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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
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  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof

Definitions

• This application relates to a medicament and its use in methods of treatment, diagnosis and imaging.
• it relates to the treatment of cancer with a cytotoxic agent linked to a targeting agent.
• Auristatins are highly potent antimitotic agents related in structure to the marine natural product, dolastatin 10.
• dolastatin 10 and MMAE The naturally occurring antimitotic pentapeptide dolastatin 10 and its synthetic analogue monomethyl auristatin E (MMAE) possess subnanomolar cytotoxicity against many human cancer cell lines and are over a hundred- to a thousand-times more potent than many pharmaceuticals, including taxol and doxorubicin, respectively, which are currently used in clinic.
• Dolastatins and auristatins inhibit tubulin polymerization in dividing cells, whereas taxol promotes it and stabilizes the microtubules. In the end, both dolastatins or auristatins and taxol inhibit cell proliferation. Additionally, auristatins function as the vascular disrupting agents and damage the established tumor vessels, thereby likely causing a more pronounced effect than taxol in vivo. Nonetheless, the therapeutic efficacies of the auristatins and dolastatins are poor as they also cause nonselective toxicity to normal cells, giving rise to significant systemic effects (Bajjuri, KM et al. ChemMedChem. (2011) 6(1): 54-59).
• ADCs Antibody-drug conjugates consist of potent anticancer drugs covalently linked to monoclonal antibodies (mAb) that bind to tumor-associated antigens, and were introduced as a more selective way to direct highly toxic drugs to cells bearing such antigens.
• ADCs offer the opportunity to widen the therapeutic window of cancer therapies by increasing the amount of drug that gets to the tumor and sparing normal tissues from systemic drug exposure, only a small percentage of administered mAb will localize within tumors (Wu AM and Senter PD (2005) Arming antibodies: prospects and challenges for
• folic acid binds to the folate receptor at the cell surface with very high affinity, and is internalized by receptor-mediated endocytosis. Furthermore, the folate receptor is overexpressed by carcinomas of the kidney, brain, lung, esophagus, and breast but has very restricted expression on most normal tissues. (Wu, W. et al. Int J Nanomedicine. 2012; 7: 3487-3502). As such, conjugates bearing folic acid may provide a mechanism to target toxic chemotherapeutics that do not have the deficiencies of ADCs.
• the present invention addresses this need by the creation of extended recombinant polypeptide (XTEN) conjugates that can be purified to homogeneity and that are amenable to chemical conjugation with folic acid and toxins such as MMAE using a wide diversity of conjugation methods.
• XTEN extended recombinant polypeptide
• the use of the purified XTEN reagents results in high -yield monodispersed products that have good solubility, that remain stable, and result in enhanced terminal half- life compared to unconjugated products, while the use of folic acid targeting moieties permits more specific delivery of anti -neoplastic agents such as MMAE and related analogs, resulting in increased efficacy and reduced non-specific toxicity.
• the present invention relates, in part, to novel anti-cancer medicaments that have enhanced properties of being specifically targeted to cancer cells by being linked to folate receptor ligands, that have enhanced pharmacokinetic properties that require less frequent dosing, and that are designed to release toxic payload drugs when internalized by the targeted cells.
• the invention provides conjugates comprising XTEN engineered for covalent linking to the one or more molecules of a first and a second payload using orthogonal pendant cross-linkers with reactive groups, resulting in XTEN-folate conjugates that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more molecules of the two types of payloads. It is an object of the present invention to provide such engineered XTEN polypeptides for use in creating conjugates with the payloads as compositions with enhanced pharmaceutical properties, including enhanced pharmacokinetic properties.
• the invention provides XTEN that are substantially homogeneous in length and sequence that are useful for preparing the conjugates comprising the XTEN linked to one or more payloads such that the resulting XTEN-folate conjugates have a high degree of purity.
• Such conjugates are useful in preparing monodispersed pharmaceutical compositions that are used in the treatment of cancer subjects for which the one or more payloads have utility in the treatment of such cancer.
• the invention provides conjugates comprising a first and a second extended recombinant polypeptide (XTEN), wherein each of said first and second XTEN is independently selected from the group consisting of the XTEN sequences set forth in Table 1 and wherein: a) the first and the second XTEN are conjugated to each other by a reaction product of an alkyne and an azide reactant, one of said alkyne and azide reactant being located at the N- terminus of the first XTEN and the other of said alkyne and azide reactant being linked to the N- terminus of the second XTEN, wherein the alkyne and azide reactants are selected from, the group consisting of the reactants set forth in Table 9; b)each of the first and the second XTEN comprises one or more cysteine residues and further comprises a first cross-linker conjugated to each cysteine residue of the first XTEN and a second cross-linker conjugated to each cysteine residue of
• the conjugate has the configuration of formula I
• Pi is the first payload
• P 2 is the second payload
• CLi is the first cross-linker
• x is an integer from 1 to about 100, or 1 to about 50, or 1 to about 40, orl to about 20, or 1 to about 10, or 1 to about 5, or is 3, or is 2, or is 1
• CL 2 is the cross-linker
• y is an integer from 1 to about 100, or 1 to about 50, or 1 to about 40, orl to about 20, or 1 to about 10, or 1 to about 5, or is 3, or is 2, or is 1 with the proviso that x + y is > 2
• 2xCL is the reaction product of the alkyne and azide reactants selected from the group consisting of the reactants set forth in Table 9
• XTENi is the first XTEN
• XTEN 2 is the second XTEN.
• the first XTEN and the second XTEN each independently have at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity or is identical to a sequence selected from the group of sequences set forth in Table 1, when optimally aligned.
• the first XTEN of the conjugate has at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity or is identical, when optimally aligned, to a sequence selected from the group of sequences consisting of Seg 174, Seg 175, Seg 176, Seg 177, Seg 186, Seg 187, Seg 188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193, Seg 194, Seg 195, Seg 196, Seg 197, Seg 198, and Seg 199 set forth in Table 1 and the second XTEN has at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%
• the first XTEN and the second XTEN of the conjugate are the identical are each has at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity or is identical, when optimally aligned, to a sequence selected from the group consisting of Seg 174, Seg 175, Seg 176, Seg 177, Seg 186, Seg 187, Seg 188, Seg 189, Seg 190, Seg 191, Seg 192, Seg 193, Seg 194, Seg 195, Seg 196, Seg 197, Seg 198, and Seg 199 set forth in Table 1.
• the first cross-linker and the second cross linker are independently selected from the group consisting of N-maleimide, N-(5-aminopentyl)maleimide, 6- maleimidocaproic acid, iodoacetyl, pyridyl disulfide and vinyl sulfone, 3-propargyloxypropanoic acid, (oxyethyl),,-acetylene where n is 1-10, dibenzylcyclooctyne (DBCO), cyclooctyne (COT), 3- azide-propionic acid, 6-azide-hexanoic acid, and (oxyethyl) n -azide where n is 1-10.
• DBCO dibenzylcyclooctyne
• COT cyclooctyne
• the first cross-linker is N-(5-aminopentyl)maleimide and the second cross linker is 6-maleimidocaproic acid.
• the alkyne reactant is selected from the group consisting of 3-propargyloxypropanoic acid NHS ester, acetylene-(oxyethyl) n -NHS ester where n is 1- 10, dibenzylcyclooctyne (DBCO)-NHS ester, DBCO-(oxyethyl) n -NHS ester where n is 1 -10, cyclooctyne (COT)-NHS ester, COT-(oxyethyl) n - NHS ester where n is 1 -10, COT-(oxyethyl) n -pentafluorophenyl (PFP) ester where n is 1-10, BCOT-NHS ester, BCOT-(oxye
• the alkyne reactant is 6-(l 1,12- didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoic acid N-hydroxysulfosuccinimide ester (DBCO-sulfo-NHS ester) and the azide reactant l-azido-3,6,9,12-tetraoxapentadecan-15-oic acid N-hydroxysuccinimide ester (azide-(oxyethyl)4-NHS ester).
• the XTEN-folate conjugate at least 90%, 91%, 92%, 93%, 94%, or 95% of individual first XTEN molecules in said conjugate have an identical sequence length and at least 90%, 91%, 92%, 93%, 94%, or 95% of individual second XTEN molecules in said conjugate have an identical sequence length.
• the first XTEN and the second XTEN are identical and the sequence is Seg 176 set forth in Table i , and x is 3 and y is 3.
• the first XTEN and the second XTEN are identical, the XTEN sequences are Seg 177 set forth in Table 1, and x is 9 and y is 9.
• the second payload is valine- citrulline- »-aminobenzyloxycarbonyl-monomethylauristatin E.
• the second payload is valine-citrulline-p-aminobenzyloxycarbonyl- monomethylauristatin F.
• the invention provides an XTEN-folate conjugate having the structure set forth in FIG. 49.
