XTEN conjugate compositions and methods of producing the same

Abstract

To provide XTEN conjugate compositions and methods of producing the same.SOLUTION: The present invention relates to an extended recombinant polypeptide (XTEN) composition, a conjugate composition comprising XTEN bound to a crosslinker useful for conjugation to XTEN and a pharmacologically active payload, a method for producing highly purified XTEN, a method for producing XTEN-linker and XTEN-payload conjugates, and a method for using a composition of an XTEN-crosslinker and an XTEN-payload. The present invention addresses this need and provides related advantages. The compositions and methods disclosed herein not only are useful as therapeutics but are also particularly useful as research tools for preclinical and clinal development of a candidate therapeutic agent.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 61 / 634,312, filed February 27, 2012. No. 61 / 690,187, filed June 18, 2012; and and priority to U.S. Patent Application No. 61 / 709,942, filed October 4, 2012. The benefit of prior art is claimed, and these applications are incorporated herein by reference. [Background technology]

[0002] Increasing the half-life of a therapeutic drug, whether it is a therapeutic protein, peptide, or Often, the therapeutic agent itself, whether it is a drug or a small molecule, requires special formulation or modification. PEGylation, the addition of antibody fragments or albumin molecules to the therapeutic agent, etc. Conventional modification methods suffer from a number of significant drawbacks. Although these conventional methods generally involve high product prices, complex manufacturing processes, and Manufacturing processes and the final product often suffer from low purity. This is difficult, if not impossible, to achieve. The reaction itself was carefully designed to generate a homogenous population of PEG-bearing PEGylation agents. This is especially true in the case of uncontrolled PEGylation. Metabolism can have serious side effects. For example, PEGylated proteins have been shown to be It has been observed to cause renal tubular vacuolation (Bendele, A., Seely , J., Richey, C., Sennello, G. & Shopp, G. Short. communication:renal tubular vacuolation in animals treated with polyethylene-gl ycol-conjugated proteins.Toxicol.Sci.199 8.42,152-157). PEGylated proteins or their substitutes that are cleared by the kidney The drug accumulates in the kidney, causing the formation of PEG hydrates that interfere with normal glomerular filtration. Additionally, animals and humans can be induced to produce antibodies against PEG ( Sroda, K. et al.Repeated injections of PEG -PE liposomes generate anti-PEG antibodi es.Cell.Mol.Biol.Lett.2005.10,37-47).

[0003] Thus, production of highly pure forms of therapeutic agents with extended half-life characteristics at reasonable prices. There remains a significant need for alternative compositions and methods useful for: [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Bendele, A., Seely, J., Richey, C., Sennello, G. & Shopp, G. Short communication:renal tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins.Toxicol.Sci.1998.42,152-157 [Non-patent document 2] Sroda,K.et al.Repeated injections of PEG-PE liposomes generate anti-PEG antibodies.Cell.Mol.Biol.Lett.2005.10,37-47 Summary of the Invention [Means for solving the problem]

[0005] The present invention addresses this need and provides related advantages. The compositions and methods are useful not only as therapeutic agents, but also as preclinical and clinical applications of candidate therapeutic agents. They are also particularly useful as research tools for development. Partially, can be purified to homogeneity in one or more simple steps and / or extensively Chemistry of reactive groups with payload peptides, proteins, and small molecules using conjugation methods by generating extended recombinant polypeptide (XTEN) reagents amenable to selective conjugation. The use of XTEN reagents has resulted in high homogeneity, high yields, and high yields compared to unconjugated products. Superior in one or more aspects, including solubility, long-term stability, and enhanced terminal half-life This results in a high yield of XTEN-conjugated drug.

[0006] The present invention relates, in part, to one or more payload pharmacologically or biologically active agents. Substances useful as conjugation partners for binding and resulting in XTEN-payload compositions The present invention relates to novel compositions comprising substantially homogeneous extended recombinant polypeptides (XTENs). In this regard, the present invention provides a method for producing a medicament comprising administering to a subject a medicament ... 1, 2, 3, 4, 5, 6, 7 or more species of payload , 8, 9, or 10 or more molecules to provide an XTEN-payload composition. The present invention provides engineered XTENs with enhanced pharmaceutical properties, including enhanced pharmacokinetic properties. for use in creating conjugates with payload drugs of interest as compositions having It is an object of the present invention to provide such engineered XTEN polypeptides. The present invention provides a method for preparing XTEN-payload conjugates using a single antibody so that the resulting XTEN-payload conjugates have a high degree of purity. It is useful for preparing conjugates containing XTEN attached to a payload of at least 1000 ketones, and XTEN that are substantially homogeneous in sequence. Such conjugates of high purity include: One or more payloads are medically useful in the prevention, treatment, or amelioration of a condition. They are useful in preparing pharmaceutical compositions for subjects with the condition.

[0007] In a first aspect, the present invention provides an XTEN-crosslinker intermediate and an XTEN-payload. Substantially homogeneous XTEN polypeptides useful as conjugation partners to create endothelial compositions. In some embodiments, the present invention provides extended recombinant polypeptide compositions. and providing a substantially homogeneous population of polypeptides containing XTEN, and identifying individual polypeptides within the population. At least 90%, 91%, 92%, 93%, 94%, or 95% of the polypeptide molecules In one such embodiment, the XTEN has the same sequence length as all XTEN amino acids. The amino acid sequence is at least 36 to about 3000 amino acid residues, and glycine (G), Alanine (A), serine (S), threonine (T), glutamate (E) and proline The total amino acid residues of XTEN are approximately >90%. The N sequence is: (i) the XTEN sequence is three adjacent sequences that are identical unless the amino acid is serine; or (ii) contains at least about 80%, or about 90%, of the XTEN sequence. %, or about 95%, consist of non-overlapping sequence motifs, and each of the sequence motifs is about 9 It contains about 14 amino acid residues, and any two adjacent amino acid residues are or (iii) the XTEN sequence does not occur more than twice in each of the sequences. and the XTEN sequence is substantially non-repetitive, having a subsequence score of less than 0. ,90%,91%,92%,93%,94%,9,determined by the GOR algorithm. >5%, >96%, >97%, >98%, or >99% random coil formation and the XTEN sequence has less than 2%, or 3%, or 4%, or 5% alpha- or alpha-transferases. The XTEN sequence was determined by the Chou-Fasman algorithm. and having less than 2%, or 3%, or 4%, or 5% β-sheets as defined by X When the TEN sequence was analyzed by the TEPITOPE algorithm, the predicted T cells The TEPITOPE algorithm for epitopes within the XTEN sequence lacks cytoplasmic epitopes. and the rhythm prediction is based on a score of -8, -9, or -10. In another embodiment of the foregoing, the XTEN is selected from the group consisting of: at least about 90%, or at least at least about 91%, or at least about 92%, or at least about 93%, or at least 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%, or 100% distribution Contains sequences with sequence identity.

[0008] In other embodiments, the substantially homogeneous XTEN polypeptide composition comprises one or more parent XTEN polypeptides. In one embodiment, the present invention provides a substantially homogeneous antibody comprising a first affinity tag. and a first affinity tag that is a hydrophobic interaction cloning agent. Chromatography (HIC), cation exchange, anion exchange, immobilized metal ion affinity chromatography chromatography (IMAC), and immobilized antibody chromatography. In one embodiment of the foregoing, the first affinity tag has a binding affinity for the base substrate. is at least about 90% to a sequence selected from the group consisting of the sequences set forth in Table 7; 91%, 92%, 93%, 94%, or at least about 95% sequence identity. In another embodiment of the XTEN and affinity tags described above, the composition may further comprise one or more helper In one embodiment, the helper sequence is selected from the sequences set forth in Table 10. at least about 90%, or at least about 91%, or or at least about 92%, or at least about 93%, or at least about 94%, or is at least about 95%, or at least about 96%, or at least about 97%, or have at least about 98%, or at least about 99%, or 100% sequence identity In another embodiment, the helper sequence comprises the sequence KNPEQAEEQX1EET ( wherein X1 is independently S or R; ANPEQAEEQX1EET (wherein X1 is independently S or R); KNPEQAEEQAEEQX1EET (wherein , X1 is independently S or R);KX2X3EQAEEQAEEQX1EET wherein X1 is independently S or R, and X2 is independently K or N; X3 is independently K, N, T, Q, H, P, E, D, A, R, or S); KX 2(X3) 10 QX1EET, where X1 is independently S or R and X2 is independently X3 is independently K, N, T, Q, H, P, E, D, A, R, or S); KX2(X3)7AEEQX1EET, wherein X1 is independently , S or R, X2 is independently K or N, and X3 is independently K, N , T, Q, H, P, E, D, A, R, or S);KX2X3EQE(X3)3A EEQREET (wherein X2 is independently K or N, and X3 is independently K, N, T, Q, H, P, E, D, A, R, or S);KX2X3EQE(X3)3 AEE(X3)5, where X2 is independently K or N, and X3 is independently K , N, T, Q, H, P, E, D, A, R, or S);KKQEQEKEQAEE Q(X4X5)2REET, where X4 is independently A or S, and X5 is independently and K, Q, or E);KKQEQEKEQAEEQ(X4X5)4REET wherein X4 is independently A or S, and X5 is independently K, Q, or E. KKQEQEKEQAEEQ(Z)4REET, wherein Z is any naturally occurring KX2(X3) n (wherein n is an integer of 10 to 40, and X X2 is independently K or N, and X3 is independently K, N, T, Q, H, P, E, D, A, R, or S); (X3)n (wherein n is an integer of 10 to 50, and X 3 is independently K, N, T, Q, H, P, E, D, A, R, or S); KX2 QEQEKEQAEEQ(X4X5) n X1EET (wherein n is zero or 1 to 10) are integers, X1 is independently S or R, and X2 is independently K or N; X4 is independently A or S, and X5 is independently K, Q, or E. );KX2(X3) n (X4X5) m X1EET (wherein n is an integer of 5 to 20, m is zero or an integer from 1 to 10, X1 is independently S or R, and X2 is , independently K or N, and X3 is independently K, N, T, Q, H, P, E, D, A, R, or S; X4 is A or S; and X5 is independently K, Q, or is E); and KX2(X3) n (Z) m X1EET (wherein n is an integer of 5 to 20) m is a number, m is zero or an integer from 1 to 10, and X1 is independently S or R. X2 is independently K or N, and X3 is independently K, N, T, Q, H, or P. , E, D, A, R, or S, and Z is any naturally occurring L amino acid; and at least 80%, 90%, 95%, 98%, or more of the aforementioned when optimally aligned. or any sequence homolog showing 99% sequence identity.

[0009] Other embodiments of the foregoing substantially homogeneous XTEN, affinity tag, and helper sequence compositions In some embodiments, the composition further comprises a first cleavage sequence. If desired, the cleavage sequence is 8 and the sequences set forth in Table 9. In one of the foregoing embodiments, The composition comprises a compound of Formula I:

[0010] [ka]

[0011] where HS is a helper sequence and AT1 is a first affinity tag. , CS1 is the first cleavage sequence, and XTEN is the extended recombinant polypeptide. In another embodiment of the composition, the composition further comprises a second cleavage sequence. the first and second cleavage sequences can be cleaved by the same protease, The composition comprises a compound of Formula II:

[0012] [ka]

[0013] where HS is a helper sequence and AT1 is a first affinity tag. CS1 is the first cleavage sequence, CS2 is the second cleavage sequence, and XTEN is the extension sequence. In another embodiment of the above composition, the first affinity tag is a long recombinant polypeptide. is the sequence RPRPRPRPRPRPPR, HHHHHH, or any sequence known in the art or comprises any affinity tag disclosed herein.

[0014] In other embodiments of the substantially homogeneous XTEN composition, the composition comprises a first and a second an affinity tag, a first and second cleavage sequence, and a helper sequence, is different from the first affinity tag and is a chromatographic substrate different from the first affinity tag. The chromatographic substrates are HIC, cation exchange, anion exchange, a first and second cleavage sequence selected from the group consisting of ion exchange, IMAC, and immobilized antibody; The sequence can be cleaved by the same protease, and the second affinity tag can be cleaved by the same protease, as described in Table 7. at least about 90%, 91%, 92%, or 93% of a sequence selected from the group consisting of the sequences listed in %, 93%, 94%, or at least about 95% sequence identity. In one embodiment, the composition has Formula III:

[0015] [ka]

[0016] where HS is a helper sequence and AT1 is a first affinity tag. where CS1 is a first cleavage sequence, CS2 is a second cleavage sequence, and XTEN is AT2 is the extended recombinant polypeptide, and AT3 is the second affinity tag. In this embodiment, the first affinity tag comprises the sequence RPRPRPRPRPRPRP and the second The second affinity tag comprises the sequence HHHHHH. One affinity tag comprises the sequence HHHHHH and the second affinity tag comprises the sequence RPRPRPR In another embodiment of the above composition, the first affinity tag comprises the sequence RPRPRPRPRPRPRPRPRPRPRPRPR, and the second affinity tag comprises the sequence H Including HHHHHHH.

[0017] In another aspect, the present invention provides a method for producing a substantially homogeneous polypeptide obtained by the process. In some embodiments, the composition comprises a population of polypeptides. host cells containing a vector encoding the and culturing the cells under conditions effective to express the polypeptide, wherein the encoded polypeptide is and culturing the polypeptide, wherein the polypeptide comprises XTEN, a first cleavage sequence, and a first affinity tag. and then chromatographically separating the crude expression product polypeptides using a first affinity chromatography method. and adsorbing the functional tag onto the first chromatographic substrate under conditions effective to capture the functional tag onto the first chromatographic substrate. and recovering the polypeptide. In some embodiments, at least one of the polypeptides in the resulting population Both have 90%, 91%, 92%, 93%, 94%, or 95% identical sequence length. In one embodiment of the foregoing composition, the first chromatographic substrate is HIC, cationic The method of the present invention is selected from the group consisting of ion exchange, anion exchange, and IMAC. In an embodiment, the affinity tag is selected from the group consisting of the affinity tags in Table 7. In another embodiment of the composition, the first chromatographic substrate is a cation exchange In another embodiment of the above composition, the first affinity tag comprises the sequence RPRPRPRPRPRPRP. In an embodiment, the first chromatographic substrate is IMAC and the first affinity tag comprises the sequence HHHHHHHH. In one embodiment of the aforementioned composition, the coding vector comprises at least an affinity tag, at least a first cleavage sequence, a helper sequence, and optionally a second and encoding any of the XTEN embodiments described herein, including In another embodiment of the above composition, the vector comprises a second cleavage sequence and a second affinity sequence. The first and second cleavage sequences further encode a sex tag, and the first and second cleavage sequences are cleaved by the same protease. The second affinity tag can be a second chromatographic tag that is different from the first affinity tag. and the composition binds the polypeptide to a second chromatographic substrate. a second affinity tag is captured onto a second chromatographic substrate; adsorbing the polypeptide under conditions effective to elute the polypeptide; and and recovering at least one of the polypeptides of the population. At least 90%, 91%, 92%, 93%, 94%, or 95% of the sequences have the same sequence length. In one embodiment of the invention, the first chromatographic substrate is Unlike the substrate, each of the first and second chromatographic substrates independently comprises H The aforementioned is selected from the group consisting of IC, cation exchange, anion exchange, and IMAC. In another embodiment of the composition, the first chromatographic substrate is a cation exchange , the first affinity tag has the sequence RPRPRPRPRPRPR or RPRPRPRPRP PRPRPRPRPRPR, and the second chromatographic substrate is IMAC; The first affinity tag comprises the sequence HHHHHHHH or HHHHHHHH. In another embodiment of the product, the first chromatographic substrate is IMAC and the first The affinity tag comprises the sequence HHHHHHHH or HHHHHHHH and is The affinity substrate is a cation exchange and the first affinity tag has the sequence RPRPRPRPRP In another embodiment, the RPR or RPRRPRPRPRPRPRPRPRPRPRPRPPR. In this case, the above-mentioned composition comprising the first affinity tag or the first and second affinity tags is treating the composition with a protease under conditions effective to cleave the cleavage sequence(s); This releases the XTEN from the affinity tag(s) and allows the XTEN to be chromatographed. On the lithography substrate, the affinity tag(s) are effective in capturing XTEN. Alternatively, adsorption and elution of XTEN can be performed under conditions that are not favorable for proteases. and recovering the XTEN in the resulting composition. At least 90%, 91%, 92%, 93%, 94%, or 95% of the individual molecules of EN have the same sequence length. In one embodiment of the above composition, the cleavage sequence(s) are Can be cleaved by a protease selected from the group consisting of the proteases in Table 9 In another embodiment of the aforementioned composition, the cleavage sequence(s) can be cleaved by trypsin. The protease can be trypsin. In some embodiments, the chromatographic substrate is anion exchange. macrocap Q, capto Q, superQ-650M, and poros Alternatively, the substrate may be selected from the group consisting of one affinity tag or two The aforementioned composition comprising an affinity tag of the present invention is effective to cleave the cleavage sequence(s). and treating the XTEN under suitable conditions, thereby releasing the XTEN from one or two affinity tags. and dissolving the protease on a chromatography substrate, the protease and the affinity tag. adsorption under conditions that are effective for capturing , but not XTEN; and recovering the XTEN from the eluate. At least 90%, 91%, 92% of the individual molecules of XTEN in the resulting eluate , 93%, 94%, or 95% have identical sequence lengths. wherein the cleavage sequence(s) is / are selected from the group consisting of the proteases in Table 9. In another embodiment of the above composition, the cleavage sequence The protease(s) can be cleaved by trypsin, and the protease utilized is Trypsin. The chromatographic substrate is cation exchange, HIC, or IMAC. You can choose from one or more of the following:

[0018] In another embodiment, the present invention provides XTEN segments of partially equal length and sequence. In one embodiment, the present invention relates to a polypeptide composition that can be cleaved into XTEN. The present invention provides a composition comprising an XTEN sequence, the XTEN sequence being capable of being cleaved by trypsin. and further comprising one or more cleavage sequences capable of cleaving the nucleotide sequence under conditions effective to cleave all of the cleavage sequences. Treatment with trypsin results in the preparation of XTEN fragments, with each XTEN fragment The fragment has at least about 99% sequence identity to every other fragment in the preparation. In one embodiment of the composition, the cleavage sequence has the sequence SASRSA or SA having at least 86% sequence identity to or identical to SKSA. In another embodiment, the cleavage sequence comprises the sequence RX or KX, where X is a proline. In one embodiment of the aforementioned composition, the XTEN composition is any L-amino acid other than ribonucleotides. is at least about 90%, 91% or more for a sequence selected from the group of sequences listed in Table 6 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% have sequence identity of

[0019] In another embodiment, the present invention provides, in part, XTEN fragments of substantially equal length and sequence. In one embodiment, the present invention relates to a method for producing XTEN fragments. A method for producing a substantially homogenous population is provided, the method comprising: A population of polypeptides comprising sequences selected from the group consisting of: Treating with trypsin under conditions effective to cleave all of the cleavage sequence(s). At least 90%, 91%, 92%, 93% of the individual molecules of the XTEN fragment , 94%, or 95% have the same sequence length. The method involves distributing the XTEN fragments onto a chromatographic substrate. Adsorption occurs under conditions that are effective for capturing catalysing agents but not for proteases. eluting the XTEN fragments; and recovering the XTEN fragments. and, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 113%, 114%, 115%, 4%, or 95% have identical sequence lengths. The chromatographic substrate is anion exchange. Q, superQ-650M, and poros D. In another embodiment of the above method, the XTEN is selected from the group of sequences set forth in Table 6. At least about 90%, 91%, 92%, 93%, 94%, 95% for the selected sequence , 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the resulting XTEN fragments may be selected from the group of sequences listed in Tables 2 or 3. At least about 90%, 91%, 92%, 93%, 94%, 95%, or %, 96%, 97%, 98%, or 99% sequence identity. Thus, the present invention provides XTEN compositions produced by the processes of the aforementioned method embodiments. do.

