutically acceptable ” and “ physiologically acceptable ” refer to carriers , diluents , excipients , and the like that can be administered to a subject , preferably without excessive adverse side effects ( e . g ., nausea , headaches , etc .). such preparations for administration include sterile aqueous or non - aqueous solutions , suspensions , and emulsions . examples of non - aqueous solvents are propylene glycol , polyethylene glycol , vegetable oils , such as olive oil , and injectable organic esters , such as ethyl oleate . aqueous carriers include water , alcoholic / aqueous solutions , emulsions , or suspensions , including saline and buffered media . vehicles include sodium chloride solution , ringer &# 39 ; s dextrose , dextrose and sodium chloride , lactated ringer &# 39 ; s or fixed oils . intravenous vehicles include fluid and nutrient replenishers , electrolyte replenishers ( such as those based on ringer &# 39 ; s dextrose ), and the like . preservatives and other additives may also be present , such as , for example , antimicrobial , anti - oxidants , chelating agents , and inert gases and the like . various pharmaceutical formulations appropriate for administration to a subject known in the art are applicable in the methods of the invention ( e . g ., remington &# 39 ; s pharmaceutical sciences , 18th ed ., mack publishing co ., easton , pa . ( 1990 ); and the merck index , 12th ed ., merck publishing group , whitehouse , n . j . ( 1996 )). controlling the duration of action or controlled delivery of an administered composition can be achieved by incorporating the composition into particles or a polymeric substance , such as polyesters , polyamine acids , hydrogel , polyvinyl pyrrolidone , ethylene - vinylacetate , methylcellulose , carboxymethylcellulose , protamine sulfate or lactide / glycolide copolymers , polylactide / glycolide copolymers , or ethylenevinylacetate copolymers . the rate of release of the composition may be controlled by altering the concentration or composition of such macromolecules . colloidal dispersion systems include macromolecule complexes , nano - capsules , microspheres , beads , and lipid - based systems , including oil - in - water emulsions , micelles , mixed micelles , and liposomes . the compositions administered by a method of the present invention can be administered parenterally by injection , by gradual perfusion over time , or by bolus administration or by a microfabricated implantable device . the composition can be administered via inhalation , intravenously , intraperitoneally , intramuscularly , subcutaneously , intracavity ( e . g ., vaginal or anal ), transdermally , topically , or intravascularly . the compositions can be administered in multiple doses . an effective amount can readily be determined by those skilled in the art . unless otherwise defined , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention , suitable methods and materials are described below . other features and advantages of the invention will be apparent from the following detailed description , and from the claims . the invention is further described in the following examples , which do not limit the scope of the invention ( s ) described in the claims . anti - egfr ( cetuximab ) has been approved for squamous head and neck cancer ( locally or regionally advanced in combination with radiotherapy and metastatic after platinum based therapy ) and egfr expressing metastatic colorectal cancer ( monotherapy in patients after failure of both oxaliplatin and irinotecan based chemo or in patients intolerant to irinotecan based chemo ). not applicable for colonrectal cancer ( crc ) patients having k - ras mutations . across various studies about 55 - 60 % of mcrc patients respond to cetuximab in first line setting , however , this response too is transient ( progression free survival ( pfs ) advantage of 1 . 