• the invention relates, in part, to XTEN-folate conjugates that are targeted and have enhance toxicity to cells bearing folate receptors.
• the invention provides XTEN-payload conjugate wherein treatment of a folate receptor-bearing cell with a conjugate results in greater cytotoxicity compared to treatment with a comparable conjugate lacking folate.
• the cytotoxicity is measured in an in vitro assay using a folate receptor-bearing cell selected from the group consisting of the cells set forth in Table 24 and the greater cytotoxicity is a reduction in IC50 by at least 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1000 nM.
• the cytotoxicity is measured in an in vitro assay using a KM folate receptor-bearing cell and the greater cytotoxicity is a reduction in IC50 by at least 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM or 1000 nM.
• the invention relates, in part, to XTEN-folate conjugates that, when administered to a subject, exhibit a prolonged terminal half-life compared to the payloads not conjugated to XTEN.
• administration of a single dose of the XTEN-folate conjugate described herein to a subject results in an increase in the terminal half- life of at least 2- fold, 4-fold, 8-fold, or 10-fold greater compared to the second payload administered singly to the subject using a comparable molar dose.
• administration of a single dose of the XTEN-folate conjugate described herein to a subject results in a terminal half-life of at least 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, or 7 days, or 10 days, or 14 days.
• the invention relates, in part, to XTEN-folate conjugates in which the XTEN component is substantially homogeneous in length.
• the first XTEN and the second XTEN utilized in the conjugates have the same XTEN sequence and are obtained from a substantially homogenous population of polypeptides prepared by the process comprising: culturing a host cell that comprises a vector encoding the polypeptide in a fermentation reaction under conditions effective to express the polypeptide as a component of a crude expression product of the host cell, wherein the encoded polypeptide comprises the XTEN, a first cleavage sequence, a first affinity tag with binding affinity to a first chromatography substrate, a second cleavage sequence, and a second affinity tag wherein the first and the second cleavage sequences are capable of being cleaved by the same protease and wherein the second affinity tag has binding affinity to a second, different chromatography substrate than the first affinity tag; adsorb
• the first chromatography substrate is selected from the group consisting of a HIC substrate, a cation exchange substrate, an anion exchange substrate, and an IMAC substrate.
• the first and the second affinity tags are independently selected from the group consisting of the affinity tags of Table 5.
• the first chromatography substrate is a cation exchange substrate and the first affinity tag comprises a sequence selected from RPRPRPRPRPR and RPRPRPRPRPRPRPRPRPRPRPRPR.
• the second chromatography substrate is an IMA C substrate and the second affinity tag comprises the sequence selected from HHHHHH and HHHHHH.
• the process further comprises treating the polypeptide with a protease under conditions effective to cleave the cleavage sequences, thereby releasing the XTEN from the poiypeptide; adsorbing the XTEN onto an anion chromatography substrate under conditions effective to capture the XTEN; eluting the XTEN; and recovering the XTEN wherein at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95% of the individual XTEN molecules have an identical sequence length.
• the anion chromatography substrate can be selected from the group consisting of macrocap Q, capto Q, superQ-650M, and poros D.
• the cleavage sequences are capable of being cleaved by trypsin wherein the cleavage sequence is selected from the group consisting of the sequences set forth in Table 8, and the protease is trypsin.
• the process further comprises treating the polypeptide with a protease such as trypsin under conditions effective to cleave the cleavage sequence(s), thereby releasing the XTEN from the polypeptide; adsorbing the protease onto a chromatography substrate under conditions effective to capture the protease but not the XTEN; and recovering the XTEN in the eluate wherein at least 90%, 91%, 92%, 93%, 94%, or 95% of the XTEN have an identical sequence length.
• the chromatography substrate is one or more of a HIC substrate, a cation exchange substrate, and an IMAC substrate.
• the invention provides pharmaceutical compositions comprising the XTEN-folate conjugate composition of any one of the preceding embodiments and a
• the pharmaceutical composition has utility for treatment of a cancer.
• the pharmaceutical composition is utilized for use in a pharmaceutical regimen for treatment of a subject with a cancer, said regimen comprising the pharmaceutical composition.
• the pharmaceutical regimen further comprises the step of determining the amount of pharmaceutical composition needed to achieve an improvement in a parameter associated with the cancer.
• the pharmaceutical regimen further comprises administering the pharmaceutical composition in an effective amount in two or more successive doses to the subject at an effective amount, wherein the administration results in at least a 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% greater improvement of at least one, two, or three parameters associated with the cancer compared to an untreated subject, wherein the parameters are selected from the group consisting of response rate as defined by the Response Evaluation Criteria in Solid Tumors (RECIST), time-to-progression of the cancer (relapse), discovery of local recurrence, discovery of regional metastasis, discovery of distant metastasis, onset of symptoms, hospitalization, increase in pain medication requirement, requirement of salvage chemotherapy, requirement of salvage surgery, requirement of salvage radiotherapy, time-to-treatment failure, and increased time of survival.
• RECIST Response Evaluation Criteria in Solid Tumors
• the invention provides a XTEN-folate conjugate for use in the preparation of a medicament for treatment of mammalian cells or treatment of a cancer.
• the invention provides an article of manufacture comprising a XTEN-folate conjugate as described herein, a container, and a package insert or label indicating that the compound can be used to treat cancer.
• the invention provides a kit comprising a pharmaceutical composition of the XTEN-folate conjugate, a container, and a package insert or label indicating that the compound can be used to treat cancer.
• the invention relates, in part, to methods of treatment utilizing the XTEN-folate conjugate described herein.
• the method of the aspect of the invention can be used to kill cancer cells in vitro or in vivo.
• the invention provides a method of treating a cancer cell in vitro, comprising administering to a culture of a cancer cell a composition comprising an effective amount of an XTEN-folate conjugate.
• the invention provides a method of treating a cancer in a subject, comprising administering to the subject a pharmaceutical composition comprising an effective amount of an XTEN-folate conjugate.
• the pharmaceutical composition comprises the conjugate having the structure set forth in FIG. 49.
• the cancer is selected from the group consisting of non-small cell lung cancer, mesothelioma, platinum-resistant ovarian cancer, endometrial cancer, adenocarcinoma of the lung, and refractory advanced tumors.
• the administration results in at least a 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90% greater improvement of at least one, two, or three parameters associated with the cancer compared to an untreated subject wherein the parameters are selected from the group consisting of response rate as defined by the Response Evaluation Criteria in Solid Tumors (RECIST), time-to-progression of the cancer (relapse), discovery of local recurrence, discovery of regional metastasis, discovery of distant metastasis, onset of symptoms, hospitalization, increase in pain medication requirement, requirement of salvage chemotherapy, requirement of salvage surgery, requirement of salvage radiotherapy, time-to-treatment failure, and increased time of survival.
• RECIST Response Evaluation Criteria in Solid Tumors
• inventive XTEN-folate conjugates can exhibit one or more or any combination of the improved properties disclosed herein.
• FIG. 1 shows schematics of XTEN suitable for conjugation with payloads.
• FIG. 1A shows unmodified XTEN.
• FIG. IB shows a cysteine-engineered XTEN with an internal cysteine with a thiol side chain; below is an XTEN with an a reactive N-terminal amino group; below is an XTEN with an N-terminal cysteine with a thiol reactive group.
• FIG. 1C shows cysteine- engineered XTEN with multiple internal cysteines.
• ID shows two variations of a cysteine- engineered XTEN with an internal cysteine with a thiol side chains and a reactive N-terminal amino group and, at the bottom, a shows a cysteine- and lysine-engineered XTEN with internal cysteines and internal lysines.
• FIG. 2 shows a conjugation reaction utilizing NHS-esters and their water soluble analogs sulfo-NHS-esters) reacting with a primary amino group to yield a stable amide XTEN- payload product.
• FIG. 3 shows a conjugation reaction utilizing thiol groups and an N-maleimide.
• the maleimide group reacts specifically with sulfhydryl groups when the pH of the reaction mixture is between pH 6.5 and 7.5, forming a stable thioether linkage that is not reversible.
• FIG. 4 shows a conjugation reaction utilizing haloacetyls.
• the most commonly used haloacetyl reagents contain an iodoacetyl group that reacts with sulfhydryl groups at physiological pH.
• the reaction of the iodoacetyl group with a sulfhydryl proceeds by nucleophilic substitution of iodine with a thiol producing a stable thioether linkage in the XTEN-payload.
• FIG. 5 shows a conjugation reaction utilizing pyridyl disulfides.
• Pyridyl disulfides react with sulfhydryl groups over a broad pH range (the optimal pH is 4-5) to form disulfide bonds linking XTEN to payloads.
• FIG. 6 shows a conjugation reaction utilizing zero-length cross-linkers wherein the cross-linkers are used to directly conjugate carboxyl functional groups of one molecule (such as a payload) to the primary amine of another molecule (such as an XTEN).
• FIG. 7 shows a conjugation reaction utilizing the Huisgen 1,3-dipolar cycloaddition of alkynes to azides to form l,4-disubsituted-l,2,3-triazoles, as shown.
• FIG. 8 shows a conjugation reaction using thio-ene based click chemistry that may proceed by free radical reaction, termed thiol-ene reaction, or anionic reaction, termed thiol Michael addition.
• FIG. 9 shows a conjugation reaction utilizing click chemistry based on reactions between hydrazides and aldehydes, resulting in the illustrated hydrazone linkage in the XTEN- payload.
• FIG. 10 shows a conjugation reaction utilizing traceless Staudinger ligation, like Native Chemical Ligation (NCL), resulting in a native amide bond at the ligation site
• FIG. 11 shows a conjugation reaction utilizing enzymatic ligation.
• Transglutaminases are enzymes that catalyze the formation of an isopeptide bond between the ⁇ -carboxamide group of glutamine of a payload peptide or protein and the ⁇ -amino group of a lysine in a lysine- engineered XTEN (or an N-terminal amino group), thereby creating inter- or intramolecular crosslinks between the XTEN and payload.
• FIG. 12 shows various XTEN-cross-linker precursor segments that are used as reactants to link to payloads or to other XTEN reactants.
• FIG. 12A is intended to show that the IB represents the remaining reactive group of the precursors on the right.
• FIG. 12B shows similar reactive precursors with either multiple (left) or single (right) payload A molecules conjugated to the XTEN.
• FIG. 13 shows exemplary permutations of XTEN-cross-linker precursor segments with two reactive groups of cross-linkers or reactive groups of an incorporated amino acid that are used as reactants to link to payloads or to other XTEN reactants.
• the IB and 2B represent reactive groups that will, in other figures, react with a like-numbered reactive group; 1 with 1 and 2 with 2, etc.
• FIG. 14 illustrates the creation of various XTEN precursor segments.
• FIG. 14A shows the steps of making an XTEN polypeptide, followed by reaction of the N-terminus with the cross- linker with 2B-1A reactive groups, with the 1A reacting with the N-terminal IB (e.g., an alpha amino acid) to create the XTEN precursor 2 with the reactive group 2B.
• FIG. 14B shows the sequential addition of two cross-linkers with 2A reactive groups to 2B reactive groups of the XTEN, resulting in XTEN precursor 4, which is then reacted with a cross-linker at the N-terminus between a reactive IB and the 1A of a cross-linker, resulting in XTEN precursor 5, with reactive groups 4B and 3B.
• the XTEN-precursor 5 then could serve as a reactant with two different payloads or XTEN.
• FIG. 15 illustrates various configurations of bispecific conjugates with two payloads.
• FIG. 15A illustrates configurations with one molecule each of two payloads, while FIG. 15B illustrates various configurations with multiple copies of one or both payloads.
• FIG. 16 illustrates various examples of conjugates with high valency. Conjugations sites of payloads can grouped (FIG. 26A) or interspersed (FIG. 26B).
• FIG. 17 illustrates the preparation of bispecific conjugates from an XTEN precursor carrying both amino and thiol groups in which many chemistries can be used and the order of payload addition can vary.
• FIG. 17A shows the creation of a single XTEN precursor to which two different payloads are attached.
• FIG. 17B shows a segment approach starting from two XTEN precursor molecules. This approach allows one to conjugate both payloads to XTEN using the same type of linker chemistry.
• the figure shows thiol as the group to which payloads are conjugated, and then the N-terminus of each segment is modified with a cross-linker to enable head-to-head segment conjugation, resulting in a dimeric, bispecific conjugate final product.
• FIG. 18 is a schematic flowchart of representative steps in the assembly, production and the evaluation of a XTEN.
• FIG. 19 is a schematic flowchart of representative steps in the assembly of an XTEN polynucleotide construct encoding a fusion protein.
• Individual oligonucleotides 501 are annealed into sequence motifs 502 such as a 12 amino acid motif ("12-mer"), which is ligated to additional sequence motifs from a library to create a pool that encompasses the desired length of the XTEN 504, as well as ligated to a smaller concentration of an oligo containing Bbsl, and Kpnl restriction sites 503.
• the resulting pool of ligation products is gel-purified and the band with the desired length of XTEN is cut, resulting in an isolated XTEN gene with a stopper sequence 505.
• the XTEN gene is cloned into a staffer vector.
• the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is then performed with Bbsl/Hindlll to remove 507 and 508 and place the stop codon. The resulting product is then cloned into a Bsal/Hindlll digested vector, resulting in gene 500 encoding an XTEN.
• FIG. 20 is a schematic flowchart of representative steps in the assembly of a gene encoding XTEN, its expression, conjugation with a payload and recovery as an XTEN-payload, and its evaluation as a candidate product.
• FIG. 21 shows generalized XTEN with either N- or C-terminal tags or N- and C- terminal sequences optimized for purification using methods illustrated in FIGS. 22.
• FIG. 22 shows a generalized scheme for purification of XTEN with, in this illusrtrative embodiment, two tags in which a two-step purification method to capture first one tag and then the second can be utilized to remove truncated XTEN from fermentation, resulting in the highly purified target XTEN entity.
• FIG. 23 shows an SDS-PAGE analysis of XTEN constructs with experimental tags after expression in E.coli as described in Example 6. Soluble lysates were loaded on the 4-12% Bis-Tris polyacrylamide gel, with amounts loaded per lane equivalent to 36 ⁇ of cell culture suspension. The gel was stained with Coomassie Blue stain using standard methods.
• FIG. 24 shows an SDS-PAGE analysis of the RP11-XTEN-His8 construct expressed in E.coli, as described in Example 6. Heat-treated soluble lysates were loaded on the 4-12% Bis- Tris polyacrylamide gel with amounts equivalent to 1 or 2 ⁇ of cell culture suspension, respectively. The gel was stained with Coomassie Blue stain. The gel demonstrates that essentially all the expressed RP11-XTEN-His8 protein was found in the pelleted fraction.
• FIG. 25 shows an SDS-PAGE analysis of the MacroCap SP purification of RP11- XTEN-His8 polypeptide described in Example 1. Fractions were analyzed by 4-12% SDS-PAGE followed by Coomassie staining.
• FIG. 26 shows an SDS-PAGE analysis of the IMAC purification of the RP11-XTEN- His8 polypeptide described in Example 1. Fractions were analyzed by 4-12% SDS-PAGE followed by Coomassie staining.
• FIG. 27 shows an SDS-PAGE analysis of the trypsin digestion of RP11-XTEN-His8 protein purified by two chromatographic steps (SP + IMAC) described in Example 1. Preparations were analyzed by 4-12% SDS-PAGE followed by Coomassie staining (FIG. 27A) and silver staining (FIG. 27B).
• FIG. 28 shows the pharmacokinetic profile (plasma concentrations) in cynomolgus monkeys after single doses of different compositions of GFP linked to unstructured polypeptides of varying length, administered either subcutaneously or intravenously, as described in Example 35.
• the compositions were GFP-L288, GFP-L576, GFP-XTEN AF576, GFP-Y576 and XTEN AD836-GFP.
• Blood samples were analyzed at various times after injection and the concentration of GFP in plasma was measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal antibody for detection.
• FIG. 29 shows an SDS-PAGE gel of samples from a stability study of the fusion protein of XTEN AE864 fused to the N-terminus of GFP.
• the GFP-XTEN was incubated in cynomolgus plasma and rat kidney lysate for up to 7 days at 37°C, as described in Example 28.
• GFP-XTEN administered to cynomolgus monkeys was also assessed. Samples were withdrawn at 0, 1 and 7 days and analyzed by SDS PAGE followed by detection using Western analysis and detection with antibodies against GFP.
• FIG. 30 shows the near UV circular dichroism spectrum of Ex4-XTEN_AE864, performed as described in Example 29.
• FIG. 31 shows results of a size exclusion chromatography analysis of glucagon-XTEN construct samples measured against protein standards of known molecular weight, with the graph output as absorbance versus retention volume, as described in Example 31.
• the glucagon-XTEN constructs are 1) glucagon- Y288; 2) glucagonY-144; 3) glucagon- Y72; and 4) glucagon-Y36.
• FIG. 32 is a schematic of the logic flow chart of the algorithm SegScore (Example 33).
• the following legend applies: i, j - counters used in the control loops that run through the entire sequence; HitCount- this variable is a counter that keeps track of how many times a subsequence encounters an identical subsequence in a block; SubSeqX - this variable holds the subsequence that is being checked for redundancy; SubSeqY - this variable holds the subsequence that the SubSeqX is checked against; BlockLen - this variable holds the user determined length of the block; SegLen - this variable holds the length of a segment.
• the program is hardcoded to generate scores for subsequences of lengths 3, 4, 5, 6, 7, 8, 9, and 10;
• Block - this variable holds a string of length BlockLen.