[0020] In another embodiment, the present invention provides, in part, a method for producing XTEN from host cells in high expression yields. In some embodiments, the present invention relates to a method for producing a gene encoding an XTEN sequence and a helicase. A host cell containing a vector encoding a polypeptide containing a loop sequence is subjected to a fermentation reaction. and producing about 2 grams of the polypeptide under conditions effective to express the polypeptide as a component of a crude expression product. / liter (g / L), or about 3 g / L, or about 4 g / L, or about 5 g / L, or The method includes culturing the culture medium at a polypeptide concentration of greater than about 6 g / L or greater than about 7 g / L. In one embodiment of the method, the expression yield is determined by the fermentation reaction. At a wavelength of 0 nm, the In another embodiment, the present invention provides a method for producing a composition comprising: A host cell containing a vector encoding a polypeptide containing a helper sequence is subjected to a fermentation reaction. in a culture medium containing a 100% ethanol-containing ... a dry weight host cell concentration of greater than 1000 mg / gram (mg / g), or at least about 15 mg / g, or at least about 20 mg / g, or at least about 25 mg / g, or at least about 30 mg / g, or at least about 40 mg / g, or less and culturing the strain at a concentration of about 50 mg / g of polypeptide. In one embodiment of the method, the high yield expression is achieved by the fermentation reaction at a wavelength of 600 nm. Reach an optical density of at least 100, or at least 130, or at least 150 In another embodiment, the present invention provides a method for producing a gene encoding an XTEN sequence and a helper sequence. and a host cell containing a vector encoding a polypeptide comprising the sequence of about 10 milligrams / under conditions effective to express the polypeptide as a component of the expression product A dry weight host cell concentration of greater than gram (mg / g), or at least about 250 micrograms mol / L, or about 300 micromol / L, or about 350 micromol / L, or about 400 micromol / L, or about 450 micromol / L, or about 50 The method further comprises culturing the strain at a concentration of 0 micromol / L of the polypeptide. In one embodiment of the method, the expression yield is determined by the fact that the fermentation reaction produces at least reach an optical density of at least 100, or at least 130, or at least 150 In one embodiment of the above method, the expressed polypeptide helper The helper sequence is present at the N-terminus of the polypeptide, and this helper sequence is one of the sequences listed in Table 10. at least about 90%, 91%, 92%, 93% , 94%, or 95% sequence identity or are identical. In an embodiment, the expression vector comprises a first affinity tag and a nucleotide sequence that binds the affinity tag to an XTE. N, and the method further comprises: and recovering the crude expression product polypeptide on a first chromatographic substrate. and a first affinity tag of the polypeptide effective to capture the first affinity tag onto a chromatographic substrate. and adsorbing the first chromatographic substrate under conditions suitable for HIC, cationic adsorbing the cations selected from the group consisting of ion exchange, anion exchange, and IMAC; and recovering the polypeptide, wherein at least 90% of the polypeptide is recovered. 0%, 91%, 92%, 93%, 94%, or 95% have the same sequence length. In another embodiment of the method, the expression vector comprises a first affinity tag and a a second affinity tag different from the first affinity tag, and a cleavage sequence between each affinity tag and the XTEN. The method further comprises recovering a crude expression product from a host cell fermentation reaction mixture; a peptide on a first chromatography substrate by coupling the peptide to a first affinity tag of the peptide. adsorbing under conditions effective to capture onto a chromatographic substrate The first chromatographic substrate is selected from HIC, cation exchange, anion exchange, and IMA. C. The peptide is then chromatographed onto a second chromatographic substrate, along with a second affinity tag for the polypeptide. and adsorbing the antibody under conditions effective to capture the antibody on the chromatographic substrate. , the second chromatographic substrate is HIC, cation exchange, anion exchange, and IM and eluting the polypeptide, and and recovering the polypeptide, wherein at least 90%, 91% or more of the polypeptide is recovered. , 92%, 93%, 94%, or 95% have the same sequence length. In one embodiment, the method comprises: subjecting the polypeptide to a cleavage sequence(s) effective to cleave the cleavage sequence(s). and treating the polypeptide with a protease under conditions that release the XTEN from the polypeptide. and XTEN was deposited onto an anionic chromatography substrate, which is effective for capturing XTEN. adsorbing under effective conditions, eluting the XTEN, and recovering the XTEN. and wherein at least 90%, or at least 91%, or or at least 92%, or at least 93%, or at least 94%, or 95% % have the same sequence length. From the group consisting of p Q, capto Q, superQ-650M, and poros D In one embodiment of the foregoing method, the cleavage sequence can be selected from trypsin The protease can be trypsin. In this embodiment, the method comprises cleaving the polypeptide at the cleavage sequence(s). and treating the polypeptide with a protease under conditions effective to release the XTEN from the polypeptide. and chromatographically separating the protease and the parent substance from the protease on a chromatographic substrate. Adsorption under conditions that are effective for capturing affinity tags but not for XTEN and recovering the XTEN in the eluate, 0%, 91%, 92%, 93%, 94%, or 95% have the same sequence length. In one embodiment of the method, the cleavage sequence is capable of being cleaved by trypsin and is useful. The protease used is trypsin. In the aforementioned method for the chromatographic substrate, the chromatographic substrate may be HIC, cation exchange, and You can choose from one or more of the IMACs.

[0021] In another embodiment, the present invention provides, in part, a method for producing a composition comprising substantially identical XTEN polymers immobilized on a substrate. In one embodiment, the present invention relates to a solid support comprising a population of polypeptide molecules. providing a solid support containing a population of immobilized substantially identical polypeptide molecules, The solid support comprises a chromatographic substrate and an immobilized polypeptide (XTEN and , a first affinity tag, and a second affinity tag), wherein the first affinity tag comprises: The XTEN is joined to the XTEN by a cleavage sequence at the N-terminus of the XTEN, and the second affinity tag is joined to the C-terminus and the second affinity tag is joined to the XTEN by a cleavage sequence at Unlike the conventional method, the chromatographic substrate may be selectively bound to either the first or second affinity tag, but not both. At least 90%, 91%, or more of the immobilized polypeptide molecules can bind to either the %, 92%, 93%, 94%, or 95% have the same sequence length. 4, and the sequences listed in Tables 22 to 25. at least about 90%, or at least about 91%, or at least about 92%, or at least 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 In one embodiment of XTEN comprising sequences with 99% or 100% sequence identity , the first and second affinity tags each independently consist of a sequence set forth in Table 7 at least about 90%, 91%, 92%, 93%, 94%, for a sequence selected from the group; or at least about 95% sequence identity, and the truncation sequences are those set forth in Tables 8 and 9. In one embodiment of the foregoing, the cleavage sequence is selected from the group consisting of the sequence SAS has at least about 86% sequence identity to RSA or SASKSA, or In one of the foregoing embodiments, the cleavage sequence comprises the sequence RX or KX, wherein , X is any L-amino acid other than proline. The body is HIC chromatography resin, cation exchange resin, anion exchange chromatograph The aforementioned one is selected from the group consisting of a cellulose acetate resin, a cellulose acetate resin, and an IMAC chromatography resin. In an embodiment, the first affinity tag has the sequence RPRPRPRPRPRPPR or RPR and the second affinity tag comprises the sequence HHHH In another embodiment of the foregoing, the first affinity tag comprises , the sequence HHHHHH or HHHHHHHH, and the second affinity tag comprises the sequence RPRP Contains RPRPRPRPR or RPRPRPRPRPRPRPRPRPRPRPRPPR.

[0022] In another aspect, the present invention relates, in part, to compositions of XTEN conjugated to a crosslinker. In some embodiments, the present invention provides a method for preparing a crosslinker comprising the steps of: and a cross-linking agent. The crosslinkers listed in Table 13, the alkyne reactants listed in Table 15, and the In one embodiment of the conjugate composition, the azide reactant is selected from the group consisting of: The crosslinker 1 is a crosslinker that binds to the α-amino group of the N-terminal amino acid residue of XTEN and each lysine residue of XTEN. the epsilon amino group, and the thiol group of each cysteine ​​residue of XTEN. Wherein the XTEN is conjugated to at least a first XTEN, if desired, this embodiment The XTEN in At least about 90%, or at least about 91%, or at least about 92%, or less at least about 93%, or at least about 94%, or at least about 95%, or at least at least about 96%, or at least about 97%, or at least about 98%, or less Both have about 99%, or 100% sequence identity. XTEN is AE144, AE288, AE432, AE576, AE864, Seg Segment 174, Segment 175, Segment 176, Segment 177, Segment 1 86, segment 187, segment 188, segment 189, segment 190, Segment 191, Segment 192, Segment 193, Segment 194, Segment 195, segment 196, segment 197, segment 198, and segment 1 99, wherein the crosslinker is conjugated to the alpha amino group of the N-terminal amino acid of XTEN. In another embodiment of the conjugate composition, the XTEN comprises a segment described in Table 3. 174, segment 175, segment 176, segment 177, segment 186, Segment 187, Segment 188, Segment 189, Segment 190, Segment Segment 191, Segment 192, Segment 193, Segment 194, Segment 195 , segment 196, segment 197, segment 198, and segment 199 and the cross-linker is conjugated to a thiol group of each cysteine ​​residue of the XTEN. In another embodiment of the conjugate composition, the first crosslinker is N-maleimide, iodoacetate. Pyridyl disulfide and vinyl sulfone, 3-propargyloxypropanoic acid , (oxyethyl) n -acetylene (n is 1-10), dibenzylcyclooctyne (DB CO), cyclooctyne (COT), 3-azido-propionic acid, 6-azido-hexane Acid, and (oxyethyl) n -azide (n is 1 to 10). In the preceding embodiment of the paragraph, the conjugate has formula IV:

[0023] [ka]

[0024] wherein, independently for each occurrence, CL1 is a crosslinker and x is from 1 to about 100, or 1 to about 50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or an integer from 1 to about 5, or 9, or 3, or 2 or 1. If desired, the XTEN in this embodiment can be any of the XTENs listed in Tables 2 and 3. at least about 80%, or at least or 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 9 In one embodiment of the conjugate of Formula IV, the conjugate comprises a sequence having 9%, 100%, or 100% sequence identity. wherein CL1 is a crosslinker selected from Table 13. In other embodiments, the composition comprises a single first payload conjugated to each first crosslinker. The compound further comprises an atomic residue, the residue being selected from the group consisting of carbon, nitrogen, oxygen, and sulfur. In one embodiment of the above, the first payload of a single atom residue is selected from the group consisting of those listed in Tables 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 40, 41, 42, 43 8, and 21. In another embodiment of the crosslinker-conjugated composition, the composition comprises a first crosslinker-conjugated 11 and 12. nothing.

[0025] In other embodiments of the XTEN-crosslinker conjugate composition, the present invention provides a method for preparing a first crosslinker comprising administering to a subject a first crosslinker comprising administering to a subject a first crosslinker comprising administering to a subject a second crosslinker comprising administering to a subject a second crosslinker comprising administering to a subject a second crosslinker comprising administering to a subject a third ... fourth cross a cross-linking agent as described herein covalently attached to one or more of the above molecules and a second cross-linking agent; An embodiment of a composition of XTEN is provided, wherein the first crosslinker is a crosslinker of each cysteine ​​residue of the XTEN. conjugated to either the thiol group or the epsilon amino group of each lysine residue of XTEN, and a second The crosslinkers are conjugated to the alpha amino group of the N-terminal amino acid of XTEN, and each crosslinker is independently From the crosslinker listed in Table 13, the alkyne reactant in Table 15, and the azide reactant in Table 15 In the foregoing embodiment, the composition is selected from the group consisting of Formula V:

[0026] [ka]

[0027] wherein, independently, for each occurrence, CL1 is linked to a cysteine ​​residue of XTEN. CL1 is a first crosslinker conjugated to XTEN at the N-terminus, and CL2 is a second crosslinker conjugated to XTEN at the N-terminus. crosslinking agent, where x is an integer from 1 to about 10, and y is the integer 1, provided that x+y > 2, wherein the XTEN is a cysteine ​​engineered XTEN containing x cysteine ​​residues. or a lysine-engineered XTEN containing x lysine residues. In another embodiment of the crosslinker-conjugated composition, the composition comprises a first crosslinker conjugated to each of the first crosslinkers. a single atom residue of the first payload, consisting of carbon, nitrogen, oxygen, and sulfur; and a single residue of the second payload conjugated to each of the second crosslinkers. a one-atom residue selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; In one embodiment of the foregoing, the first payload of single atom residues is selected from the group consisting of: , 12, 18, and 21, The second payload of single atom residues is independently listed in Tables 11, 12, 18, and 21. The payload can be selected from the group consisting of: XTEN-crosslinker-payload In some embodiments of the residue composition, the composition has Formula VI:

[0028] [ka]

[0029] wherein, independently for each occurrence, P R1 is a single atom residue of the payload wherein the residue is selected from the group consisting of carbon, nitrogen, oxygen, and sulfur, and CL1 is a crosslinking agent, wherein x is 1 to about 100, or 1 to about 50, or 1 to about 40, or 1 to about 2 is 0, or an integer of 1 to about 10, or an integer of 1 to about 5, or is 3, or is 2 or 1. If desired, the XTEN in this embodiment may be any of the XTENs listed in Table 2 and Table 3. 3. At least about 80% of the sequences selected from the group of sequences set forth in at least about 90%, or at least about 91%, or at least about 92%, or less at least about 93%, or at least about 94%, or at least about 95%, or at least or at least about 96%, or at least about 97%, or at least about 98%, or at least Containing sequences with about 99%, or 100% sequence identity. In this state, the single atom residue of the payload is as described in Tables 11, 12, 18, 19, and 21. The conjugate of Formula VI is derived from a payload selected from the group consisting of payloads carried on In one embodiment, CL1 is a cross-linker selected from Table 13. In one embodiment, each crosslinker is attached to a cysteine ​​sulfur of XTEN. In another embodiment of the complex, each crosslinker is attached to a lysine epsilon amino group of XTEN. In another embodiment of the conjugate of Formula VI, x is 1 and the crosslinker is the N-terminal amino acid of XTEN. In another embodiment of the conjugate of Formula VI, CL1 is selected from Table 15. In another embodiment, the reaction product of the first and second click chemistry reactants is The present invention provides a preparation of a conjugate of Formula VI, wherein the XTEN molecules of the preparation of the conjugate At least about 80%, or at least about 90%, or at least about 91%, or less at least about 92%, or at least about 93%, or at least about 94%, or less In another embodiment of the XTEN-crosslinker conjugate composition, about 95% of the XTEN-crosslinker conjugates have the same sequence length. wherein the composition comprises a first payload conjugated to each of the first crosslinkers, a payload selected from the group consisting of payloads described in 1, 12, 18, and 21; and a second payload different from the first payload conjugated to a second crosslinker. , a payload selected from the group consisting of those listed in Tables 11, 12, 18, and 21. and a payload of 2. One embodiment of the XTEN-crosslinker-payload conjugate composition In one embodiment, the composition comprises a first payload conjugated to each of the first crosslinkers, , a payload selected from the group consisting of the drug moieties in Table 21, and conjugated to a second crosslinker. a second payload different from the first payload, the second payload being selected from the group consisting of targeting moieties in Table 21; and a second payload selected from the XTEN having the first and second payloads. In one embodiment of the crosslinker-payload conjugate composition, the single second payload is by reaction of an alkyne reactant selected from the group consisting of 15 reactants with an azide reactant. A conjugated second crosslinker is attached to the N-terminus of XTEN. In some embodiments of the payload composition, the composition has Formula VII:

[0030] [ka]

[0031] wherein, independently for each occurrence, P1 is selected from the group consisting of Tables 11, 12, 18, 19, and and 21, wherein CL1 is a payload selected from the group consisting of the payloads described in , a crosslinking agent, and x is 1 to about 100, or 1 to about 50, or 1 to about 40, or 1 is an integer between 1 and about 20, or between 1 and about 10, or between 1 and about 5, or is 9, or is 3, or 2, or 1, and XTEN is a nucleotide sequence listed in Tables 2 and 3. at least about 80%, or at least about 90% to a sequence selected from the group of sequences %, 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%, or or a sequence having 100% sequence identity. In one embodiment of the conjugate of Formula VII and CL1 is a cross-linker selected from Table 13. In one embodiment of the conjugate of Formula VII Thus, each crosslinker is attached to a cysteine ​​sulfur of XTEN. In the form, each crosslinker is attached to a lysine epsilon amino group of XTEN. In another embodiment, x is 1 and the crosslinker is attached to the N-terminal amino group of XTEN. In one embodiment, the conjugate of formula VII consists of a conjugate set forth in Table 21. In another embodiment of the conjugate of Formula VII, CL1 is selected from the group: The reaction product of the first and second click chemistry reactants is selected from the group consisting of: The composition of the foregoing embodiment comprising a payload conjugated to an XTEN-crosslinker is It will be understood by those skilled in the art that the formula represents the reaction product and therefore may differ from the exact composition of the reactants. In another embodiment, the present invention provides the preparation of a conjugate of formula VII: , at least about 80%, or at least about 90% of the XTEN molecules in the preparation of this conjugate , 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% have the same sequence length.