5 - 2 mths ) ( epar ). significant numbers of patients either do not respond to cetuximab or become resistant to therapy . in the recurrent metastatic head and neck cancer , only 35 % patients respond to cetuximab with chemo with only 2 - 3 month overall survival ( os ) and ( pfs ) advantage . clearly , a significant unmet need exists to improve efficacy of cetuximab therapy in both these indications . moreover , egfr is also expressed in gastric cancer , non - small cell lung cancer ( nsclc ) and pancreatic cancers . however , cetuximab has failed to prove any significant benefit in these indications over standard of care . thus , the present invention provides for improvement by combining cetuximab with an immunomodulatory therapy . programmed death - 1 ( pd - 1 ) is an inhibitory receptor expressed on t cells after activation . it has been shown to down - regulate t - cell activity upon binding its ligand pd - l1 on apcs . many tumors constitutively express pd - l1 and its &# 39 ; over expression has been associated with impaired tumor immunity , more aggressive disease and decreased survival ( see thompson 2004 ). till date pd - l1 expression has been demonstrated to correlate with poor prognosis in patients with renal cell carcinoma ( rcc ), ovarian cancer and melanoma . immunohistochemical analysis of freshly isolated tumor samples from patients with ovarian , lung , and breast cancers , renal cell carcinoma , squamous cell carcinoma of the head and neck , esophageal carcinoma , glioblastoma , thymoma , colon carcinoma , pancreatic and melanoma found that the vast majority express b7 - h1 ( see flies 2011 ; nomi 2007 ). several pre - clinical studies have demonstrated increased tumor rejection by blocking pd1 - pdl1 interaction . recently , anti - pd1 and pd - l1 based therapies have demonstrated considerable activity in melanoma and some other solid tumors confirming their application as one of the most promising anti - cancer therapies . cetuximab based therapy may be improved upon by combining it with immunomodulation to remove immunosuppressive environment or delay the development of resistance . moreover , patients who develop resistance to cetuximab due to mutations in the downstream pathways may still benefit from anti - egfr1 - pd - 1 since the fusion protein of the present invention binds to the egfr receptor and negates the pd - l1 expressed by the tumors , allowing t cells to mount an anti - tumor response . accordingly , the fusion proteins of the present invention can bind to both egfr and pd - l1 on the surface of the tumor cells . the anti - egfr1 - pd1 fusion protein constructs of the present invention may be used in colorectal cancer , squamous head and neck cancer , non - small cell lung cancer , gastric cancer and pancreatic cancer . the antibody fusion molecules of the present invention have duel therapeutic properties . on one hand the molecule retains the complete activity of the anti - egfr1 ( cetuximab ) and in parallel , it has the pd - l1 receptor binding activity in the tumor environment . the new molecules of the anti - egfr1 - pd1 fusion proteins that were developed for the cancer therapies herein are devoid of the amino acid lysine ‘ k ’ from the c - terminus of heavy chain for the reasons described above . the main objective of the fusion protein design is to keep the anti - egfr1 molecule intact along with its function unaffected and allows fusion of the pd1 molecule to the various location on the anti - egfr1 antibody . that being , fusion to the hc c - terminus , lc c - terminus , hc n - terminus , and or lc n - terminus and double fusions on both the chains as shown in fig6 . the codon - optimized nucleotide sequences of the anti - egfr1 - pd1 individual domains were optimized for expression in cho cells . such optimized sequences ( seq id nos : 1 , 2 , 3 , and 4 ) were assemble in a mammalian expression vector with help of primers described in table 2 : using the shown above cdna primers set , constructs were assembled as shown in fig1 . high levels of tgfβ are produced by many types of tumors , including melanomas and cancers of the breast , colon , esophagus , stomach , liver , lung , pancreas , and prostate , as well as hematologic malignancies ( see teicher 2001 ; dong 2006 ). tgfβ is known to be immunosuppressive for t cells and nk cells through blocking of il - 2 and other mechanisms , including generation of t - regs . several lines of evidence suggest that negating tgfβ activity may enhance anti - tumor effects of t cells ( wrzesinski 2007 ). moreover , tgfβ can foster tumor growth through epithelial to mesenchymal transition and promoting angiogenesis . tgfβ expression is also associated with poor prognosis in patients and earlier recurrence . however , considering the pleotropic effects of tgfβ in controlling the immune response , it has been shown that generalized blocking of tgfβ activity may result in widespread auto - inflammatory activity . hence , localized depletion of tgfβ in the tumor vicinity may be an alternative way to modulate immunosuppressive environment . anti - egfr1 - tgfβrii fusion protein of the present invention binds to egfr on the tumor cells and ties up the tgfβ around the tumor to enhance immune response against tumor cells . the objective is to design the antibody fusion molecules which have duel therapeutic properties . on one hand the molecule should retain the complete activity of the anti - egfr1 ( cetuximab ) and in parallel ; it should have the tgfβ binding activity in the tumor environment . the amino acid sequence of the anti - egfr1 igg molecule was retained excepting that the lysine was not expressed at the c - terminus of the heavy chain . both single and double fusion and expression levels are shown in table 3 , wherein tgfβrii was fused with anti - egfr1 . the codon - optimized nucleotide sequences of the anti - egfr1 - tgfβrii individual domains were optimized for expression in cho cells . such sequences ( seq id nos : 1 , 2 , 4 , and 7 ) were assemble in a mammalian expression vector . the expression constructs are set forth in fig2 . transfection of the above vectors combination to obatin the desired cell line : the expression constructs developed above were transfected in the following combination , as set forth in table 4 , into cho cells to produce the following fusion proteins using the constructs as defined in fig2 . the procedure describes in detail the small scale purification process of igg using c10 / 10 or xk26 column and using mab select xtra affinity resin . the samples generated by this protocol can be used for various analysis the culture supernatant secreted from recombinant cell line producing monoclonal antibodies or fusion monoclonal antibodies under sterile conditions were tested for titer and endotoxins ; the affinity chromatography using mab select xtra protein a resin was washed and equilibrated with binding buffer ; the ph of the supernatant was adjusted using 0 . 5m phosphate to the same ph has the column ; the supernatant was allowed to bind to the column / pass through the column at the flow rate of 0 . 5 ml / minute to achieve the maximum binding ; all the fusion mabs binds through the fc region and rest of the impurities passed pass through as flow through ; the column was washed with equilibration buffer ; the bound fusion mabs were eluted using 0 . 1 m glycine ph 3 . 0 ; the eluted proteins were adjusted back to neutral ph or the stable formulation ph ; the purified proteins were stored at − 20 ° c . or at 2 - 8 ° c . depending on the stability . the transfected supernatants obtained were purified using proteina affinity column . later these were analyzed on reducing and non - reducing sds - page to find out the integrity of the molecule , as shown in fig2 , where in the proteina purified samples were analyzed on 12 % reducing sds - page . as expected all the fusion partners are giving the expected pattern on sds - page . the lc fusion and hc are running closely but the bands are separated . this higher mobility may be due to the 8 n - glycosylation sites ( tgbrii 3 * 2 = 6 + 2 on lc ) fig2 shows the results of the proteina purified samples that were analyzed on 6 % non - reducing sds - page and although the amino acid composition is same , there is a difference in mobility . it may be due to the variable levels of glycosylation pattern based on the tgfβrii position and access in the molecule . immunohistochemical analysis of freshly isolated tumor samples from patients with ovarian , lung , and breast cancers , renal cell carcinoma , squamous cell carcinoma of the head and neck , esophageal carcinoma , glioblastoma , thymoma , colon carcinoma , pancreatic and melanoma found that the vast majority express b7 - h1 ( see flies 2011 ; nomi 2007 ). several pre - clinical studies have demonstrated increased tumor rejection by blocking pd1 - pdl1 interaction . recently , anti - pd1 and pd - l1 based therapies have