• the string is composed of letters from an input XTEN sequence and is determined by the position of the i counter;
• SubSeqList - this is a list that holds all of the generated subsequence scores.
• FIG. 33 depicts the application of the algorithm SegScore to a hypothetical XTEN of 11 amino acids in order to determine the repetitiveness.
• a pair-wise comparison of all subsequences is performed and the average number of identical subsequences is calculated to result, in this case, in a subsequence score of 1.89.
• FIG. 34 provides the results of the assay to measure the fluorescence signal of RP11 clones pSD0107 to pSDOl 18), as described in Example 6.
• One positive control pLCW970
• two negative controls pBr322 and pLCW970+10 mM phosphate were included.
• the GFP expression level was measured using samples from 2-3 shake flasks per construct.
• FIG. 35 shows the screening results of libraries LCW1157-1159.
• FIG. 35A-C provides the fluorescence histograms of LCW1157-1159, showing the number of colonies identified for each fluorescence signal region, as described in Example 6. The average fluorescence reading of the negative control (black arrow) and positive pSDOl 16 (white arrow) are marked in the figures.
• FIG. 35D-F provides the correlation between the fluorescence reading in the original test and the retest of the select clones.
• FIG. 36 shows results of the SDS-PAGE analysis of the top 8 expression construct products and controls under unreduced conditions, as described in Example 6.
• the desired full length protein end product RP11-XTEN-GFP is indicated by an arrow, and the higher band is the dimer of the protein.
• FIG. 37 shows the SDS-PAGE evaluation of the MacroCap SP capture efficiency for the top 4 expression construct products under non-reducing conditions, as described in Example 6.
• Lanes 1-4 load, flow through, wash and elution of LCWl 159.004, 2.
• Lanes 5-8 load, flow through, wash and elution of LCWl 159.006.
• Lanes 9-12 load, flow through, wash and elution of LCWl 158.004.
• 13-16 load, flow through, wash and elution of LCWl 157.040.
• Lanes 17-20 1-4 load, flow through, wash and elution of negative control. Unmarked lanes are molecular weight standards.
• FIG. 38 shows the summary of library LCWl 163 screening results with a comparison of the fluorescence signal of the top 4 expression products and the controls in the retests, as described in Example 6. Each sample had 4 replicates, represented by 4 individual dots in the figure.
• FIG. 39 shows the summary of library LCWl 160 screening results, as described in Example 6. Fluorescence histogram of LCWl 157-1159, showing the number of colonies identified for each fluorescence signal region; average fluorescence reading of negative control (black arrow), pSDOl 16 (white arrow), and LCWl 159.004 (high expression candidates from screening LCWl 157-1159, grey arrow) were marked in the figures.
• FIG. 40 shows 4-12% SDS-PAGE/silver staining analysis of MacroCap Q fractions as described in Example 2.
• FIG. 40A Batch 2, lane 1 : molecular weight standard; lanes 2-5:
• FIG. 40B Batch 1, lane 1 : molecular weight standard; lanes 2-6: MacroCap Q flow through fractions 1-5, respectively; lanes 7-16: MacroCap Q elution fractions 1-10, respectively.
• FIG. 41 shows results of analyses of reaction mixtures from the preparation of conjugates to lxAzide,3xMMAE-XTEN analyzed by C18-RP-HPLC and mass spectroscopy, as described in Example 5.
• FIG. 41A is analysis of the initial lxAmino,3xThiol-XTEN reactant.
• FIG. 41B is analysis of the protein modification with MMAE-Maleimide, showing the mass increase corresponding to modifications of three cysteines with MMAE-Mal.
• FIG. 41 C shows the analysis of the protein modification with Azide-PEG4-NHS ester, with mass increases corresponding to the single addition of the azide-PEG4 moiety.
• FIG. 42 shows the results of screening libraries LCWl 171, 1172, 1203, and 1204, as described in Example 2.
• FIG. 42A-D Fluorescence histogram of LCWl 171, 1172, 1203, 1204, showing the number of colonies identified for each fluorescence signal region; average fluorescence reading of negative control (black arrow) and pSDOl 16 (white arrow) when screening LCWl 171-1172 were marked in the FIGS. 94A and B; average fluorescence reading of negative control (black arrow), pSDOl 16 (white arrow), and CBD control (grey arrow) when screening LCW1203-1204 are marked in FIGS. 94C and D.
• FIG. 43 shows the results of screening libraries LCW1208-1210, as described in Example 6.
• FIGS. 95A-C Fluorescence histograms of LCW1208-1210, showing the number of colonies identified for each fluorescence signal region; average fluorescence reading of negative control (black arrow) and CBD control (grey arrow) are marked in the figures.
• FIG. 44 illustrated the production of XTEN segments from a precursor that contains three repeat copies of XTEN of identical length and sequence.
• the XTEN precursor comprises three identical copies of XTEN that are flanked by identical protease cleavage sites.
• the XTEN precursor further comprises N- and C- terminal affinity purification tags to facilitate purification of full-length precursor molecules. Following purification of the precursor it is cleaved by protease that acts on all the incorporated cleavage sequences to release the tags from the XTEN, which is followed by purification to separate the individual units of XTEN, facilitating the high-yield production of XTENs with short and intermediate lengths from long- chain precursor molecules.
• FIG. 45 shows results of analyses of reaction mixtures from the preparation of conjugates to lxDBCO,3xFA(y)-XTEN analyzed by C18-RP-HPLC and mass spectroscopy, as described in Example 18.
• FIG. 45A is analysis of the initial lxAmino,3xThiol-XTEN reactant.
• FIG. 45B is analysis of the protein modification with Folate-gamma-Maleimide, showing the mass increase corresponding to modifications of three cysteines with FA(y)-Mal.
• FIG. 45 C shows the analysis of the protein modification with DBCO-sulfo-NHS ester, with mass increases corresponding to the single addition of the DBCO moiety.
• FIG. 46 shows C4 RP-HPLC analyses of the click conjugate reactants and product 3xFA(y),3xMMAE-XTEN, as described in Example 19.
• FIG. 47 shows analyses of final 3xFA(y),3xMMAE-XTEN product purified by preparative RP-HPLC, as described in Example 19.
• FIG. 47A shows size exclusion
• FIG. 47B shows RP-HPLC analysis (Phenomenex Jupiter C18 5 ⁇ 30 ⁇ 150 x 4.60mm column, Buffer A: 0.1% TFA in H20, Buffer B: 0.1% TFA in CAN, flow rate lml/min, gradient 5% to 50%B in 45min).
• FIG. 3C shows ESI-MS analysis (QSTAR-XL, calculated MW 85,085.4 Da, experimental MW 85,091 Da).
• FIG. 48 shows results of a killing assay demonstrating selective cytotoxicity of 3xFA(y),3xMMAE-XTEN on KB cells, as described in Example 69.
• the inhibitory dose response curves are shown for the groups of free MMAE (filled circles); 3xMMAE-XTEN (filled, inverted triangles) and 3xFA(y),3xMMAE-XTEN in the presence (filled triangles) and absence (filled squares) of folic acid competitor on KB cells.
• FIG. 49 shows the structure of the XTEN-payload conjugate 3xFA(y),3xMMAE- XTEN.
• FIG. 49A shows the two XTEN linked by the reaction of the azide l-azido-3,6,9,12- tetraoxapentadecan-15-oic acid, N-hydroxysuccinimide ester and the alkyne 6-(l 1,12- didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoic acid, N-hydroxysuccinimide (or N- hydroxysulfosuccinimide) ester.
• FIG. 49A shows the two XTEN linked by the reaction of the azide l-azido-3,6,9,12- tetraoxapentadecan-15-oic acid, N-hydroxysuccinimide ester and the alkyne 6-(l 1,12- didehydrodibenzo[b,
• FIG. 49B shows the X residue of Cys modified with folate-g- aminopentyl-maleimide.
• FIG. 49C shows the Z residue of Cys modified with maleimidocaproyl- valine-citrulline-p-aminobenzyloxycarbonyl-monomethylauristatin.
• FIG. 50 shows an example of an XTEN-folate conjugate comprising folate targeting moieties and toxin payloads that exert selective action on a target cell, such as a tumor cell.
• the particular design of the dimeric XTEN conjugate comprises folate and MMAE. This conjugate binds to the folate-receptor on that is over-expressed on many cancer cells. Receptor binding results in internalization followed by proteolytic break down and the intracellular liberation of MMAE, which is toxic to the cell.
• a cell includes a plurality of cells, including mixtures thereof.
• polypeptide polypeptide
• peptide protein
• polymers of amino acids of any length may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
• the terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
• amino acid refers to either natural amino acids, including L optical isomers. Standard single or three letter codes are used to designate amino acids.
• natural L-amino acid means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).
• non-naturally occurring means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally-occurring sequence found in a mammal.
• a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.