[0032] In another aspect, the present invention provides, in part, a first and second XTEN conjugated to each other. In some embodiments, the conjugate composition comprises a first and a second XT The XTEN may be the same or different, each independently as set forth in Table 3. at least about 90%, or at least about 90% for a sequence selected from the group of sequences 1%, 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%, or 100% sequence identity and the first and second XTENs are bounded by the N-terminus of the first and second XTENs. by reacting an alkyne reactant selected from the group consisting of the reactants in Table 15 with an azide reactant. The XTEN conjugates are then conjugated to one another using a crosslinker formed therefrom, resulting in a dimeric XTEN conjugate. In one embodiment of the XTEN composition, at least one of each individual molecule of the first XTEN have at least 90%, 91%, 92%, 93%, 94%, or 95% identical sequence length; at least 90%, 91%, 92%, 93% of each individual molecule of the second XTEN; 94%, or 95% have identical sequence lengths. In this case, the first XTEN is segment 174, segment 175, and segment 176 as shown in Table 3. Segment 176, Segment 177, Segment 186, Segment 187, Segment 1 88, segment 189, segment 190, segment 191, segment 192, Segment 193, Segment 194, Segment 195, Segment 196, Segment A sequence selected from the group consisting of segments 197, 198, and 199. At least about 90%, or at least about 91%, or at least about 92% for the column , 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 or at least about 99%, or 100% sequence identity with the second XTEN, Segment 174, segment 175, segment 176, and segment 17 7, Segment 186, Segment 187, Segment 188, Segment 189, Seg Segment 190, Segment 191, Segment 192, Segment 193, Segment 1 94, segment 195, segment 196, segment 197, segment 198, and and segment 199. 90%, or at least about 91%, or at least about 92%, or at least about 9 3%, 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% or 100% sequence identity. The first XTEN and the second XTEN are the same and each are listed in Table 3. at least about 90%, or at least about 91%, to a sequence selected from the group of sequences, or at least about 92%, or at least about 93%, or at least about 94%, or or at least about 95%, or at least about 96%, or at least about 97%, or have at least about 98%, or at least about 99%, or 100% sequence identity In another embodiment of the dimeric XTEN conjugate, the first XTEN and the second XTEN N is the same, and each of the segments 174 and 175 is listed in Table 3. , segment 176, segment 177, segment 186, segment 187, segment Segment 188, Segment 189, Segment 190, Segment 191, Segment 19 2, Segment 193, Segment 194, Segment 195, Segment 196, Seg Segment 197, Segment 198, and Segment 199. 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 9 5%, or at least about 96%, or at least about 97%, or at least about 98% %, or at least about 99%, or 100% sequence identity. In another embodiment of the conjugate, the first and second XTEN each comprise one or more synaptones. a first cross-linker comprising a cysteine ​​residue and conjugated to each cysteine ​​residue of the first XTEN; a second cross-linker conjugated to each cysteine ​​residue of the second XTEN, and the second crosslinker are independently selected from the group consisting of the crosslinkers set forth in Table 13. In another embodiment of the dimeric XTEN conjugate, the first and second XTEN are each , which contains one or more lysine residues, and which is covalently linked to each lysine residue of the first and second XTEN of the conjugate. The crosslinking agent may be selected from the group consisting of the crosslinkers listed in Table 13. In another embodiment of the dimeric XTEN conjugated to a crosslinker, the conjugate comprises a first and further comprising a single atom residue of a first payload conjugated to each crosslinker of one XTEN, is selected from the group consisting of carbon, nitrogen, oxygen, and sulfur, and the second XTE and further comprising a single atom residue of a second payload conjugated to each crosslinker of N, said residue comprising: In the foregoing embodiment, the single atom is selected from the group consisting of carbon, nitrogen, oxygen, and sulfur. The first payload of one atom residue is a payload listed in Tables 11, 12, 18, and 21. and the second payload of a single atom residue can be selected from the group consisting of the first payload. It is a different payload from the payloads listed in Tables 11, 12, 18, and 21. The dimer XTEN-crosslinker-payload residue can be selected from the group consisting of: In some embodiments of the base composition, the composition comprises a compound of formula X

[0033] [ka]

[0034] wherein, independently for each occurrence, P R1 is a single atom of the first payload residue, the residue being selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; P R 2 is a single atom residue of the second payload, which residue is a carbon, nitrogen, oxygen, and sulfur, CL1 is a crosslinking agent, and x is 1 to about 100, or 1 to about 50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to about 5 Is it an integer, or is it 9, or is it 3, or is it 2, or is it 1? CL2 is a cross-linking agent different from CL1, and y is 1 to about 100, or 1 to about 50. or an integer of 1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to about 5. or 9, or 3, or 2, or 1, but and x+y> 2, where 2xCL is, alternatively, a bivalent crosslinker or a crosslinker selected from Table 15 XTEN1 is the reaction product of the first and second click chemistry reactions described in Table 2 and 3 and 4, or at least 80% of the sequence selected from the group of sequences set forth in at least about 90%, or at least about 91%, or at least about 92%, or at least 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% or 100% sequence identity with the polypeptides listed in Table 2. and 3, wherein the sequence is at least 80%, or at least At least about 90%, or at least about 91%, or at least about 92%, or at least at least about 93%, or at least about 94%, or at least about 95%, or less at least about 96%, or at least about 97%, or at least about 98%, or at least A conjugate of formula X is a polypeptide having about 99%, or 100%, sequence identity. In one embodiment, CL1 and CL2 are each a group of crosslinkers listed in Table 13. In another embodiment of the conjugate of formula X, x is 1 and CL1 is selected from XT In another embodiment of the conjugate of formula X, CL1 is 15 is a reaction product of a first and a second click chemistry reactant selected from Table 15. In another embodiment of the invention, C2 is a first azide and a second azide selected from Table 15. In another embodiment of the conjugate of formula X, Each CL1 binds to a cysteine ​​sulfur of XTEN1, and each CL2 binds to a cysteine ​​sulfur of XTEN2. In another embodiment of the conjugate of Formula X, each CL is bonded to an XTEN1 sulfur. and each CL2 is bound to the lysine epsilon amino group of XTEN2. In another embodiment of the conjugate of Formula X, each CL1 is linked to a cysteine ​​sulfur of XTEN1. Each CL2 is linked to a lysine epsilon amino group of XTEN2. In this embodiment, XTEN1 and XTEN2 are the same. In one embodiment, XTEN1 and XTEN2 are different. The present invention provides a preparation of a conjugate of formula X, wherein at least one of the XTEN molecules of the preparation of the conjugate is at least about 80%, or at least about 90%, or at least about 91%, or less at least about 92%, or at least about 93%, or at least about 94%, or at least Approximately 95% of the dimeric XTENs have the same sequence length. In some embodiments, the composition further comprises a first payload conjugated to each crosslinker of the first XTEN. This first payload contains the payloads listed in Tables 11, 12, 18, and 21. and a second payload selected from the group consisting of: This second payload is conjugated to each crosslinker of the second XTEN, and the second payload is is selected from the group consisting of the payloads listed in Tables 11, 12, 18, and 21 In another embodiment of dimeric XTEN conjugated to a crosslinker, the composition comprises a first XTEN N, further comprising a first payload conjugated to each crosslinker of N, wherein the first payload is selected from the group consisting of or a targeting moiety selected from the group consisting of those listed in Table 21 and different from the first payload and a second payload comprising a crosslinker of the second XTEN. and the second payload is selected from the group of toxins listed in Table 18 or Table 21. The dimeric XTEN conjugated to a crosslinker and the first and second payloads are In an embodiment, the first XTEN is segment 176 described in Table 3, and the second XTEN is segment 176 described in Table 3. The XTEN is selected from the group consisting of segment 176 and segment 177 as described in Table 3. In some embodiments of the dimeric XTEN-crosslinker-payload composition, The composition is of formula XI

[0035] [ka]

[0036] wherein, independently for each occurrence, P1 is selected from the group consisting of Tables 11, 12, 18, 19, and and 21, and P2 is a first payload selected from the group of payloads listed in selected from the group of payloads set forth in Tables 11, 12, 18, 19, and 21; P1 CL1 is a crosslinker, and x is from 1 to about 100, or or 1 to about 50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to about is an integer of about 5, or 9, or 3, or 2, or 1, CL2 is a crosslinker different from CL1, and y is 1 to about 100, or 1 to about 100. An integer of about 50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or 1 to about 5 Is it a number, or is it a 9, or is it a 3, or is it a 2, or is it a 1? However, x+y > 2, where 2xCL is, alternatively, a bivalent crosslinker, or and XTEN1 is the reaction product of a first and second click chemistry reactant selected from: at least 80% to a sequence selected from the group of sequences listed in Tables 2 and 3, and is 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 less at least about 96%, or at least about 97%, or at least about 98%, or at least a first substantially homogeneous XTEN having at least about 99%, or 100%, sequence identity with the XTEN; and XTEN2 has a low affinity to a sequence selected from the group of sequences listed in Tables 2 and 3. At least 80%, or at least about 90%, or at least about 91%, or less at least about 92%, or at least about 93%, or at least about 94%, or at least or at least about 95%, or at least about 96%, or at least about 97%, or at least A first substance having about 98%, or at least about 99%, or 100% sequence identity In one embodiment of the conjugate of Formula XI, CL1 and CL2 are each selected from the group of crosslinkers set forth in Table 13. Another embodiment of the conjugate of Formula XI is In an embodiment, x is 1 and CL1 is attached to the N-terminal amino group of XTEN. In another embodiment of the conjugate of XI, CL1 is selected from the first and second groups selected from Table 15. In another embodiment of the conjugate of formula XI, C2 is the reaction product of a first and second click chemistry reactant selected from Table 15. In another embodiment of the conjugate of XTEN1, each CL1 is attached to a cysteine ​​sulfur of XTEN1. and each CL2 is attached to a cysteine ​​sulfur of XTEN2. In the form, each CL1 is linked to a lysine epsilon amino group of XTEN1, and each CL2 is linked to a lysine epsilon amino group of XTEN1. In another embodiment of the conjugate of formula XI, each CL1 is attached to the cysteine ​​sulfur of XTEN1, and each CL2 is attached to the lysine ε of XTEN2. In another embodiment of the conjugate of Formula XI, XTEN1 and XT In another embodiment of the conjugate of formula XI, XTEN1 and XTEN2 are identical. EN2 is different. In one embodiment, the conjugate of formula XI is a conjugate described in Table 21. In another embodiment, the present invention provides a method for preparing a conjugate of formula XI and providing at least one XTEN1 and XTEN2 molecule of each of the conjugate preparations. About 80%, or 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 9 5% have the same sequence length.

[0037] In another aspect, the present invention provides a method for producing a trimeric conjugate composition comprising: In some embodiments, the composition comprises a first, second, and third XTEN. wherein the conjugate composition comprises a first, second, and third XTEN, the XTENs being the same. The first, second, and third XTENs may be the same or different, and the first, second, and third XTENs may be the same or different, and the first, second, and third XTENs may be the same or different, and the first, second, and third XTENs may be the same or different, and the second ... 4. The crosslinking agent is selected from the group consisting of the trivalent crosslinkers described in In one embodiment of the trimeric conjugate, the first, second and third The XTENs may be identical or different, each corresponding to either Table 2 or Table 3. At least about 90% to a sequence selected from the group of sequences described, 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 9 7%, or at least about 98%, or at least about 99%, or 100% sequence identity In another embodiment of the trimeric conjugate, the first, second and third XT The XTENs may be identical or different and may be at least one of each individual molecule of the first XTEN. 90%, 91%, 92%, 93%, 94%, or 95% of the sequence length of each of the At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 113%, 114%, 115%, 11 4%, or 95%, have the same sequence length, and the minority of each individual molecule of the third XTEN At least 90%, 91%, 92%, 93%, 94%, or 95% of the sequences have the same sequence length. In another embodiment of the trimeric conjugate, the trivalent crosslinker is tris-(2-maleimido)ethoxysilane. Tris-(succinimidyl)amine (TMEA) and amine-reactive tris-(succinimidylaminotrimethyl)amine In another embodiment of the trimeric conjugate, The first, second, and third XTENs are identical and are each listed in Table 3. Segment 174, Segment 175, Segment 176, Segment 177, Segment Segment 186, Segment 187, Segment 188, Segment 189, Segment 190 , segment 191, segment 192, segment 193, segment 194, segment segment 195, segment 196, segment 197, segment 198, and segment at least about 90% to a sequence selected from the group consisting of 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 9 7%, or at least about 98%, or at least about 99%, or 100% sequence identity In another embodiment of the trimeric conjugate, the first, second and third XT EN are identical and each have segment 174, segment 17, as described in Table 3. 5, Segment 176, Segment 177, Segment 186, Segment 187, Seg Segment 188, Segment 189, Segment 190, Segment 191, Segment 1 92, segment 193, segment 194, segment 195, segment 196, segment 197, segment 198, and segment 199. At least about 90%, or at least about 91%, or at least about 92% of the sequence %, 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%, or 100% sequence identity, and the third XTEN , unlike the first and second XTENs, segment 174, segment 174, as described in Table 3 Segment 175, Segment 176, Segment 177, Segment 186, Segment 187 , segment 188, segment 189, segment 190, segment 191, segment Segment 192, Segment 193, Segment 194, Segment 195, Segment 19 6, segment 197, segment 198, and segment 199. 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 9 8%, or at least about 99%, or 100% sequence identity. In another embodiment, each XTEN comprises at least a first cysteine ​​residue and is conjugated The body comprises a first crosslinker conjugated to each cysteine ​​residue of the first XTEN and a second XTEN. a second crosslinker conjugated to each cysteine ​​residue of the XTEN; and a third crosslinker conjugated to each cysteine ​​residue of the XTEN. and a conjugated third crosslinker, which crosslinker is selected from the crosslinkers listed in Table 13. In some embodiments of the trimeric conjugate, the composition is selected from the group consisting of: II:

[0038] [ka]

[0039] wherein, independently for each occurrence, 3xCL is a trivalent crosslinker and CL1 is the first crosslinker conjugated to XTEN1, and CL2 is the second crosslinker conjugated to XTEN2. 2 crosslinker, CL3 is a third crosslinker conjugated to XTEN3, and x is 1 to y is an integer from 1 to about 10; z is an integer from 1 to about 10; , where x+y > 3, where XTEN1 is the first XTEN and XTEN2 is XTEN1 is the second XTEN, and XTEN2 is the third XTEN. In one embodiment, the conjugate comprises a first payload conjugated to each first crosslinker of a first XTEN. a single atom residue selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; and a single atom residue of a second payload conjugated to each second crosslinker of the second XTEN. a residue selected from the group consisting of carbon, nitrogen, oxygen, and sulfur, and a third XTE N, a single atom residue of a third payload conjugated to each third crosslinker, comprising carbon, nitrogen, and a residue selected from the group consisting of: , oxygen, and sulfur. In another embodiment, the composition comprises a peptide as set forth in Tables 11, 12, 18, and 21. a first peptide conjugated to each first crosslinker of a first XTEN selected from the group consisting of and a payload selected from the group consisting of those listed in Tables 11, 12, 18, and 21. a second payload conjugated to each second crosslinker of the selected second XTEN, with the same or different payload as in Tables 11, 12, 18, and 21. Each third crosslinker of the third XTEN is selected from the group consisting of the payloads described in a third payload conjugated to the first or second payload, or or a different payload. In embodiments, the first payload comprises a targeting moiety that has specific binding affinity for a target. and the targeting moiety is selected from the group consisting of the targeting moieties set forth in Tables 17-19 and 21. The second and third payloads are drugs that can be the same or different. and the drug is selected from the group consisting of drugs listed in Tables 11, 18, and 21. The first payload is a targeting moiety that has specific binding affinity for a target, and the second payload is a targeting moiety that has specific binding affinity for a target. and a trimeric XTEN-payload covalent complex, wherein the second payload and the third payload are drugs. In one embodiment of the composition, the targeting moiety is selected from the group consisting of LHRH and folate. The drugs used were doxorubicin, paclitaxel, auristatin, monomethyl auristatin, auristatin E (MMAE), monomethylauristatin F, maytansine, dolastatin, Richeamicin, vinca alkaloids, camptothecin, mitomycin C, epothilone, h TNF, I1-12, bortezomib, ranpirnase, Pseudomonas, exotoxin, SN-38, and rachelmycin. a targeting moiety having specific binding affinity for the target, and a second payload and a third payload In one embodiment of the trimeric XTEN-payload conjugate composition, the load is a drug. The target moiety and drug moiety correspond to any one of conjugates 1-290 listed in Table 21. The first payload is a targeting moiety that has specific binding affinity for a target, and the second Trimeric XTEN-payload conjugate compositions in which the payload and the third payload are drugs In another embodiment of the product, the conjugate comprises XTEN corresponding to conjugate 71 in Table 21 and a targeting and a drug moiety. wherein the composition is of formula XIII

[0040] [ka]

[0041] wherein, independently for each occurrence, 3xCL is as set forth in Tables 13 and 14. P1 is a trivalent crosslinker selected from the group of trivalent crosslinkers ... from the group consisting of payloads conjugated to the agent and set forth in Tables 11, 12, 18, and 21. P2 is a second payload conjugated to each crosslinker of the second XTEN, 11, 12, 18, and 21, P1 is the same as or different from the first payload, and P2 is the third XTEN The third payload conjugated to each of the crosslinkers is listed in Tables 11, 12, 18, and 21. a payload to be carried on the first or second payload; CL1 is the same as or different from LOAD, CL2 is a first crosslinker, and x is 1 to about 1. 00, or 1 to about 50, or 1 to about 40, or 1 to about 20, or 1 to about 10, or or an integer from 1 to about 5, or 9, or 3, or 2 , or 1, CL2 is a second crosslinker, and y is 1 to about 100, or 1 to about 50, or an integer from 1 to about 40, or from 1 to about 20, or from 1 to about 10, or from 1 to about 5 or 9, or 3, or 2, or 1, z is from 1 to about 100, or from 1 to about 50, or from 1 to about 40, or from 1 to about 20, or is an integer from 1 to about 10, or from 1 to about 5, or is 9, or is 3, or or 2, or 1, but x+y+z > Subject to 3, XTEN1 is at least 80% to a sequence selected from the group of sequences listed in Tables 2 and 3, and is 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 less at least about 96%, or at least about 97%, or at least about 98%, or at least a first XTEN having at least about 99%, or 100%, sequence identity with the XTE; N2 is at least 80 to a sequence selected from the group of sequences listed in Tables 2 and 3 %, or 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 or a second XTEN having at least about 99%, or 100%, sequence identity to the XTEN; XTEN3 has at least one sequence selected from the group of sequences set forth in Tables 2 and 3. or at least about 80%, or 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 9 5%, or at least about 96%, or at least about 97%, or at least about 98% %, or at least about 99%, or 100% sequence identity with the third XTEN. Yes, XTEN 1、 XTEN 2、 and XTEN3, which may be the same or different XT In some embodiments, the conjugate of formula XIII is a first payload. The payload is a targeting moiety that has specific binding affinity for a target. and the targeting moiety is selected from the group consisting of the targeting moieties set forth in Tables 17-19 and 21. and at least one other of the payloads is a drug, and the drug is selected from the group consisting of those listed in Tables 11, 19, and and the drugs listed in Table 21. The target moiety is LHRH or folic acid, and the drug is doxorubicin, paclitaxel, or auristatin, monomethylauristatin E (MMAE), monomethylauristatin F, Maytansine, dolastatin, calicheamicin, vinca alkaloids, camptothecin, Mitomycin C, epothilone, hTNF, I1-12, bortezomib, ranpirnase, Selected from the group consisting of Pseudomonas exotoxin, SN-38, and rachelmycin In another embodiment of the trimeric XTEN conjugate composition, the composition comprises a compound represented by Formula XIV:

[0042] [ka]

[0043] wherein, independently for each occurrence, 3xCL is a trivalent crosslinker and CL1 is the first crosslinker conjugated to XTEN1, and CL2 is the second crosslinker conjugated to XTEN2. 2, where x is an integer from 1 to about 10 and y is an integer from 1 to about 10; However, x+y > 2, where XTEN1 is the first XTEN and XTEN2 is the second XTEN. XTEN 2 is XTEN 3, and XTEN 4 is XTEN 5, which are listed in Table 2. One embodiment of the trimeric XTEN conjugate composition of Formula XVI is selected from the group consisting of: wherein the composition comprises a first payload conjugated to each first crosslinker of a first XTEN. a single atom residue selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; Each of the second XTENs was a single atom residue of a second payload conjugated to a second crosslinker. and a residue selected from the group consisting of carbon, nitrogen, oxygen, and sulfur. In another embodiment of the trimeric XTEN conjugate composition of XVI, the composition comprises the XTEN conjugates of Tables 11, 12, 18, and 21. a first payload conjugated to a first crosslinker and a and a second XTEN conjugated to each second crosslinker, wherein the second XTEN is selected from the group consisting of a payload comprising: a second payload that is the same as or different from the first payload; In one embodiment of the above, the first payload further comprises a specific a targeting moiety having a different binding affinity, the targeting moiety being one of those listed in Tables 17-19 and 21. and the second payload is selected from the group consisting of a targeting moiety ... 21. In another embodiment of the present invention, wherein the first payload is a targeting moiety selected from the group consisting of LHRH and folate. The second payload is doxorubicin, paclitaxel, auristatin, monomethicone, Luoristatin E (MMAE), monomethylauristatin F, maytansine, dras Tatin, calicheamicin, vinca alkaloids, camptothecin, mitomycin C, epo Tiron, hTNF, I1-12, bortezomib, ranpirnase, pseudomonas, exo The drug is selected from the group consisting of toxin, SN-38, and rachelmycin. In one embodiment of the above, the first payload is a drug from Table 11 and a protein from Table 12. and the second payload is a drug selected from the group consisting of: and selected from the group consisting of the drugs in Table 11 and the proteins in Table 12. In some embodiments, the first payload and the second payload are the same and are and a protein in Table 12. In another embodiment, the composition has Formula XV:

[0044] [ka]

[0045] wherein, independently for each occurrence, 3xCL is selected from XTEN1 and XTEN2 and XTEN3, and CL1 is a trivalent crosslinker that binds the first nucleotide conjugated to XTEN1. where x is an integer from 1 to about 10, and XTEN1 is a first XTEN. , XTEN is selected from the group consisting of the sequences set forth in Table 3, and XTEN2 is a second X XTEN, wherein the XTEN is selected from the group consisting of the sequences set forth in Table 2; XTEN3 is a third XTEN, wherein the XTEN is selected from the group consisting of the sequences set forth in Table 2. In one embodiment of a trimeric XTEN conjugate composition configured as Formula XVII, the composition The article comprises a first XTEN having a single atom residue of a first payload conjugated to each first crosslinker. wherein the residue is selected from the group consisting of carbon, nitrogen, oxygen, and sulfur. In one embodiment of a trimeric XTEN conjugate composition configured as XVII, the composition comprises: a first payload selected from the group consisting of those listed in Tables 11, 12, 18, and 21; and further comprising a first payload conjugated to each first crosslinker of the XTEN.

[0046] In another aspect, the present invention provides a method for producing a tetrameric conjugate composition, comprising: In some embodiments, the compositions include a first, second, third, and fourth XTEN. wherein the conjugate composition comprises first, second, third, and fourth XTENs; N is selected from the group consisting of the sequences set forth in Table 3, and XTEN can be the same or or may be different, and the first and second and third and fourth XTENs may be tetrameric crosslinkers. The tetrameric crosslinkers are conjugated to each other via their N-termini using a tetravalent maleimide cluster. In one embodiment of the tetrameric conjugate, the first and second and third and fourth X TEN may be the same or different, each listed in either Table 2 or Table 3 at least about 90%, or at least about 90% for a sequence selected from the group of sequences 1%, 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%, or 100% sequence identity In another embodiment of the tetrameric conjugate, the first, second and third XTENs have are the same or different and are present in at least one of each individual molecule of the first XTEN. 90%, 91%, 92%, 93%, 94%, or 95% have the same sequence length, and the second At least 90%, 91%, 92%, 93%, 94% of each individual molecule of XTEN or 95% have the same sequence length and at least one of each individual molecule of the third XTEN. 90%, 91%, 92%, 93%, 94%, or 95% of the sequence length of each of the At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, In another embodiment of the tetrameric conjugate, 4%, or 95% of the tetrameric conjugates have the same sequence length. The first, second, third, and fourth XTENs are the same and each are listed in Table 3. , segment 174, segment 175, segment 176, segment 177, segment Segment 186, Segment 187, Segment 188, Segment 189, Segment 19 0, segment 191, segment 192, segment 193, segment 194, segment Segment 195, Segment 196, Segment 197, Segment 198, and Segment at least about 90%, or at least about 90% to a sequence selected from the group consisting of: 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%, or 100% sequence In another embodiment of the tetramer conjugate, the first and second XTEN have the identity The segments 174, 175, and 176 are the same as those in Table 3. Segment 176, Segment 177, Segment 186, Segment 187, Segment 1 88, segment 189, segment 190, segment 191, segment 192, Segment 193, Segment 194, Segment 195, Segment 196, Segment 197, segment 198, and segment 199. at least about 90%, or at least about 91%, or at least about 92%, or is 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 less and the third and fourth XTENs have at least about 99%, or 100%, sequence identity with each other. The first and second XTENs are identical but different, each containing a sequence as set forth in Table 3. Segment 174, Segment 175, Segment 176, Segment 177, Segment 186, segment 187, segment 188, segment 189, segment 190, Segment 191, Segment 192, Segment 193, Segment 194, Segment Segment 195, Segment 196, Segment 197, Segment 198, and Segment 199, or at least about 90% 91%, or at least about 92%, or at least about 93%, or at least about 9 4%, 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%, or 100% sequence identity In another embodiment of the tetrameric conjugate, each XTEN has at least a first sequence. and the conjugate comprises a first XTEN conjugated to each cysteine ​​residue of the first XTEN. a crosslinker conjugated to each cysteine ​​residue of the second XTEN; and a second crosslinker conjugated to each cysteine ​​residue of the third XTEN. A third crosslinker conjugated to each cysteine ​​residue of the EN and a fourth crosslinker conjugated to each cysteine ​​residue of the XTEN. and a fourth crosslinker conjugated to the group, wherein each crosslinker is a crosslinker described in Table 13. In some embodiments of the tetrameric conjugate composition, the composition is selected from the group consisting of: , formula XVI

[0047] [ka]

[0048] wherein, independently for each occurrence, 4xCL is a tetravalent crosslinker and CL1 is the first crosslinker conjugated to XTEN1, and CL2 is the second crosslinker conjugated to XTEN2. 2 crosslinker, CL3 is a third crosslinker conjugated to XTEN3, and CL4 is a fourth crosslinker conjugated to XTEN4, wherein v is an integer from 1 to about 10, and x is y is an integer of 1 to about 10; z is an integer of 1 to about 10. However, x+y+z > 4, XTEN1 is the first XTEN, and XTE N2 is the second XTEN, XTEN3 is the third XTEN, and XTEN3 is In another embodiment of the tetrameric conjugate composition, the composition comprises a first XTEN and a fourth XTEN. A single atom residue of a first payload conjugated to each first crosslinker of TEN, comprising carbon, a residue selected from the group consisting of nitrogen, oxygen, and sulfur, and each second bridge of the second XTEN; A single atom residue of a second payload conjugated to a bridger, the single atom residue comprising carbon, nitrogen, oxygen, and a residue selected from the group consisting of sulfur and a third crosslinker conjugated to each third crosslinker of the third XTEN. 3 payload single atom residues selected from the group consisting of carbon, nitrogen, oxygen, and sulfur A single fourth payload conjugated to each fourth crosslinker of the fourth XTEN with the selected residue. a one-atom residue selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; In another embodiment of the tetrameric conjugate composition, the composition further comprises: 8, and 21. The first payload conjugated to the crosslinker 1 and the second payload conjugated to the crosslinker 2 are listed in Tables 11, 12, 18, and 21. and a second XTEN conjugated to each second crosslinker, wherein the second XTEN is selected from the group consisting of a payload comprising: A second payload, which may be the same as or different from the first payload. and a payload selected from the group consisting of those listed in Tables 11, 12, 18, and 21. a third payload conjugated to each third crosslinker of the third XTEN, 2 payload and any of the payloads listed in Tables 11, 12, 18, and and each fourth crosslink of a fourth XTEN selected from the group consisting of a payload described in 21. a fourth payload conjugated to the agent, the fourth payload being the same as the first, second, or third payload; The tetramer XTEN-payload covalently binds to the XTEN-binding domain and further comprises a payload, which may be the same or different. In one embodiment of the composition, the first payload has specific binding affinity for a target. a targeting moiety having a targeting moiety selected from the targeting moieties listed in Tables 17-19 and 21; and at least one other of the second, third, and fourth payloads is selected from the group consisting of: One is a drug, which consists of drugs listed in Tables 11, 18, and 21. In one embodiment of the tetrameric XTEN-payload conjugate composition, The payload of 1 is a targeting moiety, which is selected from the group consisting of LHRH and folate. and at least one of the second, third, and fourth payloads is selected from the group consisting of doxorubicin, Bisin, paclitaxel, auristatin, maytansine, dolastatin, calicheamicin vinca alkaloids, camptothecin, mitomycin C, epothilones, hTNF, I 1-12, bortezomib, ranpirnase, Pseudomonas, exotoxin, SN-38 The drug is selected from the group consisting of tetrameric XTEN-Peiro, ... In another embodiment of the polymer conjugate composition, the first payload comprises a specific binding domain for the target. a targeting moiety having affinity for the target molecules described in Tables 17-19 and 21. and at least one of the second, third, and fourth payloads is selected from the group consisting of: The other is a drug, which is a drug listed in Tables 11, 18, and 21. wherein the XTEN, targeting moiety, and drug moiety are selected from the group consisting of: It corresponds to any one of conjugates 1 to 290.

[0049] In another embodiment, the present invention provides multimeric XTEN molecules configured, in part, in a branched fashion. In one embodiment, a solution of the composition has a reduced viscosity. The present invention provides a method for producing a medicament comprising at least three XTEs linked together in a branched fashion (e.g., a trimeric fashion). A composition comprising a solution comprising multimeric XTEN having an N fragment, wherein the viscosity of the solution is , the corresponding linear XTEN at equimolar concentrations of ≥ 100, 130, or 150 mg / ml compared to a solution containing ≥ 100, 130, or 150 mg / ml of trimeric XTEN A decrease of at least 5, 6, 7, 8, 9, or 10 cP in the solution containing the preparation In another embodiment, the present invention provides a method for producing tetrameric hydroxylase inhibitors (e.g., hydroxylase inhibitors) that are linked together in a branched fashion (e.g., a tetrameric fashion). a composition comprising a solution containing multimeric XTEN having at least four XTEN fragments; and the composition comprises a corresponding linear amino acid having the same number of amino acids and the same molar concentration. The viscosity of the solution is ≥ 100, 130, or 150 ≥ 100 mg / ml compared to a solution containing the corresponding linear XTEN at an equimolar concentration , 130, or 150 mg / ml of trimeric XTEN preparation. In another embodiment, the present invention provides a method for determining whether a patient's blood glucose level is increased by 5, 6, 7, 8, 9, or 10 cP. , at least five XTEN flags linked together in a branched fashion (e.g., pentameric fashion). and a composition comprising a solution containing multimeric XTEN having an equal number of have a viscosity less than that of a solution containing an amino acid and the corresponding linear XTEN at the same molar concentration. The viscosity of the solution is ≥ 100, 130, or 150 mg / ml of the corresponding equimolar concentration. ≥ 100, 130, or 150 mg / ml compared to solutions containing linear XTEN at least 5, 6, 7, 8, 9, or 10 in a solution containing a trimeric XTEN preparation In the foregoing embodiment of this paragraph, the individual XT The EN is selected from the group consisting of the sequences set forth in Tables 2 and 3.

[0050] In another embodiment, the present invention relates to a sequence selected from the group of sequences set forth in Table 52. at least about 90%, or at least about 91%, or at least about 92%, or or at least about 93%, or at least about 94%, or at least about 95%, or is at least about 96%, or at least about 97%, or at least about 98%, or Compositions of polypeptides having at least about 99%, or 100%, sequence identity are provided. do.

[0051] In another embodiment, the present invention provides an XTEN-payload conjugate described herein. and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition is selected from the group consisting of those listed in Table 16. In another embodiment, the compound has utility in treating a condition selected from the group consisting of the following conditions: The pharmaceutical compositions described herein have utility for use in pharmaceutical regimens for the treatment of subjects. In another embodiment, the pharmaceutical regimen comprises a pharmaceutical composition. 16. Achieving a beneficial effect in a subject having a condition selected from the group of conditions described in The method further comprises determining the amount of pharmaceutical composition needed to In some embodiments, the pharmaceutical regimen used to treat a subject may comprise administering two or more pharmaceutical compositions. and administering to a subject in successive doses an amount effective to treat the disease, the administration of which is comparable to that of an untreated subject. , at least one of at least one, two, or three parameters associated with the state 0%, or 20%, or 30%, or 40%, or 50%, or 60%, or provides an improvement of over 70%, 80%, or 90%.

[0052] In another embodiment, the present invention provides a method for treating a condition selected from the group of conditions set forth in Table 16. and (iii) administering to a subject a composition comprising an XTEN-PY composition as described herein for use in the preparation of a medicament for the treatment of a subject. The conjugate of any one of the loaded conjugate embodiments is provided.

[0053] In some embodiments, the present invention provides a combination of a payload conjugated to an XTEN. The present invention provides a method for selecting an XTEN comprising a plurality of XTEN sequences as a therapeutic agent, the method comprising: providing a library, each of the XTEN sequences comprising at least a first and conjugated to at least a second payload different from the first payload. and (1) selecting from the library a first payload conjugated to a single payload. (1) XTEN sequence conjugated to a second payload alone; and (2) XTEN sequence conjugated to a second payload alone. exhibit improved in vitro or in vivo parameters compared to XTEN sequences. and selecting the first payload as a therapeutic agent. The first and second payloads may be administered to a patient with a common disease (e.g., both the first and second payloads may be administered to a patient with a common disease). In one embodiment of the method, the method is therapeutically effective for ameliorating a disease targeted by the method. The first drug and the second drug are therapeutically effective for treating different symptoms of a common disease. In one embodiment of this method, the common diseases are cancer, cancer supportive care, cardiovascular, central nervous system Endocrine disorders, gastrointestinal disorders, genitourinary disorders, hematological disorders, HIV infection, hormonal disorders, inflammation, autoimmune disorders Autoimmune diseases, infectious diseases, metabolic diseases, musculoskeletal diseases, nephrological disorders, ophthalmological diseases, pain, and In one embodiment of the method, the first payload and the second payload are selected from a respiratory system. Irode mediates their therapeutic effects through a common biological pathway. In an embodiment, the first payload and the second payload are the same as those in Table 11, Table 18, and The different drugs are selected from the group consisting of the drugs listed in Table 21. In the embodiment, the first payload and the second payload are 21. A different biologically active protein selected from the group consisting of the proteins described in In one embodiment of the method, the first payload is a protein selected from the group consisting of those listed in Table 11, Table 18, and Table 19. and a drug selected from the group consisting of a drug listed in Table 21, and the second payload is A biological agent selected from the group consisting of the proteins listed in Table 12, Table 18, and Table 21. It is an environmentally active protein.

[0054] In another embodiment, the present invention provides SASRSA or SASXSA (wherein X is R or Affinity purification via a proteolytic cleavage site having a sequence selected from An isolated polypeptide is provided, comprising an extended recombinant polypeptide attached to a tag.

[0055] In another embodiment, the present invention provides SASRSA or SASXSA (wherein X is R or and a proteolytic cleavage site at its N-terminus having a sequence selected from the group consisting of: and a first affinity purification tag, SASRSA or SASXSA (where X is R or and a C-terminus thereof, via a proteolytic cleavage site, having a sequence selected from the group consisting of: an isolated polypeptide comprising an XTEN, wherein the XTEN is bound to a second affinity purification tag at A polypeptide is provided.

[0056] In another aspect, the invention provides a method for treating a condition in a subject with an XTEN-payload conjugate composition. In one embodiment, the present invention relates to a method for treating a condition in a subject. The method further provides a method for administering an effective amount of any of the XTEN-payload embodiments described herein. to a subject in need of treatment. In accordance with the present invention, there is provided a method of treating a condition in a subject, comprising administering an effective amount of any of the compounds listed in Table 21. The method includes administering a conjugate from the group consisting of the conjugates described above to a subject in need of treatment. In the preceding embodiment of the preceding paragraph, the condition to be treated may be any of the conditions listed in Table 13. In another embodiment, the present invention provides a therapeutic method for treating a variety of conditions, including, but not limited to, Embodiments of the XTEN-payload conjugates described herein for use in imaging and a pharmaceutically acceptable carrier. The regimen comprises administering two or more sequential doses of the pharmaceutical composition.

[0057] In one embodiment, the present invention provides a method for treating a condition selected from the group of conditions set forth in Table 16.

[0033] Embodiments of the XTEN-payloads described herein for the preparation of medicaments for placement In another embodiment, the present invention provides the use of a conjugate of any one of the following: 16. A pharmaceutical composition for the treatment of a condition selected from the group described in claim 16, comprising an effective amount conjugates of any one of the XTEN-payload embodiments described herein Includes.