demonstrated considerable activity in melanoma and some other solid tumors confirming their application as one of the most promising anti - cancer therapies . although , anti - ctla4 may allow co - stimulation of t cells , they may still be inhibited by pd - l1 - pd - 1 interaction . this may be one of the reasons for only a minority of patients having response to anti - ctla4 antibody . fusion antibody of both anti - ctla4 and pd1 are more efficacious than either agent alone since anti - ctla4 allows t cell co - stimulation whereas pd1 binds to pd - l1 on tumor cells to negate the immunosuppression of t cells in tumor microenvironment . this may even be safer than the anti - ctla4 because the lone use of anti - ctla4 has led to immune breakthrough adverse events . the objective is to design the antibody fusion molecules which have duel therapeutic properties . on one hand the molecule should retain the complete activity of the anti - ctla4 ( ipilimumab ) and in parallel ; it should have the pd l1 receptor binding activity in the tumor environment . the complete amino acid sequence of the anti - ctla4 igg molecule was used except the removal of the lysine at the c - terminus of the heavy chain . a 15 amino acid linker was positioned between the pd1 and anti - ctla4 . the following combinations of constructs , as setforth in table 5 , were designed as shown in fig2 . the details of the above fusion protein constructs are given below . the complete nucleotide sequence of the anti - ctla4 - pd1 individual domains were codon optimized for expression in cho cells ( seq id nos : 3 , 4 , 5 and 6 ). the cdnas were synthesized . the constructs were assembled in mammalian expression vectors . the expression of anti - ctla4 - pd1 fusion proteins using the constructs as set forth in fig3 . the expression constructs developed and shown in fig3 were transfected in the following combination into cho cells ( table 6 ) to produce the following fusion proteins . the titer obtained for each constructs are mentioned in the last column . the procedure describes the use of small scale purification process of igg using c10 / 10 or xk26 column and using mab select xtra affinity resin . the samples generated by this protocol can be used for various analysis . the culture supernatant secreted from recombinant cell line producing monoclonal antibodies or fusion monoclonal antibodies under sterile conditions were tested for titer and endotoxins ; the affinity chromatography using mab select xtra proteina resin was washed and equilibrated with binding buffer ; the ph of the supernatant was adjusted using 0 . 5m phosphate to the same ph has the column ; the supernatant was allowed to bind to the column / pass through the column at the flow rate of 0 . 5 ml / minute to achieve the maximum binding ; all the fusion mabs binds through the fc region and rest of the impurities passed through as flow through ; the column was washed with equilibration buffer ; the bound fusion mabs were eluted using 0 . 1 m glycine ph 3 . 0 ; the eluted proteins were adjusted back to neutral ph or the stable formulation ph ; and the purified proteins are stored at − 20 ° c . or at 2 - 8 ° c . depending on the stability . the fusion mab was tested for its ability to bind to its targets in three different elisas : 1 ) egfr1 target - binding elisa , 2 ) tgfβ - target binding elisa and 3 ) bifunctional elisa . for the target binding elisas , the targets ( rhegfr - fc chimera or tgfβ ) were coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . binding of the fusion mab was detected by the addition of a biotinylated anti - human igg f ( ab ) 2 secondary antibody , followed by a 1 hr incubation with peroxidase - conjugated streptavidin at room temperature . tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . for the bifunctional elisa , rhegfr - fc chimera was coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . after washing , tgfβ was added and the plate was incubated at room temperature for 1 hr . the plate was washed and anti - tgfβ - biotin was added and the plate incubated at room temperature for 1 hr . the plate was washed and streptavidin - hrp was added and the plate incubated at room temperature for 1 hr . after washing , tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . the binding of the anti - egfr1 hc - tgfβrii + anti - egfr1 lc fusion mab to both the targets egfr1 ( fig3 ) and tgfβ ( fig3 ) was comparable with anti - egfr1 - tgfβrii . the anti - egfr1 hc - tgfβrii + anti - egfr1 lc fusion mab was also tested in a bifunctional elisa to determine whether the anti - egfr1 and tgfβrii domains of the mab can bind to their respective targets without interfering with each other . as seen in fig3 , the anti - egfr1 hc - tgfβrii + anti - egfr1 lc fusion mab binds to both its targets , suggesting that there is no interference in binding to either target dues to the construction of the fusion mab . a - 431 cells were grown in flasks until they reached 70 - 80 % confluency . the cells were trypsinized and harvested . the cells were stained with different dilutions of the anti - egfr1 hc - tgfβrii + anti - egfr1 lc fusion mab or control ig at 2 - 8 ° c . for 30 minutes . the cells were washed and incubated with anti - human igg - fitc conjugate at 2 - 8 ° c . for 30 minutes . after washing , the cells were analyzed on a flow cytometer . live cells were gated based on their fsc vs ssc profiles . the total mfi for the gated population were recorded . the anti - egfr1 hc - tgfβrii + anti - egfr1 lc fusion binds to egfr - expressing a - 431 cells in a dose dependent manner ( fig3 ). the binding of anti - egfr1 hc - tgfβrii + anti - egfr1 lc is comparable to the binding of anti - egfr1 - tgfβrii . a - 431 cells were grown in flasks until they reached 70 - 80 % confluency . the cells were trypsinized , harvested and plated into 96 - well plates . the cells were labeled with different dilutions of the anti - egfr1 hc - tgfβrii + anti - egfr1 lc fusion mab or control ig at 2 - 8 ° c . for 30 minutes . the labeled cells were co - incubated with freshly isolated human pbmc at 37 ° c ., 5 % co 2 for 24 hours . cytotoxicity was measured using the cyto - tox - glo cytotoxicity assay kits . the anti - egfr1 hc - tgfβrii + anti - egfr1 lc fusion mediates adcc of egfr - expressing a - 431 cells by human pbmc effector cells . the adcc is dose dependent ( fig3 ). these results suggest that the fc portion of the fusion mab is intact and functional . a - 431 cells were grown in flasks until they reached 70 - 80 % confluency . the cells were trypsinized , harvested and plated into 96 - well plates . different dilutions of the anti - egfr1 hc - tgfβrii + anti - egfr1 lc fusion mab or control ig were added to the cells . the plates were incubated at 37 ° c ., 5 % co 2 for three days . on the third day , cell proliferation was measured by the alamarblue method . anti - egfr antibodies such as cetuximab are known to inhibit the proliferation of egfr1 - expressing cells . as seen in fig3 , the anti - egfr portion of the anti - egfr1 hc - tgfβrii + anti - egfr1 lc fusion mab is intact and has anti - proliferative activity . the anti - egfr1 hc + anti - egfr1 lc - tgfβrii fusion mab was tested for its ability to bind to its targets in three different elisas : 1 ) egfr1 target - binding elisa , 2 ) tgfβ - target binding elisa and 3 ) bifunctional elisa . for the target binding elisas , the targets ( rhegfr - fc chimera or tgfβ ) were coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . binding of the fusion mab was detected by the addition of a biotinylated anti - human igg f ( ab ) 2 secondary antibody , followed by a 1 hr incubation with peroxidase - conjugated streptavidin at room temperature . tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . for the bifunctional elisa , rhegfr - fc chimera was coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . after washing , tgfβ was added and the plate is incubated at room temperature for 1 hr . the plate was washed and anti - tgfβ - biotin was added and the plate incubated at room temperature for 1 hr . the plate was washed and streptavidin - hrp was added and the plate incubated at room temperature for 1 hr . after washing , tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . the binding of anti - egfr1 hc + anti - egfr1 lc - tgfβrii fusion mab to both the targets egfr1 ( fig4 ) and tgfβ ( fig4 ) was comparable to anti - egfr1 - tgfβrii . the anti - egfr1 hc + anti - egfr1 lc - tgfβrii fusion mab was also tested in a bifunctional elisa to determine whether