• hydrophilic and hydrophobic refer to the degree of affinity that a substance has with water.
• a hydrophilic substance has a strong affinity for water, tending to dissolve in, mix with, or be wetted by water, while a hydrophobic substance substantially lacks affinity for water, tending to repel and not absorb water and tending not to dissolve in or mix with or be wetted by water.
• Amino acids can be characterized based on their hydrophobicity. A number of scales have been developed.
• hydrophilic amino acids are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine.
• hydrophobic amino acids are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.
• a "fragment" when applied to a biologically active protein is a truncated form of a the biologically active protein that retains at least a portion of the therapeutic and/or biological activity.
• a “variant,” when applied to a biologically active protein is a protein with sequence homology to the native biologically active protein that retains at least a portion of the therapeutic and/or biological activity of the biologically active protein.
• a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared with the reference biologically active protein.
• the term "biologically active protein variant” includes proteins modified deliberately, as for example, by site directed mutagenesis, synthesis of the encoding gene, insertions, or accidentally through mutations and that retain activity.
• sequence variant means polypeptides that have been modified compared to their native or original sequence by one or more amino acid insertions, deletions, or substitutions. Insertions may be located at either or both termini of the protein, and/or may be positioned within internal regions of the amino acid sequence.
• a non-limiting example is insertion of an XTEN sequence within the sequence of the biologically-active payload protein.
• Another non-limiting example is substitution of an amino acid in an XTEN with a different amino acid.
• deletion variants one or more amino acid residues in a polypeptide as described herein are removed. Deletion variants, therefore, include all fragments of a payload polypeptide sequence.
• substitution variants one or more amino acid residues of a polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature and conservative substitutions of this type are well known in the art.
• moiety means a component of a larger composition or that is intended to be incorporated into a larger composition, such as a functional group of a drug molecule or a targeting peptide joined to a larger polypeptide.
• XTEN release site refers to a cleavage sequence in XTEN-payload that can be recognized and cleaved by a protease, effecting release of an XTEN or a portion of an XTEN from the XTEN-payload polypeptide.
• protease means a protease that normally exists in the body fluids, cells or tissues of a mammal. XTEN release sites can be engineered to be cleaved by various mammalian proteases (a.k.a.
• XTEN release proteases such as trypsin, FXIa, FXIIa, kallikrein, FVIIIa, FVIIIa, FXa, Flla (thrombin), Elastase-2, MMP-12, MMP13, MMP-17, MMP-20, or any protease that is present in a subject.
• Other equivalent proteases endogenous or exogenous that are capable of recognizing a defined cleavage site can be utilized. The cleavage sites can be adjusted and tailored to the protease utilized.
• Activity refers to an action or effect, including but not limited to receptor binding, antagonist activity, agonist activity, a cellular or physiologic response, or an effect generally known in the art for the payload, whether measured by an in vitro, ex vivo or in vivo assay or a clinical effect.
• ELISA refers to an enzyme-linked immunosorbent assay as described herein or as otherwise known in the art.
• a "host cell” includes an individual cell or cell culture which can be or has been a recipient for the subject vectors such as those described herein.
• Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
• a host cell includes cells transfected in vivo with a vector of this invention.
• isolated when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart.
• a “concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart.
• a polypeptide made by recombinant means and expressed in a host cell is considered to be “isolated.”
• An "isolated" nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid.
• an isolated polypeptide- encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells.
• an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal or extra-chromosomal location different from that of natural cells.
• a "chimeric" protein contains at least one fusion polypeptide comprising at least one region in a different position in the sequence than that which occurs in nature.
• the regions may normally exist in separate proteins and are brought together in the fusion polypeptide; or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide.
• a chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
• Fusion refers to the joining together of two or more peptide or polypeptide sequences by recombinant means.
• operably linked means that the DNA sequences being linked are contiguous, and in reading phase or in-frame.
• An "in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs.
• ORFs open reading frames
• a promoter or enhancer is operably linked to a coding sequence for a polypeptide if it affects the transcription of the polypeptide sequence.
• the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).
• Crosslinking refers to the covalent joining of two different molecules by a chemical reaction.
• the crosslinking can occur in one or more chemical reactions, as described more fully, below.
• conjugation partner refers to the individual components that can be linked or are linked in a conjugation reaction.
• conjugation is intended to refer to the heterogeneous molecule formed as a result of covalent linking of conjugation partners one to another, e.g., a biologically active payload covalently linked to a XTEN molecule or a cross-linker covalently linked to a reactive XTEN.
• Cross-linker and “linker” and “cross-linking agent” are used interchangeably and in their broadest context to mean a chemical entity used to covalently join two or more entities.
• a cross-linker joins two, three, four or more XTEN, or joins a payload to an XTEN, as the entities are defined herein.
• a cross-linker includes, but is not limited to, the reaction product of small molecule zero-length, homo- or hetero-bifunctional, and multifunctional cross-linker compounds, the reaction product of two click-chemistry reactants. It will be understood by one of skill in the art that a cross-linker can refer to the covalently -bound reaction product remaining after the crosslinking of the reactants.
• the cross-linker can also comprise one or more reactants which have not yet reacted but which are capable to react with another entity.
• a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
• a “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.
• Heterologous means derived from a genotypically distinct entity from the rest of the entity to which it is being compared.
• a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence.
• heterologous as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
• polynucleotides refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three- dimensional structure, and may perform any function, known or unknown.
• polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched
• polynucleotides plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
• a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
• the sequence of nucleotides may be interrupted by non-nucleotide components.
• a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
• complement of a polynucleotide denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence, such that it could hybridize with a reference sequence with complete fidelity.
• Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of recombination steps which may include cloning, restriction and/or ligation steps, and other procedures that result in expression of a recombinant protein in a host cell.
• gene and “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.
• a gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof.
• a “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together.
• homology refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences.
• sequence identity refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences.
• BestFit a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences
• the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.
• polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity compared to those sequences.
• Polypeptides that are homologous preferably have sequence identities that are at least 70%, preferably at least 80%, even more preferably at least 90%, even more preferably at least 95- 99% identical.
• the ends of the DNA must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation.
• stringent conditions or “stringent hybridization conditions” includes reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Generally, stringency of hybridization is expressed, in part, with reference to the temperature and salt concentration under which the wash step is carried out.
• stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short polynucleotides (e.g., 10 to 50 nucleotides) and at least about 60°C for long polynucleotides (e.g., greater than 50 nucleotides)— for example, "stringent conditions” can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and three washes for 15 min each in
• temperatures of about 65°C, 60°C, 55°C, or 42°C may be used.
• SSC concentration may be varied from about 0.1 to 2*SSC, with SDS being present at about 0.1%.
• wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
• the Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• blocking reagents are used to block non-specific hybridization.
• blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
• Organic solvent such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
• percent identity refers to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
• Percent identity may be measured over the length of an entire defined polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polynucleotide sequence, for instance, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 contiguous residues.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• the percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of matched positions (at which identical residues occur in both polypeptide sequences), dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• the shortest sequence defines the length of the window of comparison. Conservative substitutions are not considered when calculating sequence identity.
• Percent (%) sequence identity is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity, thereby resulting in optimal alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
• Percent identity may be measured over the length of an entire defined polypeptide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
• Repetitiveness used in the context of polynucleotide sequences refers to the degree of internal homology in the sequence such as, for example, the frequency of identical nucleotide sequences of a given length. Repetitiveness can, for example, be measured by analyzing the frequency of identical sequences.
• a "vector” is a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid molecule into and/or between host cells.
• the term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions.
• An "expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a
• polypeptide(s) polypeptide(s).
• An "expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.
• serum degradation resistance refers to the ability of the polypeptides to withstand degradation in blood or components thereof, which typically involves proteases in the serum or plasma.
• the serum degradation resistance can be measured by combining the protein with human (or mouse, rat, monkey, as appropriate) serum or plasma, typically for a range of days (e.g. 0.25, 0.5, 1, 2, 4, 8, 16 days), typically at about 37°C.
• the samples for these time points can be run on a Western blot assay and the protein is detected with an antibody.
• the antibody can be to a tag in the protein. If the protein shows a single band on the western, where the protein's size is identical to that of the injected protein, then no degradation has occurred.
• the time point where 50% of the protein is degraded is the serum degradation half-life or "serum half- life" of the protein.
• circulating half-life are used interchangeably herein and, as used herein means the terminal half- life calculated as ln(2)/K e i .
• K d is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration vs. time curve.
• Half-life typically refers to the time required for half the quantity of an administered substance deposited in a living organism to be metabolized or eliminated by normal biological processes.
• Active clearance means the mechanisms by which a protein is removed from the circulation other than by filtration, and which includes removal from the circulation mediated by cells, receptors, metabolism, or degradation of the protein.