[0058] In another embodiment, the present invention provides a composition having the structure depicted in Figure 117. do. The present invention also provides, for example, the following items. (Item 1) 1. A method for producing a composition comprising a substantially homogeneous population of XTEN sequences, comprising: a. an amino acid sequence selected from the group of sequences set forth in Table 6, comprising at least one a composition comprising at least one polypeptide having an amino acid sequence comprising a cleavage sequence of To provide and b. treating said polypeptide composition with tryptophan under conditions effective to cleave said cleavage sequence. and treating with cin, At least 95%, 96% of the individual sequences in the resulting substantially homogeneous population of XTEN sequences , 97%, 98%, or 99% have identical sequence lengths. (Item 2) a. Capturing the XTEN sequences onto a chromatographic substrate and adsorption under conditions that are effective for catalyzing but not for proteases. b. eluting the XTEN sequences; c. recovering the XTEN sequences, further comprising: At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the sequences have the same sequence length. (Item 3) 3. The method according to item 2, wherein the chromatographic substrate is an anion exchange substrate. (Item 4) The anion exchange substrate is macrocap Q, capto Q, superQ-6 50M, and poros D. (Item 5) the XTEN sequence is selected from the group of amino acid sequences set forth in Table 6 At least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 5. The method according to any one of items 1 to 4, wherein the sequence has 99% or more sequence identity. (Item 6) The XTEN sequence is selected from the group of amino acid sequences set forth in Table 2 or 3. At least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 9. The method according to any one of items 1 to 5, having 98%, 99%, or 100% sequence identity. Law. (Item 7) A composition produced by the method according to any one of items 1 to 6. (Item 8) A substantially homogeneous population of polypeptides, including extended recombinant polypeptides (XTENs) 10. A composition comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 12 A composition in which 2%, 93%, 94%, or 95% have identical sequence lengths. (Item 9) wherein the XTEN a. the XTEN comprises about 36 to about 3000 amino acid residues; b. Glycine (G), Alanine (A), Serine (S), Threonine (T), Glutamine The total of the amino acid residues of the XTEN is about 9 of the total amino acid residues of the XTEN. Constituting more than 0%; c. the XTEN sequence is: (i) the XTEN sequence is (ii) does not contain three adjacent amino acids that are more identical than the XTEN sequence; At least about 80% of the sequence motifs are non-overlapping, and each of said sequence motifs is about 9 and any two adjacent amino acid residues are identical to the sequence moiety. or (iii) the XTEN distribution does not occur more than twice in each of the the sequence is substantially non-repetitive, such that it has a subsequence score of less than 10; d. The XTEN sequence has greater than 90% randomness as determined by the GOR algorithm. having a dumb coil formation; e. The XTEN sequence is determined by the Chou-Fasman algorithm. % alpha helices and 2% beta sheets; f. The XTEN sequence, when analyzed by the TEPITOPE algorithm, The XTEN sequence lacks a T cell epitope detected by the T cell antigen binding assay. Item 8, characterized in that the PITOPE algorithm prediction is based on a score of -9. The composition described in (Item 10) The XTEN is a group consisting of sequences listed in Table 2, Table 3, Table 4, and Tables 22 to 25. 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 9 ... at least about 92%, or at least about 93%, or at least about 94%, or at least at least about 95%, or at least about 96%, or at least about 97%, or less sequences that share about 98%, or at least about 99%, or 100% sequence identity with each other. 9. The composition according to item 8, comprising (Item 11) 9. The composition of claim 8, further comprising a first affinity tag. (Item 12) The first affinity tag is a hydrophobic interaction chromatography (HIC) substrate, a cation Ion exchange substrates, anion exchange substrates, immobilized metal ion affinity chromatography (IMAC) ) substrate, and an immobilized antibody substrate. 12. The composition according to item 11, having a binding affinity thereto. (Item 13) Item 11, wherein the first affinity tag is selected from the group consisting of the sequences listed in Table 7. The composition described in (Item 14) 14. The composition according to any one of items 11 to 13, further comprising a helper sequence. (Item 15) The helper sequence is selected from the group consisting of the sequences listed in Table 10. At least about 90%, or at least about 91%, or at least about 92%, or less at least about 93%, or at least about 94%, or at least about 95%, or at least at least about 96%, or at least about 97%, or at least about 98%, or less 15. The composition according to item 14, comprising sequences having about 99% or 100% sequence identity with each other. thing. (Item 16) The helper sequence is a. KNPEQAEEQX1EET, where X1 is independently S or R; b. ANPEQAEEQX1EET, where X1 is independently S or R; c. KNPEQAEEQAEEQX1EET (wherein X1 is independently S or R) be), d. KX2X3EQAEEQAEEQX1EET, where X1 is independently S or R, X2 is independently K or N, and X3 is independently K, N, T, Q, H, P, E, D, A, R, or S), e. KX2(X3)10QX1EET, wherein X1 is independently S or R; X2 is independently K or N, and X3 is independently K, N, T, Q, H, P, E , D, A, R, or S), f. KX2(X3)7AEEQX1EET, where X1 is independently S or R X2 is independently K or N, and X3 is independently K, N, T, Q, H, or P. , E, D, A, R, or S), g. KX2X3EQE(X3)3AEEQREET (wherein X2 is independently K or is N and X3 is independently K, N, T, Q, H, P, E, D, A, R, or S be), h. KX2X3EQE(X3)3AEE(X3)5 (wherein X2 is independently K or is N and X3 is independently K, N, T, Q, H, P, E, D, A, R, or S be), i. KKQEQEKEQAEEQ(X4X5)2REET, wherein X4 is independently A or S, and X5 is independently K, Q, or E);KKQEQEKEQ AEEQ(X4X5)4REET, where X4 is independently A or S, and X5 is , independently K, Q, or E), j. KKQEQEKEQAEEQ(Z)4REET, where Z is any naturally occurring L-amino acids), k. KX2(X3)n (wherein n is an integer of 10 to 40, and X2 is independently K or N, and X3 is independently K, N, T, Q, H, P, E, D, A, R, or S), l. (X3)n (wherein n is an integer of 10 to 50, and X3 is independently K, N, T, Q, H, P, E, D, A, R, or S), m. KX2QEQEKEQAEEQ(X4X5)nX1EET (where n is zero or is an integer from 1 to 10, X1 is independently S or R, and X2 is independently K or N, X4 is independently A or S, and X5 is independently K, Q, or or E), n. KX2(X3)n(X4X5)mX1EET (wherein n is an integer of 5 to 20) , m is zero or an integer from 1 to 10, X1 is independently S or R, and X2 is independently K or N, and X3 is independently K, N, T, Q, H, P, E, D , A, R, or S; X4 is independently A or S; and X5 is independently K, Q, or E), o. KX2(X3)n(Z)mX1EET (wherein n is an integer of 5 to 20 and m is , zero or an integer from 1 to 10, X1 is independently S or R, and X2 is independently X3 is independently K, N, T, Q, H, P, E, D, A, R, or S, and Z is any naturally occurring L amino acid. Item 15. The composition according to item 14, selected from the group consisting of: (Item 17) The method further comprises the steps of: (a) providing a first cleavage sequence; (b) providing a first cleavage sequence; and (c) providing a first cleavage sequence; 17. The composition according to any one of items 11 to 16, selected from the group consisting of (Item 18) The composition comprises a compound of Formula I: [ka] wherein: a. HS is the helper sequence; b. AT1 is the first affinity tag; c. CS1 is the first cleavage sequence; d. The composition of claim 17, wherein XTEN is the extended recombinant polypeptide. (Item 19) and a second cleavage sequence, wherein the first and second cleavage sequences are from the same protease. and the composition can be cleaved by a cleavage enzyme, and the composition has Formula II: [ka] wherein: a. HS is a helper sequence; b. AT1 is the first affinity tag; c. CS1 is the first cleavage sequence; d. CS2 is the second cleavage sequence; e. The composition of claim 17, wherein XTEN is the extended recombinant polypeptide. (Item 20) the first affinity tag comprises the sequence RPRPRPRPRPRPPR or HHHHHH 19. The composition according to item 18 or item 19. (Item 21) further comprising a second affinity tag and a second cleavage sequence, wherein said second affinity tag is The first affinity tag has a binding affinity for a different chromatographic substrate than the second affinity tag, Chromatography substrates include HIC substrates, cation exchange substrates, anion exchange substrates, and IMAC. a substrate, and an immobilized antibody substrate, wherein the first and second cleavage sequences are selected from the group consisting of: 18. Any one of items 11 to 17, wherein the sequences can be cleaved by the same protease. The composition according to item . (Item 22) the second affinity tag is different from the first affinity tag, and the second affinity tag is 22. The composition according to item 21, wherein the sequence is selected from the group of sequences listed in Table 7. (Item 23) The composition comprises a compound of Formula III: [ka] wherein: a. HS is the helper sequence; b. AT1 is the first affinity tag; c. CS1 is the first cleavage sequence; d. CS2 is the second cleavage sequence; e. XTEN is the extended recombinant polypeptide; f. The composition of claim 21 or 22, wherein AT2 is the second affinity tag. . (Item 24) the first affinity tag comprises the sequence RPRPRPRPRPRPRP and the second parent 24. The composition of claim 23, wherein the affinity tag comprises the sequence HHHHHH. (Item 25) The first affinity tag comprises the sequence HHHHHH and the second affinity tag comprises the sequence 24. The composition of claim 23, comprising the sequence RPRPRPRPRPRPRP. (Item 26) a. host cells containing a vector encoding a polypeptide, and culturing the host cell under conditions effective to express the polypeptide as a component of the crude expression product of the host cell. wherein the encoded polypeptide comprises XTEN, a first cleavage sequence, and a second cleavage sequence. and culturing the antibody containing the affinity tag of 1. b. pre-coating the crude expression product polypeptide onto a first chromatographic substrate. conditions effective to capture the first affinity tag onto the first chromatographic substrate. and adsorbing the c. eluting the polypeptide; d. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 113%, 114%, 115%, and recovering said polypeptides having at least 95% or 100% identical sequence length. A composition comprising a substantially homogeneous population of polypeptides obtained by the process. (Item 27) The first chromatographic substrate may be an HIC substrate, a cation exchange substrate, an anion exchange substrate, or a cation exchange substrate. 27. The composition of claim 26, wherein the substrate is selected from the group consisting of an IMAC substrate. (Item 28) Item 26. The affinity tag is selected from the group consisting of the affinity tags listed in Table 7. The composition described in (Item 29) the first chromatographic substrate is a cation exchange and the first affinity tag is 28. The composition according to item 27, comprising the sequence RPRPRPRPRPRPRP. (Item 30) The first chromatographic substrate is IMAC and the first affinity tag is 28. The composition of item 27, comprising the sequence HHHHHHHH. (Item 31) The vector encodes the polypeptide according to any one of items 11 to 19. Item 27. The composition according to item 26. (Item 32) the vector further encodes a second cleavage sequence and a second affinity tag, and the second cleavage sequence can be cleaved by the same protease, The second affinity tag is directed to a second chromatographic substrate that is different from the first affinity tag. and wherein the composition has a binding affinity to a. Attaching the polypeptide to a second chromatographic substrate, the second affinity tag, and adsorbing the antibody under conditions effective to capture the antibody onto said second chromatographic substrate. And, b. eluting the polypeptide; c. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 113%, 114%, 115%, and recovering said polypeptides having at least 95% or 95% identical sequence length. 29. The composition according to any one of items 26 to 28, obtained by a process comprising: (Item 33) Item 33. The composition according to Item 32, wherein the vector encodes the polypeptide according to Item 23. thing. (Item 34) The first chromatographic substrate is different from the second chromatographic substrate. wherein each of the first and second chromatographic substrates is independently HIC; 32. The method according to claim 32, wherein the method is selected from the group consisting of cation exchange, anion exchange, and IMAC. The composition described above. (Item 35) the first chromatographic substrate is a cation exchange and the first affinity tag is , the sequence RPRPRPRPRPRPR, and the second chromatographic substrate comprises Item 32. IMAC, wherein the first affinity tag comprises the sequence HHHHHHHH. The composition described in (Item 36) The first chromatographic substrate is IMAC and the first affinity tag is the second chromatographic substrate comprises the sequence HHHHHHHH, and the second chromatographic substrate is a cation exchange Item 32. wherein the first affinity tag comprises the sequence RPRPRPRPRPRPRP. The composition described in (Item 37) The process comprises: a. cleaving said composition with a proteasome under conditions effective to cleave said cleavage sequence(s); treating the XTEN with an enzyme, thereby releasing the XTEN from the affinity tag(s). and, b. To capture the XTEN onto a chromatographic substrate conditions that are effective but not for said affinity tag(s) or said protease and adsorbing the c. eluting the XTEN; d. recovering said XTEN, further comprising: At least 90%, 91%, 92%, 93%, 94%, or 95% of the sequences have the same sequence length. , The composition according to any one of items 23 to 28 or 32 to 34. (Item 38) The cleavage sequence(s) may be cleaved by a protease selected from the group consisting of the proteases in Table 9. 38. The composition according to item 37, which can be cleaved by enzymes. (Item 39) The cleavage sequence(s) can be cleaved by trypsin, and the sequence is selected from the group consisting of the sequences set forth in Table 8, wherein the protease is trypsin. 39. The composition according to item 38. (Item 40) 39. The composition according to item 37 or 38, wherein the chromatographic substrate is anion exchange. Finished product. (Item 41) The anion exchange chromatography substrate is macrocap Q, capto Q Item 40 is selected from the group consisting of superQ-650M, and poros D. The composition described in (Item 42) The process comprises: a. cleaving said composition with a proteasome under conditions effective to cleave said cleavage sequence(s); treating the XTEN with an enzyme, thereby releasing the XTEN from the affinity tag(s). and, b. Capturing the protease on a chromatographic substrate. and adsorbing under conditions that are effective for the production of hydroxybenzoates but not for the XTEN. c. recovering the XTEN from the eluate, At least 90%, 91%, 92%, 93%, 94%, or 95% of the molecules have identical sequences 35. The composition according to any one of items 23 to 28 or 32 to 34, having a length of (Item 43) The cleavage sequence(s) may be cleaved by a protease selected from the group consisting of the proteases in Table 9. 43. The composition according to item 42, which can be cleaved by enzymes. (Item 44) The cleavage sequence(s) can be cleaved by trypsin, and the sequence is selected from the group consisting of the sequences set forth in Table 8, wherein the protease is trypsin. 44. The composition according to item 42 or item 43. (Item 45) The chromatography substrate is a cation exchange substrate, an HIC substrate, or an IMAC substrate. 45. The composition according to any one of items 42 to 44, wherein the composition is one or more of the following: (Item 46) A composition comprising an XTEN sequence, wherein the XTEN sequence is cleaved by trypsin. and a cleavage sequence capable of cleaving all of said cleavage sequences. Treatment with trypsin under effective conditions results in the preparation of XTEN fragments, Each XTEN fragment is at least as strong as every other fragment in the preparation. The composition has about 95%, 96%, 97%, 98%, or 99% sequence identity. (Item 47) The truncated sequence has at least 86% sequence identity with the sequence SASRSA or SASKSA. 47. The composition according to item 46, having or being identical to the sequence identity. (Item 48) The cleavage sequence comprises the sequence RX or KX, where X is any L amino acid other than proline. 47. The composition according to item 46, wherein the amino acid is (Item 49) The composition is at least Approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 47. The composition of claim 46, having 100% sequence identity. (Item 50) 1. A method for producing an XTEN sequence in a host cell, comprising: a) the XTEN sequence; b) host cells containing a vector encoding a polypeptide comprising a helper sequence; under conditions effective to express said polypeptide as a component of a crude expression product, About 2 grams per liter (g / L), or about 3 g / L, or about 4 g / L, or about 5 g / L, or about 6 g / L, or about 7 g / L of the polypeptide. A method comprising: (Item 51) 1. A method for producing an XTEN sequence in a host cell, comprising: a) the XTEN sequence; b) host cells containing a vector encoding a polypeptide comprising a helper sequence; under conditions effective to express said polypeptide as a component of a crude expression product, A dry weight host cell concentration of greater than about 10 milligrams / gram (mg / g), or at least or at least about 15 mg / g, or at least about 20 mg / g, or at least about 25 mg / g, or at least about 30 mg / g, or at least about 40 mg / g, or at a concentration of at least about 50 mg / g of said polypeptide. (Item 52) 1. A method for producing an XTEN sequence in a host cell, comprising: a) the XTEN sequence; b) host cells containing a vector encoding a polypeptide comprising a helper sequence; under conditions effective to express said polypeptide as a component of a crude expression product, A dry weight host cell concentration of greater than about 10 milligrams / gram (mg / g), or at least about 250 micromol / L, or about 300 micromol / L, or about 350 micromol / L micromol / L, or about 400 micromol / L, or about 450 micromol / L or about 500 micromoles / L of the polypeptide. ,method. (Item 53) When the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, 53. The method according to any one of items 50 to 52, wherein the concentration is measured. (Item 54) The helper sequence of the expressed polypeptide is present at the N-terminus of the polypeptide. wherein the sequence is at least one sequence selected from the group consisting of the sequences listed in Table 10. have approximately 90%, 91%, 92%, 93%, 94%, or 95% sequence identity with each other 53. The method according to any one of items 50 to 52, wherein the method is the same as or (Item 55) The vector encodes the polypeptide according to any one of items 14 to 17. Item 55. The method according to item 54. (Item 56) a. recovering the crude expression product of the host cell fermentation reaction mixture; b. pre-coating the crude expression product polypeptide onto a first chromatographic substrate. capturing the first affinity tag of the polypeptide onto the chromatography substrate. wherein the first chromatographic substrate is adsorbed under conditions effective to C substrate, cation exchange substrate, anion exchange substrate, and IMAC substrate and c. eluting and recovering the XTEN sequence; At least 90%, 91%, 92%, 93%, 94%, or 95% of the sequences are identical in length. Item 56. The method of Item 55, comprising: (Item 57) The vector encodes the polypeptide according to any one of items 21 to 23. 53. The method according to any one of Items 50 to 52. (Item 58) a. recovering the crude expression product of the host cell fermentation reaction mixture; b. chromatographically dispersing the polypeptide onto a first chromatographic substrate; under conditions effective to capture the first affinity tag onto the chromatographic substrate. wherein the first chromatographic substrate is a HIC substrate, a cation anion exchange substrates, anion exchange substrates, and IMAC substrates. And, c. eluting the polypeptide; d. Placing the polypeptide on a second chromatographic substrate. under conditions effective to capture the second affinity tag onto the chromatographic substrate. wherein the second chromatographic substrate is a HIC substrate, a cation anion exchange substrates, anion exchange substrates, and IMAC substrates. And, e. eluting the polypeptide; f. recovering said XTEN sequence, 90%, 91%, 92%, 93%, 94%, or 95% of the sequences have the same sequence length. The method described in item 57. (Item 59) a. cleaving the polypeptide with a protease under conditions effective to cleave the cleavage sequence thereby releasing the XTEN from the polypeptide; b. Capturing the XTEN onto an anionic chromatography matrix and adsorbing under conditions effective to c. eluting the XTEN; and d. recovering said XTEN, further comprising recovering at least one of said individual XTEN molecules. At least 90%, or at least 91%, or at least 92%, or at least 93% %, or at least 94%, or at least 95% have the same sequence length, item 5 6 or the method according to item 58. (Item 60) The anion chromatography substrate is macrocap Q, capto Q, s Item 59, selected from the group consisting of superQ-650M, and poros D How to post. (Item 61) The protease is trypsin, and the sequence is a group consisting of the sequences set forth in Table 8. 61. The method of claim 59 or 60, wherein the method is selected from the group consisting of: (Item 62) a. cleaving said polypeptide under conditions effective to cleave said cleavage sequence(s); treating the polypeptide with protease, thereby releasing the XTEN from the polypeptide; , b. Capturing the protease on a chromatographic substrate. and adsorbing under conditions that are effective for the production of hydroxybenzoates but not for the XTEN. c. recovering the XTEN in the eluate, At least 90%, 91%, 92%, 93%, 94%, or 95% of the sequence is identical in length The method according to item 56 or 58, (Item 63) The protease is trypsin, and the sequence is a group consisting of the sequences set forth in Table 8. Item 63. The method of item 62, wherein the method is selected from the group consisting of: (Item 64) The chromatographic substrates include HIC substrates, cation exchange substrates, and IMAC substrates. Item 64. The method according to item 62 or 63, wherein the method is one or more of: (Item 65) a solid support comprising a population of substantially identical polypeptides immobilized thereon, The solid support comprises a chromatographic substrate, and the immobilized polypeptide is each comprising an XTEN, a first affinity tag, and a second affinity tag; a. the first affinity tag is bound by a cleavage sequence at the N-terminus of the XTEN; conjugated to the XTEN, b. the second affinity tag is linked by a cleavage sequence at the C-terminus of the XTEN; conjugated to the XTEN, c. the second affinity tag is different from the first affinity tag; d. the chromatography substrate is chromatographically isolated from either the first or the second parent molecule, but not both. can be linked to any of the affinity tags, e. at least 90%, 91%, 92%, 93%, 94%, or %, or 95% have identical sequence lengths. (Item 66) The immobilized polypeptide molecule is a polypeptide according to any one of items 21 to 25. Item 66. The fixed support according to item 65, comprising: (Item 67) The truncated sequence has at least 86% sequence identity with the sequence SASRSA or SASKSA. 67. The fixed support according to item 65 or item 66, having or being identical in sequence. (Item 68) The cleavage sequence comprises the sequence RX or KX, where X is any L amino acid other than proline. 67. The solid support according to item 65 or 66, which is an amino acid. (Item 69) The solid support may be a HIC chromatography resin, a cation exchange resin, an anion exchange resin, or the like. chromatography resin, and IMAC chromatography resin. 69. The solid support according to any one of items 65 to 68, (Item 70) the first affinity tag comprises the sequence RPRPRPRPRPRPRP and the second parent 70. The composition of claim 69, wherein the affinity tag comprises the sequence HHHHHH. (Item 71) The first affinity tag comprises the sequence HHHHHH and the second affinity tag comprises the sequence 70. The composition of claim 69, comprising the sequence RPRPRPRPRPRPRP. (Item 72) a conjugate composition comprising XTEN covalently bonded to one or more molecules of at least a first crosslinker; wherein the crosslinker is a crosslinker listed in Table 13, an alkyne reactant listed in Table 15 and an azide reactant listed in Table 15, wherein the XTEN is selected from the group consisting of: XTEN described in item 37, XTEN described in item 42, XTEN described in item 7, XTEN described in Table 2 The conjugate composition is selected from the group consisting of the sequences described in Table 3 and the sequences described in Table 3. (Item 73) The first cross-linking agent is a. the alpha amino group of the N-terminal amino acid residue of the XTEN; b. the epsilon amino group of each lysine residue of said XTEN; c. a thiol group of each cysteine ​​residue of said XTEN 73. The conjugate composition of claim 72, wherein said at least first XTEN is conjugated to said at least first XTEN. (Item 74) The XTEN is directed to a sequence selected from the group consisting of the sequences set forth in Table 2 and Table 3. at least about 90%, or at least about 91%, or at least about 92%, or is 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 less 72 or 73, having at least about 99%, or 100%, sequence identity with The conjugate composition described above. (Item 75) The XTEN is AE144, AE288, AE432, AE576, AE864, Segment 174, Segment 175, Segment 176, Segment 177, Segment 186, segment 187, segment 188, segment 189, segment 190, Segment 191, Segment 192, Segment 193, Segment 194, Segment Segment 195, Segment 196, Segment 197, Segment 198, and Segment 199, wherein the cross-linking agent is selected from the group consisting of: 75. The conjugate composition according to item 74, conjugated to the alpha amino group. (Item 76) The XTEN may be a sequence of segments 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 1 176, segment 177, segment 186, segment 187, segment 188, Segment 189, Segment 190, Segment 191, Segment 192, Segment Segment 193, Segment 194, Segment 195, Segment 196, Segment 197 , segment 198, and segment 199, wherein the crosslinker is selected from the group consisting of: 75. The conjugate of claim 74, wherein the conjugate is conjugated to the thiol group of each cysteine ​​residue of the XTEN. composition. (Item 77) The first cross-linking agent may be an N-maleimide, an iodoacetyl reagent, a pyridyl disulfide reagent, or Drug, vinyl sulfone reagent, 3-propargyloxypropanoic acid, (oxyethyl)n- Cetylene (n is 1-10), dibenzylcyclooctyne (DBCO), cyclooctyne ( COT), 3-azido-propionic acid, 6-azido-hexanoic acid, and (oxyethyl 77. Any one of items 72 to 76, selected from the group consisting of n-azides (n is 1 to 10). Item 1. The conjugate according to item 1. (Item 78) the first cross-linker is conjugated to the thiol group of each cysteine ​​residue of the XTEN; the conjugate comprises a second amino acid conjugated to the alpha amino group of the N-terminal amino acid of the XTEN. The crosslinker may further comprise a crosslinker selected from the group consisting of a crosslinker listed in Table 13, an alkyl group listed in Table 15, and the like. 76. The azide reactants described in Table 15. The conjugate composition described above. (Item 79) Formula V: wherein, independently for each occurrence: a. CL1 is the first crosslinker conjugated to a cysteine ​​residue of the XTEN; b. CL2 is the second crosslinker conjugated to XTEN at the N-terminus; c. x is an integer from 1 to about 10; d. y is an integer 1, provided that x+y>2; e. the XTEN is a cysteine ​​engineered XTEN comprising x cysteine ​​residues; 79. The conjugate composition according to item 78. (Item 80) and further comprising a single atom residue of a first payload conjugated to each first crosslinker, said residue is selected from the group consisting of carbon, nitrogen, oxygen, and sulfur. 1. The conjugate composition according to claim 1. (Item 81) The first payload of the single atom residue is listed in Tables 6, 7, 18, and 21. 81. The conjugate composition of item 80, wherein the payload is selected from the group consisting of: (Item 82) From the payloads listed in Tables 6, 7, 18, and 21 conjugated to each first crosslinker 78. The method according to any one of items 72 to 77, further comprising a payload selected from the group consisting of: Conjugate composition. (Item 83) a single atom residue of a first payload conjugated to each of the first crosslinkers, the single atom residue comprising a carbon atom; a residue selected from the group consisting of nitrogen, oxygen, and sulfur, and conjugated to said second crosslinker; a single atom residue of a second payload, consisting of carbon, nitrogen, oxygen, and sulfur; 80. The conjugate composition of claim 78 or 79, further comprising a residue selected from the group: (Item 84) The first payload of the single atom residue is listed in Tables 6, 7, 18, and 21. and said second payload of said single atom residue is selected from the group consisting of: A payload different from the first payload and listed in Tables 6, 7, 18, and 21. 