the anti - egfr1 and tgfβrii domains of the mab can bind to their respective targets without interfering with each other . as seen in fig4 , the anti - egfr1 hc + anti - egfr1 lc - tgfβrii fusion mab binds to both its targets , suggesting that there is no interference in binding to either target dues to the construction of the fusion mab . a - 431 cells were grown in flasks until they reached 70 - 80 % confluency . the cells were trypsinized , harvested and plated into 96 - well plates . different dilutions of the anti - egfr1 hc + anti - egfr1 lc - tgfβrii fusion mab or control ig were added to the cells . the plates were incubated at 37 ° c ., 5 % co 2 for three days . on the third day , cell proliferation was measured by the alamarblue method . anti - egfr antibodies such as cetuximab are known to inhibit the proliferation of egfr1 - expressing cells . as seen in fig4 , the anti - egfr portion of the anti - egfr1 hc + anti - egfr1 lc - tgfβrii fusion mab is intact and has anti - proliferative activity . the fusion mab was tested for its ability to bind to its targets in three different elisas : 1 ) egfr1 target - binding elisa , 2 ) tgfβ - target binding elisa and 3 ) bifunctional elisa . for the target binding elisas , the targets ( rhegfr - fc chimera or tgfβ ) were coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . binding of the fusion mab was detected by the addition of a biotinylated anti - human igg f ( ab ) 2 secondary antibody , followed by a 1 hr incubation with peroxidase - conjugated streptavidin at room temperature . tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . for the bifunctional elisa , rhegfr - fc chimera was coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . after washing , tgfβ was added and the plate was incubated at room temperature for 1 hr . the plate was washed and anti - tgfβ - biotin was added and the plate incubated at room temperature for 1 hr . the plate was washed and streptavidin - hrp was added and the plate incubated at room temperature for 1 hr . after washing , tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . the binding of the tgfβrii - anti - egfr1 hc + anti - egfr1 lc fusion mab to both the targets egfr1 ( fig4 ) and tgfβ ( fig4 ) was comparable to anti - egfr1 - tgfβrii . the tgfβrii - anti - egfr1 hc + anti - egfr1 lc fusion mab was also tested in a bifunctional elisa to determine whether the anti - egfr1 and tgfβrii domains of the mab can bind to their respective targets without interfering with each other . as seen in fig4 , the binding of tgfβrii - anti - egfr1 hc + anti - egfr1 lc fusion mab is reduced as compared to anti - egfr1 - tgfβrii , suggesting that there is some interference in binding to either target due to the construction of the fusion mab . a - 431 cells were grown in flasks until they reached 70 - 80 % confluency . the cells were trypsinized and harvested . the cells were stained with different dilutions of the tgfβrii - anti - egfr1 hc + anti - egfr1 lc fusion mab or control ig at 2 - 8 ° c . for 30 minutes . the cells were washed and incubated with anti - human igg - fitc conjugate at 2 - 8 ° c . for 30 minutes . after washing , the cells were analyzed on a flow cytometer . live cells were gated based on their fsc vs ssc profiles . the total mfi for the gated population were recorded . the tgfβrii - anti - egfr1 hc + anti - egfr1 lc fusion binds to egfr - expressing a - 431 cells in a dose dependent manner . the binding of tgfβrii - anti - egfr1 hc + anti - egfr1 lc is comparable to the binding of anti - egfr1 - tgfβrii . a - 431 cells were grown in flasks until they reached 70 - 80 % confluency . the cells were trypsinized , harvested and plated into 96 - well plates . different dilutions of the tgfβrii - anti - egfr1 hc + anti - egfr1 lc fusion mab or control ig were added to the cells . the plates were incubated at 37 ° c ., 5 % co 2 for three days . on the third day , cell proliferation was measured by the alamarblue method . anti - egfr antibodies such as cetuximab are known to inhibit the proliferation of egfr1 - expressing cells . as seen in fig4 , the anti - egfr portion of the tgfβrii - anti - egfr1 hc + anti - egfr1 lc fusion mab is intact and has anti - proliferative activity . the fusion mab was tested for its ability