• Apparent molecular weight factor and "apparent molecular weight” are related terms referring to a measure of the relative increase or decrease in apparent molecular weight exhibited by a particular amino acid or polypeptide sequence. The apparent molecular weight is determined using size exclusion chromatography (SEC) or similar methods by comparing to globular protein standards, and is measured in "apparent kD" units.
• the apparent molecular weight factor is the ratio between the apparent molecular weight and the actual molecular weight; the latter predicted by adding, based on amino acid composition, the calculated molecular weight of each type of amino acid in the composition or by estimation from comparison to molecular weight standards in an SDS electrophoresis gel. Determination of both the apparent molecular weight and apparent molecular weight factor for representative proteins is described in the Examples.
• hydrodynamic radius or "Stokes radius” is the effective radius (3 ⁇ 4 in nm) of a molecule in a solution measured by assuming that it is a body moving through the solution and resisted by the solution's viscosity.
• the hydrodynamic radius measurements of the XTEN polypeptides correlate with the "apparent molecular weight factor” which is a more intuitive measure.
• the "hydrodynamic radius” of a protein affects its rate of diffusion in aqueous solution as well as its ability to migrate in gels of macromolecules.
• the hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape and compactness.
• Physiological conditions refers to a set of conditions in a living host as well as in vitro conditions, including temperature, salt concentration, pH, that mimic those conditions of a living subject.
• a host of physiologically relevant conditions for use in in vitro assays have been established.
• a physiological buffer contains a physiological concentration of salt and is adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5.
• a variety of physiological buffers are listed in Sambrook et al. (2001).
• Physiologically relevant temperature ranges from about 25°C to about 38°C, and preferably from about 35°C to about 37°C.
• a "single atom residue of a payload” means the atom of a payload that is chemically linked to XTEN after reaction with the subject XTEN or XTEN-linker compositions; typically a sulfur, an oxygen, a nitrogen, or a carbon atom.
• an atom residue of a payload could be a sulfur residue of a cysteine thiol reactive group in a payload, a nitrogen molecule of an amino reactive group of a peptide or polypeptide or small molecule payload, a carbon or oxygen residue or a reactive carboxyl or aldehyde group of a peptide, protein or a small molecule or synthetic, organic drug.
• a "reactive group” is a chemical structure that can be coupled to a second reactive group.
• reactive groups are amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups, aldehyde groups, azide groups.
• Some reactive groups can be activated to facilitate conjugation with a second reactive group, either directly or through a cross-linker.
• a reactive group can be a part of an XTEN, a cross-linker, an azide/alkyne click- chemistry reactant, or a payload so long as it has the ability to participate in a chemical reaction. Once reacted, a conjugation bond links the residues of the payload or cross-linker or XTEN reactants.
• Controlled release agent “slow release agent”, “depot formulation” and “sustained release agent” are used interchangeably to refer to an agent capable of extending the duration of release of a polypeptide of the invention relative to the duration of release when the polypeptide is administered in the absence of agent.
• Different embodiments of the present invention may have different release rates, resulting in different therapeutic amounts.
• payload refers to a moiety as described herein which has a biological, pharmacological or therapeutic activity or beneficial effect when administered in a subject or that can be demonstrated in vitro. Payload also includes a molecule that can be used for imaging or in vivo diagnostic purposes.
• antagonist includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein.
• Methods for identifying antagonists of a polypeptide may comprise contacting a native polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide.
• antagonists may include proteins, nucleic acids, carbohydrates, antibodies or any other molecules that decrease the effect of a biologically active protein.
• agonist is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of a native polypeptide may comprise contacting a native polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide.
• “Inhibition constant”, or "Ki” are used interchangeably and mean the dissociation constant of the enzyme-inhibitor complex, or the reciprocal of the binding affinity of the inhibitor to the enzyme.
• a "defined medium” refers to a medium comprising nutritional and hormonal requirements necessary for the survival and/or growth of the cells in culture such that the components of the medium are known. Traditionally, the defined medium has been formulated by the addition of nutritional and growth factors necessary for growth and/or survival.
• the defined medium provides at least one component from one or more of the following categories: a) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; b) an energy source, usually in the form of a carbohydrate such as glucose; c) vitamins and/or other organic compounds required at low concentrations; d) free fatty acids; and e) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.
• the defined medium may also optionally be supplemented with one or more components from any of the following categories: a) one or more mitogenic agents; b) salts and buffers as, for example, calcium, magnesium, and phosphate; c) nucleosides and bases such as, for example, adenosine and thymidine, hypoxanthine; and d) protein and tissue hydrolysates.
• treat or “treating,” or “palliating” or “ameliorating” are used interchangeably and mean administering a drug or a biologic to achieve a therapeutic benefit, to cure or reduce the severity of an existing condition, or to achieve a prophylactic benefit, prevent or reduce the likelihood of onset or severity the occurrence of a condition.
• therapeutic benefit is meant eradication or amelioration of the underlying condition being treated or one or more of the physiological symptoms associated with the underlying condition such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying condition.
• a “therapeutic effect” or “therapeutic benefit,” as used herein, refers to a physiologic effect, including but not limited to the mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, resulting from administration of a polypeptide of the invention other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein.
• the compositions may be administered to a subject at risk of recurrence of a particular cancer, even though a diagnosis of the recurrence may not have been made.
• the term "therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal.
• the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
• the drug may inhibit the growth of and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
• efficacy can, for example, be measured by assessing the time to disease progression and/or determining the response rate. Such effect need not be absolute to be beneficial. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• terapéuticaally effective dose regimen refers to a schedule for consecutively administered multiple doses (i.e., at least two or more) of a biologically active protein, either alone or as a part of a polypeptide composition, wherein the doses are given in therapeutically effective amounts to result in sustained beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition.
• Host cells can be cultured in a variety of media.
• Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing eukaryotic cells.
• animal cells can be grown in a defined medium that lacks serum but is supplemented with hormones, growth factors or any other factors necessary for the survival and/or growth of a particular cell type. Whereas a defined medium supporting cell survival maintains the viability, morphology, capacity to metabolize and potentially, capacity of the cell to differentiate, a defined medium promoting cell growth provides all chemicals necessary for cell proliferation or multiplication.
• the general parameters governing mammalian cell survival and growth in vitro are well established in the art.
• Physicochemical parameters which may be controlled in different cell culture systems are, e.g., pH, pC>2, temperature, and osmolarity.
• the nutritional requirements of cells are usually provided in standard media formulations developed to provide an optimal environment. Nutrients can be divided into several categories: amino acids and their derivatives, carbohydrates, sugars, fatty acids, complex lipids, nucleic acid derivatives and vitamins.
• hormones from at least one of the following groups: steroids, prostaglandins, growth factors, pituitary hormones, and peptide hormones to proliferate in serum-free media (Sato, G.
• cells may require transport proteins such as transferrin (plasma iron transport protein), ceruloplasmin (a copper transport protein), and high-density lipoprotein (a lipid carrier) for survival and growth in vitro.
• transferrin plasma iron transport protein
• ceruloplasmin a copper transport protein
• high-density lipoprotein a lipid carrier
• Growth media for growth of prokaryotic host cells include nutrient broths (liquid nutrient medium) or LB medium (Luria Bertani). Suitable media include defined and undefined media. In general, media contains a carbon source such as glucose needed for bacterial growth, water, and salts. Media may also include a source of amino acids and nitrogen, for example beef or yeast extract (in an undefined medium) or known quantities of amino acids (in a defined medium).
• the growth medium is LB broth, for example LB Miller broth or LB Lennox broth. LB broth comprises peptone (enzymatic digestion product of casein), yeast extract and sodium chloride.
• a selective medium is used which comprises an antibiotic. In this medium, only the desired cells possessing resistance to the antibiotic will grow.
• the present invention relates, in part, to compositions in which one or more XTEN are chemically linked to one or more payloads, including combinations of different payloads, in defined numbers in either monomeric or multimeric configurations to provide compositions with enhanced pharmaceutical, pharmacokinetic, and pharmacologic properties.
• Such compositions linked to such payloads may have utility, when administered to a subject, in the prevention, treatment or amelioration of cancer for which a payload has a pharmacologic or biologic effect.
• the invention provides compositions in which XTEN with defined numbers of orthogonal pendant reactive groups are conjugated to one or more molecules of a targeting moiety that serves as a ligand to a cell-surface receptor and one or more molecules of an effector drug.
• the resulting compositions when administered in vitro or in vivo, can selectively deliver the effector drug to cells bearing such receptors, resulting in inhibition of cell division, growth, or metastasis.
• the targeting moieties of the subject compositions exhibit a binding specificity to a given target or another desired biological characteristic when used in vivo or when utilized in an in vitro assay.
• the targets to which the subject binding fusion protein compositions are generally associated with a disease, disorder or condition; e.g., a cancer.