84. The conjugate composition according to item 83, wherein the payload is selected from the group consisting of: (Item 85) a first payload conjugated to each of said first crosslinkers, 21, and a first payload selected from the group consisting of the payloads described in a second payload different from the first payload conjugated to the second crosslinker; and a payload selected from the group consisting of those listed in Tables 6, 7, 18, and 21. 80. The conjugate composition of claim 78 or 79, further comprising a payload of 2. (Item 86) a first payload conjugated to each of said first crosslinkers, said first payload comprising a drug moiety as set forth in Table 21; a first payload selected from the group consisting of a first payload and a second payload conjugated to the second crosslinker; a second payload different from the first payload, the second payload being selected from the group consisting of targeting moieties in Table 21; 80. The covalent bond according to claim 78 or 79, further comprising a second payload selected from Role composition. (Item 87) an alkyne reactant, wherein the second payload is selected from the group consisting of the reactants in Table 15; and the second crosslinker conjugated by reaction with an azide reactant. 87. The conjugate composition according to claim 85 or 86, wherein the conjugate composition is attached to the N-terminus. (Item 88) 1. A conjugate composition comprising at least a first and a second XTEN, wherein the XTEN comprises: The XTEN described in Item 37 and the XTEN described in Item 42 may be the same or different, respectively. XTEN, XTEN as described in item 7, and a sequence as described in Table 3. and the first and second XTENs are an alkyne reactant and an azide reactant selected from the group consisting of the reactants in Table 15, depending on the terminus; The conjugated compositions are conjugated to each other using a crosslinker formed by reaction with (Item 89) The first XTEN and the second XTEN are different and each independently have a sequence as set forth in Table 3. At least about 90% to a sequence selected from the group of sequences described, 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 9 7%, or at least about 98%, or at least about 99%, or 100% sequence identity 89. The conjugate composition according to item 88, having a uniformity. (Item 90) The first XTEN comprises segment 174, segment 175, segment 176, and segment 177 as set forth in Table 3. Segment 176, Segment 177, Segment 186, Segment 187, Segment 1 88, segment 189, segment 190, segment 191, segment 192, Segment 193, Segment 194, Segment 195, Segment 196, Segment A sequence selected from the group consisting of segments 197, 198, and 199. At least about 90%, or at least about 91%, or at least about 92% for the column , 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 or at least about 99%, or 100% sequence identity to the second XTEN, Segment 174, segment 175, segment 176, and segment 177 are listed in Table 3. 177, segment 186, segment 187, segment 188, segment 189, Segment 190, Segment 191, Segment 192, Segment 193, Segment Segment 194, Segment 195, Segment 196, Segment 197, Segment 198 and a sequence of at least one different sequence selected from the group consisting of segments 199. or 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 9 90. The conjugate composition of item 89, having 9% or 100% sequence identity. (Item 91) The first XTEN and the second XTEN are the same and each have a structure as set forth in Table 3. at least about 90%, or at least about 90% to a sequence selected from the group of sequences listed 91%, or at least about 92%, or at least about 93%, or at least about 9 4%, 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%, or 100% sequence identity 89. The conjugate composition according to item 88, having the property (Item 92) The first XTEN and the second XTEN are each a segment described in Table 3. Segment 174, Segment 175, Segment 176, Segment 177, Segment 18 6, Segment 187, Segment 188, Segment 189, Segment 190, Seg Segment 191, Segment 192, Segment 193, Segment 194, Segment 1 95, segment 196, segment 197, segment 198, and segment 19 at least about 90% to a sequence selected from the group of sequences consisting of: 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 9 7%, or at least about 98%, or at least about 99%, or 100% sequence identity 92. The conjugate composition according to item 91, having a uniformity. (Item 93) the first and second XTEN each comprise one or more cysteine ​​residues; a first cross-linker conjugated to each cysteine ​​residue of the first XTEN; and and a second cross-linker conjugated to each cysteine ​​residue of N, The second crosslinker is independently selected from the group consisting of the crosslinkers listed in Table 13. 88 to 92. The conjugate composition according to any one of items 88 to 92. (Item 94) the first and second XTENs each comprise one or more lysine residues, and a crosslinker conjugated to each lysine residue of the first and / or second XTEN of the conjugate. Item 8 further comprises, wherein the cross-linking agent is selected from the group consisting of the cross-linking agents listed in Table 13. 93. The conjugate composition according to any one of items 8 to 92. (Item 95) and further comprising a single atom residue of a first payload conjugated to each crosslinker of said first XTEN. wherein the residue is selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; and a single atom residue of a second payload conjugated to each crosslinker of XTEN, said residue being Item 93 or 94, selected from the group consisting of carbon, nitrogen, oxygen, and sulfur. The conjugate composition described herein. (Item 96) The first payload of the single atom residue is listed in Tables 6, 7, 18, and 21. and said second payload of said single atom residue is selected from the group consisting of: A payload different from the first payload and listed in Tables 6, 7, 18, and 21. 96. The conjugate composition of item 95, wherein the payload is selected from the group consisting of: (Item 97) further comprising a first payload conjugated to each crosslinker of said first XTEN, The payload is selected from the group consisting of payloads listed in Tables 6, 7, 18, and 21. and further comprising a second payload selected from the first payload and different from the first payload, The payload is selected from the group consisting of the payloads listed in Tables 6, 7, 18, and 21. 95. The conjugate composition according to item 93 or item 94, (Item 98) further comprising a first payload conjugated to each crosslinker of said first XTEN, the payload of said first payload is selected from the group consisting of the targeting moieties listed in Table 18, and a second payload different from the load, said second payload being one of the payloads listed in Table 18. 95. The conjugate composition according to item 93 or item 94, wherein the toxin is selected from the group of toxins listed in (Item 99) further comprising a first payload conjugated to each crosslinker of said first XTEN, the payload of said first payload is selected from the group consisting of the targeting moieties listed in Table 21; and a second payload different from the load, said second payload being one of the payloads listed in Table 21. 95. The conjugate composition according to item 93 or item 94, wherein the toxin is selected from the group of toxins listed in (Item 100) The first XTEN is segment 176 of Table 3 and the second XTEN is segment 176 of Table 3 Item 176 and 177 are selected from the group consisting of 99. The conjugate composition according to claim 99. (Item 101) Conjugate compositions comprising at least a first XTEN, a second XTEN, and a third XTEN 37. The method of claim 36, wherein the XTEN is selected from the group consisting of: 2, the XTEN described in item 7, and the sequence described in Table 3. wherein the first, second, and third XTENs are selected from the group consisting of: and cross-linking the N-terminus of the cross-linker using a trivalent cross-linker selected from the group consisting of the trivalent cross-linkers described herein. A conjugated composition conjugated to (Item 102) The trivalent crosslinker is tris-(2-maleimidoethyl)amine (TMEA) and amine From the group consisting of amine-reactive tris-(succinimidyl aminotriacetate (TSAT) 101. The conjugate composition according to item 100, selected from (Item 103) The first, second, and third XTEN are identical and each has a sequence as set forth in Table 3. Segment 174, segment 175, segment 176, and segment 177 are described. , segment 186, segment 187, segment 188, segment 189, segment Segment 190, Segment 191, Segment 192, Segment 193, Segment 19 4, segment 195, segment 196, segment 197, segment 198, and and segment 199, or At least about 91%, or at least about 92%, or at least about 93%, or less At least about 94%, or at least about 95%, or at least about 96%, or at least at least about 97%, or at least about 98%, or at least about 99%, or 100% % sequence identity. (Item 104) The first and second XTEN are the same and each have a sequence as set forth in Table 3. Segment 174, Segment 175, Segment 176, Segment 177, Segment 186, segment 187, segment 188, segment 189, segment 190, Segment 191, Segment 192, Segment 193, Segment 194, Segment Segment 195, Segment 196, Segment 197, Segment 198, and Segment 199, or at least about 90% 91%, or at least about 92%, or at least about 93%, or at least about 9 4%, 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%, or 100% sequence identity wherein the third XTEN is different from the first and second XTENs and is as described in Table 3. Segment 174, segment 175, segment 176, and segment 17 7, Segment 186, Segment 187, Segment 188, Segment 189, Seg Segment 190, Segment 191, Segment 192, Segment 193, Segment 1 94, segment 195, segment 196, segment 197, segment 198, and and segment 199, and is 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 less at least about 97%, or at least about 98%, or at least about 99%, or 10 103. The conjugate composition according to any one of items 100 to 102, having 0% sequence identity. (Item 105) the conjugate comprises a first cross-linker conjugated to each cysteine ​​residue of the first XTEN; a second cross-linker conjugated to each cysteine ​​residue of the second XTEN; and and a third cross-linker conjugated to each cysteine ​​residue of N, wherein the cross-linker is selected from the group consisting of those listed in Table 1. Item 103 or 104, selected from the group consisting of crosslinkers according to item 3. Conjugate composition. (Item 106) Formula XII: [ka] wherein, independently for each occurrence: a. 3xCL is the trivalent crosslinker; b. CL1 is the first crosslinker conjugated to XTEN1; c. CL2 is the second crosslinker conjugated to XTEN2; d. CL3 is the third crosslinker conjugated to XTEN3; e. x is an integer from 1 to about 10; f. y is an integer from 1 to about 10; g. z is an integer from 1 to about 10, provided that x+y>3; h. XTEN1 is the first XTEN; i. XTEN2 is the second XTEN; j. The conjugate composition of claim 105, wherein XTEN3 is the third XTEN. (Item 107) a. a single atom residue of a first payload conjugated to each first crosslinker of said first XTEN; a radical selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; b. a single atom residue of a second payload conjugated to each second crosslinker of said second XTEN; a radical selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; c. a single atom residue of a third payload conjugated to each third crosslinker of said third XTEN; a group selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; 107. The conjugate composition according to item 105 or item 106, comprising: (Item 108) a. selected from the group consisting of payloads listed in Tables 6, 7, 18, and 21 a first payload conjugated to each first crosslinker of said first XTEN; b. Selected from the group consisting of payloads set forth in Tables 6, 7, 18, and 21; a second payload conjugated to each second crosslinker of said second XTEN; c. Selected from the group consisting of payloads set forth in Tables 6, 7, 18, and 21; a third payload conjugated to each third crosslinker of said third XTEN. 107. The conjugate composition according to item 105 or item 106. (Item 109) the first payload is a targeting moiety that has specific binding affinity for a target, the targeting moiety is selected from the group consisting of the targeting moieties set forth in Tables 17-19 and 21; The second and third payloads may be the same or different drugs. wherein the drug is selected from the group consisting of drugs listed in Table 6, Table 18, and Table 21. 109. The conjugate composition according to item 108. (Item 110) The targeting moiety is selected from the group consisting of LHRH and folic acid, and the drug is doxorubicin. Bicine, paclitaxel, auristatin, monomethyl auristatin E (MMAE), Monomethyl auristatin F, maytansine, dolastatin, calicheamicin, vincaine Alkaloids, camptothecin, mitomycin C, epothilones, hTNF, I1-12, Lutezomib, ranpirnase, pseudomonas, exotoxin, SN-38, and laccase 110. The conjugate composition according to item 109, selected from the group consisting of rumin and thiamin. (Item 111) the targeting moiety and the drug moiety are selected from the group consisting of conjugates 1-290 listed in Table 21 Item 109. The conjugate composition according to item 108, corresponding to any one of (Item 112) The conjugate comprises the XTEN, the targeting moiety, and a prepolymer corresponding to conjugate 71 in Table 21. 112. The conjugate composition of claim 111, having the drug moiety. (Item 113) A conjugate composition comprising at least first, second, third, and fourth XTENs. The XTEN is the XTEN described in Item 37, the XTEN described in Item 42, or the XTEN described in Item 7. and a sequence set forth in Table 3, wherein the XTEN is selected from the group consisting of: may be the same or different, and said first and said second and said third and said A fourth XTEN is conjugated to each other by the N-terminus using a tetravalent crosslinker, A conjugate composition wherein the crosslinker is a tetravalent maleimide cluster. (Item 114) the first, second, third, and fourth XTENs are the same, and each The segments 174, 175, 176, and 177 are listed in Table 3. Segment 177, Segment 186, Segment 187, Segment 188, Segment 1 89, segment 190, segment 191, segment 192, segment 193, Segment 194, Segment 195, Segment 196, Segment 197, Segment 198, and at least about 9 for a sequence selected from the group consisting of segment 199 0%, 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%, or 100% sequence identity. (Item 115) The first and second XTEN are the same and each have a sequence as set forth in Table 3. Segment 174, Segment 175, Segment 176, Segment 177, Segment 186, segment 187, segment 188, segment 189, segment 190, Segment 191, Segment 192, Segment 193, Segment 194, Segment Segment 195, Segment 196, Segment 197, Segment 198, and Segment 199, or at least about 90% 91%, or at least about 92%, or at least about 93%, or at least about 9 4%, 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%, or 100% sequence identity the third and fourth XTENs are the same, but the first and 2 XTEN, each of which has a segment 174, a segment 175, and a segment 176, as described in Table 3. 175, segment 176, segment 177, segment 186, segment 187, Segment 188, Segment 189, Segment 190, Segment 191, Segment Segment 192, Segment 193, Segment 194, Segment 195, Segment 196 , segment 197, segment 198, and segment 199. 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 9 5%, or at least about 96%, or at least about 97%, or at least about 98% %, or at least about 99%, or 100% sequence identity with any of the sequences of item 113 The conjugate composition according to any one of claims 1 to 4. (Item 116) the conjugate comprises a first cross-linker conjugated to each cysteine ​​residue of the first XTEN; a second cross-linker conjugated to each cysteine ​​residue of the second XTEN; and a third crosslinker conjugated to each cysteine ​​residue of N and each cysteine ​​residue of the fourth XTEN; and a fourth crosslinker conjugated to the residue, wherein each crosslinker is selected from the group consisting of crosslinkers listed in Table 13. 116. The conjugate composition according to claim 114 or 115, wherein the conjugate composition is selected from the group consisting of: (Item 117) Formula XIV [ka] wherein, independently for each occurrence: a. 4xCL is the tetravalent crosslinker; b. CL1 is the first crosslinker conjugated to XTEN1; c. CL2 is the second crosslinker conjugated to XTEN2; d. CL3 is the third crosslinker conjugated to XTEN3; e. CL4 is the fourth crosslinker conjugated to XTEN4; f. v is an integer from 1 to about 10; g. x is an integer from 1 to about 10; h. y is an integer from 1 to about 10; i. z is an integer from 1 to about 10, provided that x+y>4; j. XTEN1 is the first XTEN; k. XTEN2 is the second XTEN; l. XTEN3 is the third XTEN; m. The co-expression of any one of items 116, wherein XTEN4 is the fourth XTEN. Role composition. (Item 118) a. a single atom residue of a first payload conjugated to each first crosslinker of said first XTEN; a radical selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; b. a single atom residue of a second payload conjugated to each second crosslinker of said second XTEN; a radical selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; c. a single atom residue of a third payload conjugated to each third crosslinker of said third XTEN; a radical selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; d. a single atom residue of a fourth payload conjugated to each fourth crosslinker of said fourth XTEN. a group selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; 118. The conjugate composition according to item 116 or 117, comprising: (Item 119) a. selected from the group consisting of payloads set forth in Tables 6, 7, 18, and 21; a first payload conjugated to each first crosslinker of said first XTEN; b. Selected from the group consisting of payloads set forth in Tables 6, 7, 18, and 21; a second payload conjugated to each second crosslinker of the second XTEN, a payload that is the same as or different from the payload of c. Selected from the group consisting of payloads set forth in Tables 6, 7, 18, and 21; a third payload conjugated to each third crosslinker of the third XTEN, or a payload that is the same as or different from the second payload; d. selected from the group consisting of payloads set forth in Tables 6, 7, 18, and 21; a fourth payload conjugated to each fourth crosslinker of the fourth XTEN, or a payload that is the same as or different from the second or third payload. 118. The conjugate composition according to claim 116 or 117, further comprising: (Item 120) the first payload is a targeting moiety that has specific binding affinity for a target, the targeting moiety is selected from the group consisting of the targeting moieties set forth in Tables 17-19 and 21; At least one other of the second, third, and fourth payloads is a drug, The drug is selected from the group consisting of drugs listed in Tables 6, 18, and 21. 119. The conjugate composition according to claim 119. (Item 121) The targeting moiety is selected from the group consisting of LHRH and folic acid, and the drug is doxorubicin. Bisin, paclitaxel, auristatin, maytansine, dolastatin, calicheamicin vinca alkaloids, camptothecin, mitomycin C, epothilones, hTNF, I 1-12, bortezomib, ranpirnase, Pseudomonas, exotoxin, SN-38 121. The conjugate composition according to item 120, wherein the conjugate composition is selected from the group consisting of: (Item 122) the first payload is a targeting moiety that has specific binding affinity for a target, the targeting moiety is selected from the group consisting of the targeting moieties set forth in Tables 17-19 and 21; At least one other of the second, third, and fourth payloads is a drug, The drug is selected from the group consisting of drugs listed in Tables 6, 18, and 21; XTEN, the targeting moiety, and the drug moiety are conjugates 1-290 listed in Table 21. Item 120. The conjugate composition according to item 119, which corresponds to any one of (Item 123) Formula XVI: [ka] wherein, independently for each occurrence: a. 3xCL is the trivalent crosslinker; b. CL1 is the first crosslinker conjugated to XTEN1; c. CL2 is the second crosslinker conjugated to XTEN2; d. x is an integer from 1 to about 10; e. y is an integer from 1 to about 10, provided that x+y>2; f. XTEN1 is the first XTEN; g. XTEN2 is the second XTEN; h. XTEN3 is the third XTEN, and the XTEN has a sequence set forth in Table 2. Item 102. The conjugate composition according to item 101, selected from the group consisting of: (Item 124) a. a single atom residue of a first payload conjugated to each first crosslinker of said first XTEN; a radical selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; b. a single atom residue of a second payload conjugated to each second crosslinker of said second XTEN; a group selected from the group consisting of carbon, nitrogen, oxygen, and sulfur; Item 124. The conjugate composition according to item 123, comprising: (Item 125) a. selected from the group consisting of payloads set forth in Tables 6, 7, 18, and 21; a first payload conjugated to each first crosslinker of said first XTEN; b. Selected from the group consisting of payloads set forth in Tables 6, 7, 18, and 21; a second payload conjugated to each second crosslinker of the second XTEN, and a payload that is the same as or different from the payload of item 123. The conjugate composition according to claim 1. (Item 126) the first payload is a targeting moiety that has specific binding affinity for a target, the targeting moiety is selected from the group consisting of the targeting moieties set forth in Tables 17-19 and 21; The second payload is a drug, which may be the same or different, and the drug is Item 125 is selected from the group consisting of drugs listed in Table 6, Table 18, and Table 21. The conjugate composition described herein. (Item 127) The targeting moiety is selected from the group consisting of LHRH and folic acid, and the drug is doxorubicin. Bicine, paclitaxel, auristatin, monomethyl auristatin E (MMAE), Monomethyl auristatin F, maytansine, dolastatin, calicheamicin, vincaine Alkaloids, camptothecin, mitomycin C, epothilones, hTNF, I1-12, Lutezomib, ranpirnase, pseudomonas, exotoxin, SN-38, and laccase 127. The conjugate composition according to item 126, selected from the group consisting of rumin and thromycin. (Item 128) the first payload is selected from the group consisting of a drug in Table 11 and a protein in Table 12; wherein the second payload is different from the first payload and is a drug and 126. The conjugate composition according to item 125, selected from the group consisting of the proteins of table 12. (Item 129) The first payload and the second payload are the same and are selected from the group consisting of drugs and and the proteins of Table 12. (Item 130) Formula XXVII: [ka] wherein, independently for each occurrence: a. 3xCL is the trivalent crosslinker; b. CL1 is the first crosslinker conjugated to XTEN1; c. x is an integer from 1 to about 10; d. XTEN1 is the first XTEN, and the XTEN has a sequence listed in Table 3. selected from the group consisting of: e. XTEN2 is the second XTEN, and the XTEN has a sequence listed in Table 2. selected from the group consisting of: f. XTEN3 is the third XTEN, and the XTEN has a sequence set forth in Table 2. Item 102. The conjugate composition according to item 101, selected from the group consisting of: (Item 131) a single atom residue of a first payload conjugated to each first crosslinker of said first XTEN; wherein the residue is selected from the group consisting of carbon, nitrogen, oxygen, and sulfur. 130. The conjugate composition according to claim 130. (Item 132) selected from the group consisting of payloads set forth in Tables 6, 7, 18, and 21; Item 13, further comprising a first payload conjugated to each first crosslinker of the first XTEN. 10. The conjugate composition according to claim 0. (Item 133) 1. A composition comprising multimeric branched XTEN molecules, wherein a solution of said composition comprises a plurality of branched XTEN molecules each having the same number of amino acids. and has a lower viscosity than a solution containing the corresponding linear XTEN having the same molar concentration. composition. (Item 134) 134. The method of claim 133, wherein the XTEN molecule has a trimeric, tetrameric, or pentameric configuration. Composition of. (Item 135) The XTEN molecule is selected from the group consisting of the sequences set forth in Table 2 and Table 3. The composition according to item 133. (Item 136) The clay of the solution is ≥ 10 in a solution containing ≥ 100 mg / ml of multimeric XTEN. at least 10 mg / ml of the corresponding linear XTEN solution at an equimolar concentration of 0 mg / ml. Any one of items 133-135, which decreases by 5, 6, 7, 8, 9, or 10 cP The composition according to item . (Item 137) at least about 90% to a sequence selected from the group of sequences set forth in Table 52; or At least about 91%, or at least about 92%, or at least about 93%, or less At least about 94%, or at least about 95%, or at least about 96%, or at least at least about 97%, or at least about 98%, or at least about 99%, or 100% % sequence identity. (Item 138) Items 85~87, 97~99, 108~112, 118~122, 125~127, 1 29 or 132 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising: (Item 139) The drug according to item 138 for the treatment of a condition selected from the group of conditions listed in table 16. chemical composition. (Item 140) 2. For use in a pharmaceutical regimen for the treatment of a subject, the regimen comprising the pharmaceutical composition Item 139. The pharmaceutical composition according to item 138, comprising (Item 141) The pharmaceutical regimen is administered to a subject having a condition selected from the group of conditions set forth in Table 16. determining the amount of pharmaceutical composition required to achieve a beneficial effect. 141. The pharmaceutical composition of item 140, further comprising: (Item 142) The pharmaceutical regimen for treating the subject comprises administering the pharmaceutical composition in two or more consecutive doses. and administering to said subject an effective amount of At least one of at least one, two, or three parameters associated with the state 0%, or 20%, or 30%, or 40%, or 50%, or 60%, or results in an improvement of 60%, 70%, 80%, or more than 90%, item 141 The pharmaceutical composition according to claim 1. (Item 143) For use in the preparation of a medicament for the treatment of a condition selected from the group of conditions set out in Table 16. For use in items 85-87, 97-99, 108-112, 118-122, and 125 132. The conjugate of any one of claims 127, 129, or 132. (Item 144) 1. A method for selecting a combination of payloads conjugated to XTEN as a therapeutic agent, comprising: a. providing a library of XTENs comprising a plurality of XTEN sequences, Each of the XTEN sequences comprises at least a first payload and a second payload. and providing a second payload conjugated to at least one of the first and second payloads, the second payload being different from the first payload; b. from said library, (1) an XTEN sequence conjugated to said first payload alone; sequence, and (2) compared to that of the XTEN sequence conjugated to the second payload alone. , treating XTEN sequences when they exhibit improved in vitro or in vivo parameters selecting the compound as a drug. (Item 145) The first payload and the second payload are used to treat a common disease. The method according to Item 144, which is effective in (Item 146) The first and second drugs are used therapeutically to treat different symptoms of a common disease. The method according to item 144, which is effective. (Item 147) The common diseases include cancer, cancer supportive care, cardiovascular, central nervous system, endocrine disorders, gastrointestinal, and hematologic disorders. Humoral, HIV infection, hormonal disorders, inflammation, autoimmune disorders, infectious diseases, metabolic disorders, musculoskeletal system Item 145 or selected from diseases, nephrological disorders, ophthalmological diseases, pain, and respiratory Item 146. The method according to item 146. (Item 148) The first payload and the second payload are transported to each other via a common biological pathway. The method according to item 144, wherein the therapeutic effect of the (Item 149) The first payload and the second payload are listed in Table 6, Table 18, and Table 21. 145. The method of claim 144, wherein the two or more drugs are different drugs selected from the group consisting of drugs that are (Item 150) The first payload and the second payload are listed in Table 7, Table 18, and Table 21. and wherein the different biologically active proteins are selected from the group consisting of proteins Item 145. The method according to item 144. (Item 151) The first payload is selected from the group consisting of drugs listed in Table 6, Table 18, and Table 21. and the second payload is a drug selected from the group consisting of those listed in Tables 7, 18, and 21. Item 1, wherein the biologically active protein is selected from the group consisting of proteins 44. The method according to claim 44. (Item 152) SASRSA or SASXSA (X is R or K). The extended recombinant polypeptide is linked to an affinity purification tag via a proteolytic cleavage site. An isolated polypeptide comprising an XTEN. (Item 153) SASRSA or SASXSA (X is R or K). The first affinity purification tag is linked at its N-terminus via a proteolytic cleavage site. , SASRSA, or SASXSA (wherein X is R or K). It is linked at its C-terminus to a second affinity purification tag via a proteolytic cleavage site A polypeptide, including an extended recombinant polypeptide (XTEN). (Item 154) A composition having the structure depicted in FIG.