to bind to its targets in three different elisas : 1 ) egfr1 target - binding elisa , 2 ) tgfβ - target binding elisa and 3 ) bifunctional elisa . for the target binding elisas , the targets ( rhegfr - fc chimera or tgfβ ) were coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . binding of the fusion mab was detected by the addition of a biotinylated anti - human igg f ( ab ) 2 secondary antibody , followed by a 1 hr incubation with peroxidase - conjugated streptavidin at room temperature . tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . for the bifunctional elisa , rhegfr - fc chimera was coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . after washing , tgfβ was added and the plate was incubated at room temperature for 1 hr . the plate was washed and anti - tgfβ - biotin was added and the plate incubated at room temperature for 1 hr . the plate was washed and streptavidin - hrp was added and the plate incubated at room temperature for 1 hr . after washing , tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . the binding of the anti - egfr1 hc + tgfβrii - anti - egfr1 lc fusion mab to both the targets egfr1 ( fig4 ) and tgfβ ( fig4 ) was comparable with anti - egfr1 - tgfβrii . the anti - egfr1 hc + tgfβrii - anti - egfr1 lc fusion mab was also tested in a bifunctional elisa to determine whether the anti - egfr1 and tgfβrii domains of the mab can bind to their respective targets without interfering with each other . as seen in fig5 , the binding of anti - egfr1 hc + tgfβrii - anti - egfr1 lc fusion mab is reduced as compared to anti - egfr1 - tgfβrii , suggesting that there is some interference in binding to either target due to the construction of the fusion mab . a - 431 cells were grown in flasks until they reached 70 - 80 % confluency . the cells were trypsinized and harvested . the cells were stained with different dilutions of anti - egfr1 hc + tgfβrii - anti - egfr1 lc fusion mab or control ig at 2 - 8 ° c . for 30 minutes . the cells were washed and incubated with anti - human igg - fitc conjugate at 2 - 8 ° c . for 30 minutes . after washing , the cells were analyzed on a flow cytometer . live cells were gated based on their fsc vs ssc profiles . the total mfi for the gated population were recorded . the anti - egfr1 hc + tgfβrii - anti - egfr1 lc fusion binds to egfr - expressing a - 431 cells in a dose dependent manner ( fig5 ). the binding of anti - egfr1 hc + tgfβrii - anti - egfr1 lc is reduced compared to the binding of anti - egfr1 - tgfβrii . the fusion mab was tested for its ability to bind to its targets in three different elisas : 1 ) egfr1 target - binding elisa , 2 ) tgfβ - target binding elisa and 3 ) bifunctional elisa . for the target binding elisas , the targets ( rhegfr - fc chimera or tgfβ ) were coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . binding of the fusion mab was detected by the addition of a biotinylated anti - human igg f ( ab ) 2 secondary antibody , followed by a 1 hr incubation with peroxidase - conjugated streptavidin at room temperature . tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . for the bifunctional elisa , rhegfr - fc chimera was coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . after washing , tgfβ was added and the plate was incubated at room temperature for 1 hr . the plate was washed and anti - tgfβ - biotin was added and the plate incubated at room temperature for 1 hr . the plate was washed and streptavidin - hrp was added and the plate incubated at room temperature for 1 hr . after washing , tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . the binding of anti - egfr1 hc - tgfβrii + tgfβrii - anti - egfr1 lc fusion mab to the target egfr1 was slightly reduced ( fig5 ) but was higher for tgfβ ( fig5 ) when compared to the binding of anti - egfr1 - tgfβrii . the anti - egfr1 - tgfβrii + tgfβrii - anti - egfr1 lc fusion mab was also tested in a bifunctional elisa to determine whether the anti - egfr1 and tgfβrii domains of the mab can bind to their respective targets without interfering with each other . as seen in fig5 , the binding of anti - egfr1 hc - tgfβrii + tgfβrii - anti - egfr1 lc fusion mab is comparable to anti - egfr1 - tgfβrii , suggesting that there is no interference in binding to either target due to