• a target associated with a disease, disorder or condition means that the target is either expressed or overexpressed by disease cells or tissues, the target causes or is a mediator the disease, disorder or condition, or the target is generally found in higher concentrations in a tissue, organ or a localized region within a tissue or in an organ in a subject.
• XTEN extended recombinant polypeptides
• the invention provides substantially homogeneous XTEN polypeptides that are useful as conjugation partners to link to one or more payloads via a cross- linker reactant resulting in an XTEN-payload conjugate.
• XTEN are polypeptides with non-naturally occurring, substantially non-repetitive sequences having a low degree or no secondary or tertiary structure under physiologic conditions. XTEN typically have from about 36 to about 3000 amino acids, of which the majority or the entirety are small hydrophilic amino acids. As used herein, "XTEN” specifically excludes whole antibodies or antibody fragments (e.g. single-chain antibodies and Fc fragments). XTEN polypeptides have utility as a conjugation partners in that they serve in various roles, conferring certain desirable properties when linked to a payload, described more fully, below.
• the resulting XTEN-payload conjugates have enhanced properties, such as enhanced pharmacokinetic, physicochemical, pharmacologic, and pharmaceutical properties compared to the corresponding payload not linked to XTEN, making them useful in the treatment of certain conditions for which the payload is known in the art to be used.
• the unstructured characteristic and physicochemical properties of the XTEN result, in part, from the overall amino acid composition that is disproportionately limited to 4-6 types of hydrophilic amino acids, the linking of the amino acids in a quantifiable non-repetitive design, and the length of the XTEN polypeptide.
• the properties of XTEN disclosed herein are not tied to absolute primary amino acid sequences, as evidenced by the diversity of the exemplary sequences of Table 1 that, within varying ranges of length, possess similar properties, many of which are documented in the Examples. Accordingly, XTEN have properties more like non-proteinaceous, hydrophilic polymers than they do proteins.
• the XTEN of the present invention exhibit one or more of the following advantageous properties: conformational flexibility, reduced or lack of secondary structure, high degree of aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, a defined degree of charge, and increased hydrodynamic (or Stokes) radii; properties that are similar to certain hydrophilic polymers (e.g., polyethylene glycol) that make them particularly useful as conjugation partners.
• hydrophilic polymers e.g., polyethylene glycol
• the XTEN component(s) of the subject conjugates are designed to behave like denatured peptide sequences under physiological conditions, despite the extended length of the polymer.
• "Denatured” describes the state of a peptide in solution that is characterized by a large conformational freedom of the peptide backbone. Most peptides and proteins adopt a denatured conformation in the presence of high concentrations of denaturants or at elevated temperature. Peptides in denatured conformation have, for example, characteristic circular dichroism (CD) spectra and are characterized by a lack of long-range interactions as determined by NMR.
• CD characteristic circular dichroism
• the invention provides XTEN sequences that, under physiologic conditions, resemble denatured sequences that are largely devoid of secondary structure. In other cases, the XTEN sequences are substantially devoid of secondary structure under physiologic conditions. "Largely devoid,” as used in this context, means that less than 50% of the XTEN amino acid residues of the XTEN sequence contribute to secondary structure as measured or determined by the means described herein.
• substantially devoid means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or at least about 99% of the XTEN amino acid residues of the XTEN sequence do not contribute to secondary structure, as measured or determined by the methods described herein.
• Such properties include but are not limited to secondary or tertiary structure, solubility, protein aggregation, stability, absolute and apparent molecular weight, purity and uniformity, melting properties, contamination and water content.
• the methods to measure such properties include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion chromatography (SEC), HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visible spectroscopy.
• secondary structure can be measured
• Secondary structure elements such as alpha-helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra, as does the lack of these structure elements. Secondary structure can also be predicted for a polypeptide sequence via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and the Garnier-Osguthorpe-Robson algorithm (“Gor algorithm”) (Gamier J, Gibrat JF, Robson B.
• Chou-Fasman algorithm Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45
• Garnier-Osguthorpe-Robson algorithm Garnier-Osguthorpe-Robson algorithm
• the XTEN sequences used in the subject conjugates have an alpha-helix percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In another embodiment, the XTEN sequences have a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In one
• the XTEN sequences of the conjugates have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In one embodiment, the XTEN sequences of the conjugates have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2%.
• the XTEN sequences of the conjugate compositions have a high degree of random coil percentage, as determined by the GOR algorithm.
• an XTEN sequence has at least about 80%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% random coil, as determined by the GOR algorithm.
• the XTEN sequences of the conjugate compositions have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm and at least about 90% random coil, as determined by the GOR algorithm.
• the XTEN sequences of the disclosed compositions have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2% at least about 90% random coil, as determined by the GOR algorithm.
• the XTEN sequences of the compositions are substantially lacking secondary structure as measured by circular dichroism.
• the selection criteria for the XTEN to be linked to the payload used to create the conjugate compositions generally relate to attributes of physicochemical properties and conformational structure of the XTEN that is, in turn, used to confer enhanced pharmaceutical, pharmacologic, and pharmacokinetic properties to the compositions.
• XTEN are used as a carrier in the compositions, the invention taking advantage of the discovery that increasing the length of the non-repetitive, unstructured polypeptides enhances the unstructured nature of the XTENs and correspondingly enhances the physicochemical and pharmacokinetic properties of constructs comprising the XTEN carrier.
• XTEN as monomers or as multimers with cumulative lengths longer that about 400 residues incorporated into the conjugates result in longer half-life compared to shorter cumulative lengths, e.g., shorter than about 280 residues.
• proportional increases in the length of the XTEN result in a sequence with a higher percentage of random coil formation, as determined by GOR algorithm, or reduced content of alpha-helices or beta-sheets, as determined by Chou-Fasman algorithm, compared to shorter XTEN lengths.
• increasing the length of the unstructured polypeptide fusion partner, as described in the Examples results in a construct with a
• the invention encompasses XTEN conjugate compositions comprising two, three, four or more XTEN wherein the cumulative XTEN sequence length of the XTEN proteins is greater than about 100, 200, 400, 500, 600, 800, 900, or 1000 to about 3000 amino acid residues, wherein the construct exhibits enhanced pharmacokinetic properties when administered to a subject compared to a payload not linked to the XTEN and administered at a comparable dose.
• the two or more XTEN sequences each exhibit at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or more identity to a sequence selected from Table 1, and the remainder, if any, of the carrier sequence(s) contains at least 90% hydrophilic amino acids and less than about 2% of the overall sequence consists of hydrophobic or aromatic amino acids.
• the enhanced pharmacokinetic properties of the XTEN-payload conjugate, in comparison to payload not linked to XTEN, are described more fully, below.
• the subject XTEN sequences included in the subject XTEN-folate conjugates are substantially non-repetitive.
• repetitive amino acid sequences have a tendency to aggregate or form higher order structures, as exemplified by natural repetitive sequences such as collagens and leucine zippers. These repetitive amino acids may also tend to form contacts resulting in crystalline or pseudocrystaline structures.
• the low tendency of non -repetitive sequences to aggregate enables the design of long-sequence XTENs with a relatively low frequency of charged amino acids that would otherwise be likely to aggregate if the sequences were repetitive.
• the non-repetitiveness of a subject XTEN can be observed by assessing one or more of the following features.
• a substantially non-repetitive XTEN sequence has no three contiguous amino acids in the sequence that are identical amino acid types unless the amino acid is serine, in which case no more than three contiguous amino acids are serine residues.
• a substantially non-repetitive XTEN sequence in which 80-99% of the sequence is comprised of motifs of 9 to 14 amino acid residues wherein the motifs consist of 3, 4, 5 or 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), and wherein the sequence of any two contiguous amino acid residues in any one motif is not repeated more than twice in the sequence motif.
• the degree of repetitiveness of a polypeptide or a gene can be measured by computer programs or algorithms or by other means known in the art. According to the current invention, algorithms to be used in calculating the degree of repetitiveness of a particular polypeptide, such as an XTEN, are disclosed herein, and examples of sequences analyzed by algorithms are provided (see Examples, below). In one embodiment, the repetitiveness of a polypeptide of a predetermined length can be calculated (hereinafter "subsequence score") according to the formula given by Equation I:
• Subsequence score ⁇ & ⁇ ⁇ ' ⁇ I
• SegScore An algorithm termed “SegScore” was developed to apply the foregoing equation to quantitate repetitiveness of polypeptides, such as an XTEN, providing the subsequence score wherein sequences of a predetermined amino acid length "n" are analyzed for repetitiveness by determining the number of times (a "count") a unique subsequence of length "s" appears in the set length, divided by the absolute number of subsequences within the predetermined length of the sequence.
• FIG. 32 depicts a logic flowchart of the SegScore algorithm, while FIG.
• a subsequence score is derived for a fictitious XTEN with 11 amino acids and a subsequence length of 3 amino acid residues.