[0059] In particular, embodiments of the conjugates may exhibit one or more of the properties disclosed herein, or It is contemplated that any combination of the X disclosed herein may be present. Any of the TEN compositions may be used in any of the methods disclosed herein. can be utilized. (Incorporated by reference) All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. or patent application(s) to the same extent as if the application(s) were specifically and individually indicated to be incorporated by reference. and the like, all of which are incorporated herein by reference. [Brief explanation of the drawings]

[0060] The features and advantages of the present invention will become more apparent from the following detailed description and accompanying drawings, which describe exemplary embodiments. The following description may be further interpreted with reference to the accompanying drawings. [Figure 1]Schematics of XTEN suitable for conjugation with a payload are shown. (Figure 1A) Unmodified XTEN is shown. (Figure 1B) Cysteine-engineered XTEN containing an internal cysteine ​​with a thiol side chain is shown. Below that is an XTEN with a reactive N-terminal amino group, and below that is an XTEN containing an N-terminal cysteine ​​with a thiol-reactive group. (Figure 1C) Cysteine-engineered XTEN with multiple internal cysteines is shown. (Figure 1D) Cysteine-engineered XTEN containing an internal cysteine ​​with a thiol side chain and two variations of reactive N-terminal amino groups is shown. At the bottom is a cysteine- and lysine-engineered XTEN with an internal cysteine ​​and an internal lysine. [Figure 2] We demonstrate conjugation reactions utilizing NHS-esters and their water-soluble analogs (sulfo-NHS-esters) to react with primary amino groups to yield stable amide XTEN-payload products. [Figure 3] This conjugation reaction utilizes a thiol group and N-maleimide. The maleimide group reacts specifically with sulfhydryl groups to form a stable, non-reversible thioether bond when the pH of the reaction mixture is between 6.5 and 7.5. [Figure 4] Figure 1 shows a conjugation reaction utilizing haloacetyl. The most commonly used haloacetyl reagents contain an iodoacetyl group, which reacts with sulfhydryl groups at physiological pH. The reaction of the iodoacetyl group with sulfhydryl proceeds by nucleophilic displacement of the iodo with a thiol, producing a stable thioether bond in the XTEN-payload. [Figure 5] We demonstrate a conjugation reaction utilizing pyridyl disulfide, which reacts with sulfhydryl groups over a wide pH range (optimal pH 4-5) to form a disulfide bond linking XTEN to the payload. [Figure 6] Conjugation reactions utilizing zero-length crosslinkers are demonstrated, where these crosslinkers are used to directly conjugate the carboxyl functionality of one molecule (e.g., a payload) to the primary amine of another molecule (e.g., XTEN). [Figure 7]Figure 1 shows different configurations of multifunctional (or multivalent) XTEN precursors, including dendrimers. Non-limiting examples of trifunctional linkers are "Y-shaped" sulfhydryl-reactive (TMEA) (tris-(2-maleimidoethyl)amine) and amine-reactive TSAT (tris(succinimidylaminotriacetate). Any combination of reactive moieties can be designed for multivalent display using either linear (forming a "comb" configuration) or branched (forming a "dendrimer" configuration) scaffold polymers. [Figure 8] As shown, a conjugation reaction utilizing Huisgen 1,3-dipolar cycloaddition of an alkyne to an azide to form a 1,4-disubstituted-1,2,3-triazole is shown. [Figure 9] We demonstrate conjugation reactions using thio-ene-based click chemistry that can proceed via a free radical reaction called the thiol-ene reaction, or via an anionic reaction called the thiol-Michael addition. [Figure 10] Utilizing click chemistry based on the reaction between hydrazides and aldehydes, conjugation reactions leading to hydrazone linkages in exemplary XTEN-payloads are shown. [Figure 11] 1 shows the reaction between a C-terminal acyl azide and a primary amino group, resulting in the formation of an amide bond. [Figure 12] 1 shows a conjugation reaction utilizing Native Chemical Ligation (NCL), which requires a C-terminal thioester as the electrophile and an N-terminal cysteine ​​as the nucleophile. The result of this reaction is a native amide bond at the ligation site of the XTEN-payload composition. [Figure 13]