the construction of the fusion mab . the fusion mab was tested for its ability to bind to its targets in three different elisas : 1 ) egfr1 target - binding elisa , 2 ) tgfβ - target binding elisa and 3 ) bifunctional elisa . for the target binding elisas , the targets ( rhegfr - fc chimera or tgfβ ) were coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . binding of the fusion mab was detected by the addition of a biotinylated anti - human igg f ( ab ) 2 secondary antibody , followed by a 1 hr incubation with peroxidase - conjugated streptavidin at room temperature . tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . for the bifunctional elisa , rhegfr - fc chimera was coated onto nunc maxisorb plates overnight at 4 ° c . the plates were washed and then blocked with superblock at room temperature for 2 hr . different dilutions of the fusion mab or the negative control antibody was added to the plate . the plate was incubated at room temperature for 1 hr . after washing , tgfβ was added and the plate was incubated at room temperature for 1 hr . the plate was washed and anti - tgfβ - biotin was added and the plate incubated at room temperature for 1 hr . the plate was washed and streptavidin - hrp was added and the plate incubated at room temperature for 1 hr . after washing , tmb substrate solution was added and the reaction stopped with 1n h 2 so 4 . the absorbance was measured at 450 nm on a biotek synergy h4 hybrid reader . the binding of tgfβrii - anti - egfr1 hc + tgfβrii - anti - egfr1 lc fusion mab to the target egfr1 was slightly reduced ( fig5 ) but was higher for tgfβ ( fig5 ) when compared to the binding of anti - egfr1 - tgfβrii . the tgfβrii - anti - egfr1 hc + tgfβrii - anti - egfr1 lc fusion mab was also tested in a bifunctional elisa to determine whether the anti - egfr1 and tgfβrii domains of the mab can bind to their respective targets without interfering with each other . as seen in fig5 , the binding of tgfβrii - anti - egfr1 hc + tgfβrii - anti - egfr1 lc fusion mab is reduced compared to anti - egfr1 - tgfβrii , suggesting that there is interference in binding to either target due to the construction of the fusion mab . the contents of all references cited herein are incorporated by reference herein for all purposes . altschul , et al ., 1990 , basic local alignment search tool , j . mol . biol . 15 : 403 - 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544 . lee , et al ., 1994 , delivery of liposomes into cultured kb cells via folate receptor - mediated endocytosis . j biol . chem ., 269 : 3 198 . lee , et al ., 1995 , folate - mediated tumor cell targeting of liposome - entrapped doxorubicin in vitro . biochem . biophys . actu , 1233 : 134 - 144 . mackett , et al ., 1982 , vaccinia virus : a selectable eukaryotic cloning and expression vector , proc . natl . acad . sci . usa , 79 : 7415 - 7419 . mackett , et al ., 1984 , general method for production and selection of infectious vaccinia virus , j . virol ., 49 : 857 - 864 . nomi , et al ., 2007 , clinical significance and therapeutic potential of the programmed death - 1 ligand / programmed death - 1 pathway in human pancreatic cancer . clin cancer res ., 13 : 2151 - 7 . okazaki , et al ., 2007 , pd - 1 and pd - 1 ligands : from discovery to clinical application . international immunology , vol . 19 , no . 7 , pp . 813 - 824 . panicali , et al ., 1982 , construction of poxviruses as cloning vectors : insertion of the thymidine , proc . natl . acad . sci . usa , 79 : 4927 - 4931 . pardoll , d m ., 2012 , the blockage of immune checkpoints in cancer immunotherapy . nat . rev . cancer , 12 ( 4 ): 252 - 64 . strathem , et al ., 1982 , the molecular biology of the yeast saccharomyces , cold spring harbor press , vols . i and ii . thompson r h , et al ., 2004 , costimulatory b7 - h1 in renal cell carcinoma patients : indicator of tumor aggressiveness and potential therapeutic target . proc natl acad sci usa ., 101 ( 49 ): 17174 - 9 . teicher b a ., 2001 , malignant cells , directors of the malignant process : role of transforming growth factor beta . cancer metastasis rev ; 20 : 133 - 143 . wrzesinski , et al ., 2007 , transforming growth factor - i3 and the immune response : implications for anticancer therapy , clin cancer res , 13 ; 5262 .