• a predetermined polypeptide length of 200 amino acid residues has 192 overlapping 9-amino acid subsequences and 198 3-mer subsequences, but the subsequence score of any given polypeptide will depend on the absolute number of unique subsequences and how frequently each unique subsequence (meaning a different amino acid sequence) appears in the predetermined length of the sequence.
• subsequence score means the sum of occurrences of each unique 3-mer frame across 200 consecutive amino acids of the cumulative XTEN polypeptide divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence. Examples of such subsequence scores derived from 200 consecutive amino acids of repetitive and non-repetitive polypeptides are presented in Example 45.
• the invention provides a XTEN-folate comprising one XTEN in which the XTEN has a subsequence score less than 12, more preferably less than 10, more preferably less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and most preferably less than 5.
• the invention provides XTEN-cross-linker conjugates comprising an XTEN in which the XTEN have a subsequence score of less than 10, more preferably less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and most preferably less than 5.
• the invention provides XTEN-click-chemistry conjugates comprising an XTEN in which the XTEN have a subsequence score of less than 10, more preferably less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and most preferably less than 5.
• the invention provides XTEN conjugate compositions comprising at least two linked XTEN in which each individual XTEN has a subsequence score of less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less.
• the invention provides XTEN conjugate compositions comprising at least three linked XTEN in which each individual XTEN has a subsequence score of less than 10, or less than 9, or less than 8, or less than 7, or less than 6, or less than 5, or less.
• an XTEN with a subsequence score of 10 or less i.e., 9, 8, 7, etc. is characterized as substantially non-repetitive.
• the present invention encompasses XTEN used as conjugation partners that comprise multiple units of shorter sequences, or motifs, in which the amino acid sequences of the motifs are substantially non-repetitive. The non-repetitive property can be met even using a "building block” approach using a library of sequence motifs that are multimerized to create the XTEN sequences, as shown in FIGS. 18-19.
• XTEN sequence may consist of multiple units of as few as four different types of sequence motifs, because the motifs themselves generally consist of non-repetitive amino acid sequences, the overall XTEN sequence is designed to render the sequence substantially non-repetitive.
• amino acids that are included in XTEN are, e.g., arginine, lysine, threonine, alanine, asparagine, glutamine, aspartate, glutamate, serine, and glycine.
• XTEN sequences have predominately four to six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P) that are arranged in a substantially non-repetitive sequence that is about 36 to about 3000, or about 100 to about 2000, or about 144 to about 1000 amino acid residues in length.
• G glycine
• A alanine
• S serine
• T threonine
• E glutamate
• P proline
• the XTEN has less than 100% of its amino acids consisting of 4, 5, or 6 types of amino acid selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P)
• the other amino acid residues of the XTEN are selected from any of the other 14 natural L-amino acids, but are preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids.
• an individual amino acid or a short sequence of amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) may be incorporated into the XTEN to achieve a needed property, such as to permit incorporation of a restriction site by the encoding nucleotides, or to facilitate linking to a payload component, or incorporation of a cleavage sequence.
• the invention provides XTEN that incorporates from 1 to about 20, or 1 to about 15, or 1 to about 10, or 1 to 5 lysine residues wherein the reactive lysines are utilized for linking to cross-linkers or payloads, as described herein.
• the XTEN incorporates from 1 to about 20, or 1 to about 15, or 1 to about 10, or 1 to 5 cysteine residues wherein the reactive cysteines are utilized for linking to cross-linkers or payloads, as described herein.
• the XTEN incorporates from 1 to about 20 cysteine and lysine residues wherein the lysines and cysteines are utilized for linking to different cross-linkers or payloads, as described herein.
• the XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) are either interspersed throughout the XTEN sequence, are located within or between the sequence motifs, or are concentrated in one or more short stretches of the XTEN sequence such as at or near the N- or C-terminus.
• hydrophobic amino acids impart structure to a polypeptide, the invention provides that the content of hydrophobic amino acids in the XTEN utilized in the conjugation constructs will typically be less than 5%, or less than 2%, or less than 1%
• hydrophobic amino acid content Hydrophobic residues that are less favored in construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, one can design the XTEN sequences to contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of the following amino acids: methionine (to avoid oxidation), asparagine and glutamine (to avoid desamidation). In other embodiments, the amino acid content of methionine and tryptophan in the XTEN component used in the conjugation constructs is typically less than 5%, or less than 2%, and most preferably less than 1%.
• the XTEN of the subject XTEN conjugates will have a sequence that has less than 10% amino acid residues with a positive charge, or less than about 7%, or less that about 5%, or less than about 2% amino acid residues with a positive charge, the sum of methionine and tryptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 5% of the total XTEN sequence.
• the invention provides XTEN-folate conjugates comprising XTEN with defined numbers of incorporated cysteine or lysine residues; "cysteine-engineered XTEN” and “lysine-engineered XTEN", respectively. It is an object of the invention to provide XTEN with defined numbers of cysteine and/or lysine residues to permit conjugation between the thiol group of the cysteine or the epsilon amino group of the lysine and a reactive group on a payload or a cross-linker to be conjugated to the XTEN backbone.
• the XTEN of the invention has between about 1 to about 100 lysine residues, or about 1 to about 70 lysine residues, or about 1 to about 50 lysine residues, or about 1 to about 30 lysine residues, or about 1 to about 20 lysine residues, or about 1 to about 10 lysine residues, or about 1 to about 5 lysine residues, or 1 to about 3 lysine residues, or alternatively only a single lysine residue.
• the XTEN of the invention has between about 1 to about 100 cysteine residues, or about 1 to about 70 cysteine residues, or about 1 to about 50 cysteine residues, or about 1 to about 30 cysteine residues, or about 1 to about 20 cysteine residues, or about 1 to about 10 cysteine residues, or about 1 to about 5 cysteine residues, or 1 to about 3 cysteine residues, or alternatively only a single cysteine residue. In another embodiment of the foregoing, the XTEN of the invention has about 1 to about 10 lysine residues and about 1 to about 10 cysteine residues.
• conjugates can be constructed that comprise XTEN, a cross-linker, plus one or more types of payloads useful in the treatment of a cancer in a subject wherein the maximum number of molecules of the payload agent linked to the XTEN component is determined by the numbers of lysines or cysteines with a reactive side group (i.e., a terminal amino or a thiol) incorporated into the XTEN.
• a reactive side group i.e., a terminal amino or a thiol
• the invention provides polynucleotides encoding cysteine- engineered XTEN wherein nucleotides encoding one or more amino acids of an XTEN are replaced with nucleotides encoding a cysteine amino acid to create a gene encoding the cysteine- engineered XTEN.
• the invention provides polynucleotides encoding cysteine-engineered XTEN where nucleotides encoding one or more cysteine amino acids are inserted into an-XTEN encoding gene to create the cysteine-engineered XTEN gene.
• oligonucleotides encoding one or more motifs of about 9 to about 14 amino acids comprising codons encoding one or more cysteines are linked in frame with other oligos encoding
• nucleotides encoding cysteine can be linked to codons encoding amino acids used in XTEN to create a cysteine-XTEN motif with the cysteine(s) at a defined position using the methods described herein (see Example 2 and FIG. 1), or by standard molecular biology techniques, and the motifs subsequently assembled into the gene encoding the full-length cysteine-engineered XTEN.
• a cysteine-motif can be created de novo and be of a pre-defined length and number of cysteine amino acids by linking nucleotides encoding cysteine to nucleotides encoding one or more amino acid residues used in XTEN (e.g., G, S, T, E,
• XTEN motif can be created that could comprise about 9-14 amino acid residues and have one or more reactive amino acids; i.e., cysteine or lysine.
• motifs suitable for use in an engineered XTEN that contain a single cysteine or lysine are:
• the invention contemplates motifs of different lengths, such as those of Table 3, for incorporation into XTEN.
• the gene can be designed and built by linking existing "building block" polynucleotides encoding both short- and long-length XTENs; e.g., AE48, AE144, AE288, AE432, AE576, AE864, AM48, AM875, AE912, AG864, which can be fused in frame with the nucleotides encoding the cysteine- and/or lysine-containing motifs or, alternatively, the cysteine- and/or lysine-encoding nucleotides can be PCR'ed into an existing XTEN sequence (as described more fully below and in the Examples) using, for example, nucleotides encoding the islands of
• Non-limiting examples of such engineered XTEN are provided in Table 1.
• the invention provides an XTEN sequence having at least about 80% sequence identity, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% sequence identity, or is identical to a sequence or a fragment of a sequence selected from of Table 1, when optimally aligned.
• application of the cysteine- or lysine-engineered methodology to create XTEN encompassing cysteine or lysine residues is not meant to be constrained to the precise compositions or range of composition identities of the foregoing embodiments.
• the precise location and numbers of incorporated cysteine or lysine residues in an XTEN can be varied without departing from the invention as described.

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