[0023] A conjugation reaction utilizing the expressed protein ligation (EPL) method is shown. The EPL method is based on protein splicing, a process in which proteins undergo intramolecular rearrangement, resulting in the protrusion of an internal sequence (intein) and the joining of an external sequence (extein). In this method, the fusion protein undergoes an N-S shift when the side chain of the first cysteine ​​residue in the intein portion of the precursor protein nucleophilically attacks the peptide bond of the immediately upstream residue (i.e., the final residue of XTEN), creating a linear thioester intermediate, followed by rearrangement to form an amide bond between the XTEN-crosslinker and the payload. [Figure 14] A traceless Staudinger ligation, such as native chemical ligation (NCL), is utilized to demonstrate a conjugation reaction that results in a native amide bond at the ligation site. [Figure 15] 1 shows a conjugation reaction utilizing enzymatic ligation. Transglutaminase is an enzyme that catalyzes the formation of an isopeptide bond between the γ-carboxyamide group of glutamine in a payload peptide or protein and the ε-amino group of lysine (or the N-terminal amino group) in the lysine-engineered XTEN, thereby creating an inter- or intramolecular crosslink between the XTEN and the payload. [Figure 16] We demonstrate enzymatically created XTEN-payload compositions utilizing the sortase A transpeptidase enzyme from Staphylococcus aureus to catalyze the cleavage of a short five-amino acid recognition sequence, LPXTG, between threonine and glycine residues in protein 1, followed by the transfer of the acyl fragment to the N-terminal oligoglycine nucleophile of protein 1. By functionalizing protein 2 to contain oligoglycines, enzymatic conjugation of the two proteins is achieved in a site-specific manner, resulting in the desired XTEN-payload composition. [Figure 17]Various XTEN-crosslinker precursor segments are shown to be used as reactants to attach to payload or XTEN reactants. (Figure 17A) 1B is intended to indicate that the remaining reactive group of the precursor on the right represents the remaining reactive group. (Figure 17B) Similar reactive precursors with multiple (left) or single (right) Payload A molecules conjugated to XTEN are shown. [Figure 18] 1B and 2B represent reactive groups that react with similarly numbered reactive groups, such as 1 and 1, and 2 and 2, in other figures. [Figure 19] These are intended to illustrate various reactant and nomenclature examples for the configurations shown elsewhere in the figures. (Figure 19A) Various forms of reactive XTEN segment precursors are shown, each with a different reactive group on the N-terminus. (Figure 19B) Various crosslinkers with two, three, or four reactive groups are shown. In the first example, the bivalent crosslinker is a heterofunctional linker that reacts with two different types of reactive groups, represented by "2" and "1." In the case of trivalent and tetravalent crosslinkers, each reacts with only one type of reactive group, represented by "1." (Figure 19C) The nomenclature of the reaction product of two XTEN segment precursors is shown. In the top version, 1A reacts with 1B to create an N-terminally linked dimeric XTEN, with crosslinker residues represented by 1AR-1BR, while the bottom version is also an N-terminally linked dimeric XTEN, with crosslinker residues represented by 2AR-2BR. [Figure 20]The creation of various XTEN precursor segments is shown. (Figure 20A) Shows steps for producing an XTEN polypeptide, where 1A reacts with the N-terminus of the crosslinker bearing a 2B-1A reactive group, followed by reaction of the N-terminus with a crosslinker bearing a 2B-1A reactive group, to create XTEN precursor 2 bearing reactive group 2B. (Figure 20B) Shows sequential addition of two crosslinkers bearing 2A reactive groups to the 2B reactive group of XTEN, resulting in XTEN precursor 4, which then reacts with a crosslinker at the N-terminus between the crosslinker's reactive groups 1B and 1A, to create XTEN precursor 5 bearing reactive groups 4B and 3B. In such cases, XTEN precursor 5 can serve as a reactant with two different payloads or XTENs. [Figure 21] Examples of multimeric conjugates are shown. (Figure 21A) Shows how three molecules of XTEN with conjugated payload A can be conjugated to a trimeric crosslinker, resulting in a trimeric XTEN-payload conjugate with three A payloads. (Figure 21B) Shows how three molecules of a polypeptide with payload A can be conjugated to a trimeric crosslinker, resulting in a trimeric XTEN-payload conjugate with three polypeptides with A payloads. [Figure 22] Figure 22A shows examples of multimeric XTEN conjugates that can originate from an XTEN precursor with a single cysteine. The amino group in the XTEN precursor serves as reactive group 2B, and the thiol group serves as reactive group 1B. The XTEN precursor can be crosslinked using a crosslinker that can react with group 1B. The valence of the crosslinker controls the valence of the resulting intermediate. This crosslinked intermediate can react with a payload bearing reactive group 2A, which can react with the amino group to form conjugate bond 2A-BR. (Figure 22A) XTEN precursor with a single thiol group. (Figure 22B) Divalent conjugate. (Figure 22C) Trimeric conjugate. (Figure 22D) Tetrameric conjugate. [Figure 23]Figure 23A shows an example of a multimeric XTEN conjugate that can originate from an XTEN precursor with a single cysteine. The amino group in the XTEN precursor serves as reactive group 1B, and the thiol group serves as reactive group 2B. The XTEN precursor can be crosslinked using a crosslinker capable of reacting with group 1B. The valence of the crosslinker controls the valence of the resulting intermediate. This crosslinked intermediate can then react with a payload bearing reactive group 2A, which can react with the thiol group to form conjugate bond 2A-BR. (Figure 23A) shows the thiol group positioned near the C-terminus of the XTEN. As a result, the payload is positioned at the distal end of the final trimer conjugate. (Figure 23B) shows the thiol group positioned near the N-terminus of the XTEN. As a result, the payload is positioned at the distal end of the final trimer conjugate, resulting in increased payload shielding by the XTEN. [Figure 24] Examples of the creation of a "comb" architecture are shown. (Figure 24A) An XTEN-payload precursor containing a linker reactive group 1A. This payload can be recombinantly fused or conjugated to an XTEN. (Figure 24B) An XTEN precursor with a comb-like crosslinker is shown. This can be an XTEN carrying multiple reactive groups B. (Figure 24C) The final product of a "comb" architecture with five payloads A is shown. Valence is controlled by the number of reactive groups in the comb-like precursor. [Figure 25] Various configurations of bispecific conjugates with two payloads are shown: (Figure 25A) shows a configuration with one molecule of each of the two payloads, while Figure 25B shows various configurations with multiple copies of one or both payloads. [Figure 26] Various examples of high valency conjugates are shown. Payload conjugation moieties can be clustered (Figure 26A) or interspersed (Figure 26B). [Figure 27]Figure 27 shows the preparation of bispecific conjugates from XTEN precursors bearing both amino and thiol groups; many chemistries can be used, and the order of payload addition can vary. Linker conjugates can be generated as precursors. (Figure 27A) Shows the creation of a single XTEN precursor to which two different payloads are attached. (Figure 27B) Shows a segment approach starting with two XTEN precursor molecules. This approach allows both payloads to be conjugated to the XTEN using the same type of linker chemistry. In this case, the figure shows a thiol as the group to which the payload is conjugated; the N-terminus of each segment is then modified with a crosslinker to allow head-to-head segment conjugation, resulting in a dimeric bispecific conjugate final product. [Figure 28] Examples of multimeric conjugates combining antibodies, XTENs, and payloads are shown. Such constructs can offer many advantages: they can have different valencies, the XTENs can have cleavable linkers, the XTENs can provide solubility to the composition, allow for tuning of drug loading per IgG, and the XTENs can be pre-conjugated with drugs to simplify manufacturing. (Figure 28A) Two XTENs are shown conjugated to IgG at Cys residues in the hinge region. (Figure 28B) Four XTENs are shown conjugated to IgG using Cys in the hinge region. (Figure 28C) XTENs conjugated outside the hinge are shown. This can be done by inserting Cys to control the conjugation site or by random conjugation to Lys side chains. [Figure 29] An example of constructing a conjugate combining an antibody, XTEN, and a payload is shown. The antibody can have one or more reactive groups 1B. The XTEN can be conjugated to one or more payloads A. In addition, the XTEN can carry reactive groups 1A that preferentially react with reactive groups 1B on the antibody. The location of reactive groups 1B in the antibody controls the number and location of XTEN conjugated to the antibody, resulting in the final product. [Figure 30]30A-30C show examples of conjugates comprising a targeting moiety, XTEN, and a payload. The targeting moiety can be a peptide, peptoid, or receptor ligand. (Figure 30A) shows a 1x (1x3) conjugate. (Figure 30B) shows a 1x2 (1x3) conjugate. (Figure 30C) shows a 3x1 (1x3) conjugate. [Figure 31] 1 shows an example of a conjugate comprising multiple different targeting moieties, XTEN, and a payload. The targeting moiety can be a peptide, peptoid, or receptor ligand. [Figure 32] 1 shows examples of conjugates that include a targeting moiety, an XTEN, and multiple different payloads. [Figure 33] An example of a combinatorial XTEN conjugate is shown. Payloads A, B, C, and D carry reactive group 2A, which reacts with reactive group 2B on the XTEN precursor. In the next step, payloads E and F carry reactive group 1A, which reacts with reactive group 1B on the XTEN, resulting in a library of differently substituted bispecific conjugates. In this case, reactive groups 1B and 2B are thiol and amino groups, respectively. [Figure 34] An example of the creation of a combinatorial XTEN conjugate library is shown. Payloads A, B, and C are conjugated to XTEN bearing reactive group 1A, resulting in one set of XTEN precursor segments. Payloads E, F, and G are conjugated to XTEN bearing reactive group 1B, resulting in a second set of XTEN precursor segments. These segments are subjected to combinatorial conjugation and then purified from the reaction mixture. This allows for the formation of combinatorial products that can be immediately subjected to in vitro and in vivo testing. In this case, reactive groups 1A and 1B are the alpha amino groups of XTEN with or without a bispecific crosslinker. In one example, 1A is an azide and 1B is an alkyne, or vice versa, while the payloads are attached to the XTEN via thiol groups in the XTEN. [Figure 35] An example is shown of the creation of a combinatorial XTEN conjugate library that optimizes the ratio between two payloads, with each library member carrying a different ratio of payload A and payload E. [Figure 36]An example of creating a combinatorial XTEN conjugate library, creating combinations of targeting moieties and payloads, is shown. Targeting moieties 1, 2, and 3 are conjugated to XTENs bearing reactive group 1A. Payloads E, F, and G are conjugated to XTENs bearing reactive group 1B. These segments are subjected to combinatorial conjugation to form combinatorial products, each library member containing a targeting moiety and a payload. All XTEN segments bearing a payload and a conjugation group can be purified as combinatorial products ready for in vitro and in vivo testing. [Figure 37]

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[0085] [Figure 38] 1 is a schematic flow chart of representative steps in the construction, production, and evaluation of XTEN. [Figure 39]1 is a schematic flowchart of representative steps in the assembly of an XTEN polynucleotide construct encoding a fusion protein. Individual oligonucleotides 501 are annealed to a sequence motif 502, such as a 12-amino acid motif ("12-mer"), and ligated to additional sequence motifs from a library to create a pool encompassing XTEN of the desired length 504, while simultaneously ligating to oligos containing lower concentrations of BbsI and KpnI restriction sites 503. The resulting pool of ligation products is gel purified, and the band with the desired XTEN length is excised, resulting in an isolated XTEN gene with a stopper sequence 505. The XTEN gene is cloned into a stuffer vector. In this case, the vector encodes an optional CBD sequence 506 and a GFP gene 508. Digestion is then performed with BbsI / HindIII to remove 507 and 508 and place a stop codon. The resulting product is then cloned into a BsaI / HindIII digested vector, resulting in gene 500 encoding the XTEN. [Figure 40] 1 is a schematic flow chart of representative steps in the assembly of a gene encoding an XTEN, its expression, conjugation to a payload, and recovery as an XTEN-payload, and its evaluation as a candidate product. [Figure 41] Using the method shown in Figure 42, generalized XTEN with either N- or C-terminal tags or N- and C-terminal sequences optimized for purification are shown. [Figure 42] This exemplary embodiment shows a generalized scheme for the purification of dual-tagged XTENs, where a two-step purification method to capture the first tag and then the second tag can be used to remove cleaved XTENs from the fermentation, resulting in highly purified target XTEN entities. [Figure 43]1 shows an SDS-PAGE gel of CBD-TEV site-XTEN_AE864 and CBD-TEV site-XTEN_AE864-GFP constructs expressed in E. coli BL21 DE3rne-131 and E. coli BL21 DE3 cells from shake flask cultures, as described in Example 10. Proteins expressed from the gel lanes containing MW markers are: 1) MW marker; 2–5) lysates from four independent flasks expressing the CBD-TEV site-XTEN_AE864-GFP fusion protein in E. coli BL21 DE3; 6–9) lysates from four independent flasks expressing the CBD-TEV site-XTEN_AE864-GFP fusion protein in E. coli BL21 DE3rne-131; 10–13) lysates from four independent flasks expressing the CBD-TEV site-XTEN_AE864 fusion protein in E. coli BL21 DE3rne-131; and 14–17) lysates from four independent flasks expressing the CBD-TEV site-XTEN_AE864 fusion protein in E. coli BL21 DE3rne-131. The full-length protein spot appears within the outer box. Lower molecular weight bands represent host cell proteins. [Figure 44] 1 shows the relative GFP fluorescence of CBD-TEV site-XTEN_AE864-GFP expressed in E. coli BL21 DE3rne-131 and E. coli BL21 DE3 cells from shake flask cultures as described in Example 10. [Figure 45]1 shows an SDS-PAGE gel of CBD-RC-XTEN_AE864-RH8 (EC682) and CBD-R-XTEN_AE864-RH8 (EC683) constructs expressed in E. coli fermentation as described in Example 17. Gel lanes with MW markers show samples and proteins expressed from the constructs: 1) MW marker; 2) 16-hour, 3) 24-hour, 4) 40-hour, and 5) 45-hour post-inoculation E. coli fermentation #EC682 clarified soluble lysate; 6) 16-hour, 7) 24-hour, 8) 40-hour, and 9) 45-hour post-inoculation E. coli fermentation #EC683 clarified soluble lysate; 10) 1 microgram, 11) 2 micrograms, and 12) 4 micrograms of purified CBD-R-XTEN_AE864-RH8 reference standard. For E. coli fermentation clarified soluble lysate, each lane represents 3 microliters of fermenter culture. Full-length protein spots appear within the outline. Lower molecular weight bands are host cell proteins. [Figure 46] 1 shows the trace output of Toyopearl Phenyl 650M hydrophobic interaction chromatography, as described in Example 18. [Figure 47] Figure 1 shows non-reducing 4-12% Bis-Tris SDS-PAGE analysis of Toyopearl Phenyl 650M hydrophobic interaction chromatography fractions as shown in the figure and described in Example 18. Material per lane: Lane 1: Marker; Lane 2: Load 7.5 μl; Lane 3: Flow-through 1; Lane 4: Flow-through 2; Lane 5: Elution fraction E1; Lane 6: Elution fraction E2; Lane 7: Elution fraction E3; Lane 8: Elution fraction E4; Lane 9: Elution fraction E5; Lane 10: Elution fraction E6; Lane 11: Elution fraction E7; Lane 12: Elution fraction E8. [Figure 48] Figure 48 shows non-reducing 4-12% Bis-Tris SDS-PAGE analysis of Toyopearl IMAC chromatography flow-through, wash (Figure 48A), and elution fractions (Figure 48B) (non-reduced), as described in Example 18. [Figure 49]10 shows a non-reducing SDS-PAGE analysis of the trypsin-digested IMAC pool described in Example 18. [Figure 50] 1 shows the elution profile of MacroCap Q chromatography described in Example 18. [Figure 51] Figure 51 shows 4-12% Bis-Tris SDS-PAGE analysis of MacroCap Q elution fractions as described in Example 18. (Figure 51A) Flow-through, Coomassie stain. (Figure 51B) Elution fractions, Coomassie stain. (Figure 51C) Elution fractions, silver stain. [Figure 52] 1 shows traces from C18 RP-HPLC analysis of MacroCap Q elution fractions, as described in Example 18. [Figure 53] 1 shows a trace from C18 RP-HPLC of the MacroCap Q elution pool as described in Example 18. [Figure 54] 1 shows non-reducing SDS-PAGE analysis of Toyopearl Phenyl 650M hydrophobic interaction chromatography fractions as described in Example 19. [Figure 55] 1 shows non-reducing SDS-PAGE analysis of Toyopearl IMAC chromatography fractions as described in Example 19. [Figure 56] Non-reducing 4-12% Bis-Tris SDS-PAGE / silver stain analysis of MacroCap Q elution fractions as described in Example 19 is shown. [Figure 57] 1 shows traces from C18 RP-HPLC analysis of MacroCap Q elution fractions, as described in Example 19. [Figure 58] 1 shows a trace from a C18 RP-HPLC analysis of the MacroCap Q elution pool described in Example 19. [Figure 59]Figure 2 shows SDS-PAGE analysis of XTEN constructs with experimental tags after expression in E. coli, as described in Example 20. Soluble lysates were loaded onto a 4-12% Bis-Tris polyacrylamide gel, with the amount loaded per lane equivalent to 36 μl of cell culture suspension. Gels were stained with Coomassie blue using standard methods. [Figure 60] Figure 2 shows SDS-PAGE analysis of the RP11-XTEN-His8 construct expressed in E. coli as described in Example 20. Heat-treated soluble lysates were loaded onto a 4-12% Bis-Tris polyacrylamide gel in an amount equivalent to 1 or 2 μl of cell culture suspension, respectively. The gel was stained with Coomassie blue. The gel demonstrates that essentially all of the expressed RP11-XTEN-His8 protein was found in the pellet fraction. [Figure 61] 1 shows SDS-PAGE analysis of MacroCap SP purification of RP11-XTEN-His8 polypeptide as described in Example 21. Fractions were analyzed by 4-12% SDS-PAGE followed by Coomassie staining. [Figure 62] 1 shows SDS-PAGE analysis of IMAC purification of RP11-XTEN-His8 polypeptide described in Example 21. Fractions were analyzed by 4-12% SDS-PAGE followed by Coomassie staining. [Figure 63] Figure 63 shows SDS-PAGE analysis of trypsin digestion of RP11-XTEN-His8 protein purified by two chromatography steps (SP+IMAC) as described in Example 21. Preparations were analyzed by 4-12% SDS-PAGE followed by Coomassie staining (Figure 63A) and silver staining (Figure 63B). [Figure 64]Figure 64A shows the results of an analysis of the conjugation reaction of DBCO-Mal to 3x thiol-XTEN, as described in Example 23. (Figure 64A) C18 RP-HPLC analysis of the reaction mixture. A 20 μg protein sample was loaded onto a Phenomenex Jupiter C18 5 μM 300A 4.6 mm x 150 mm column. Protein was eluted with a 5-50% gradient of acetonitrile in 0.1% trifluoroacetic acid. (Figure 64B) HIC purification of the DBCO-XTEN reaction product. (Figure 64C) C18 RP-HPLC analysis of the HIC-purified DBCO-XTEN reaction product. [Figure 65] Figure 65A shows results from trypsin cleavage of dual-tagged precursor XTEN as described in Example 24. (Figure 65A) Shows 4-12% Bis-Tris SDS-PAGE analysis of protein samples loaded at 2 μg per lane. Gels were stained with Invitrogen SimplyBlue SafeStain. (Figure 65B) Shows 4-12% Bis-Tris SDS-PAGE analysis of protein samples loaded at 0.5 μg per lane. Gels were stained with Pierce Silver Stain Kit. [Figure 66] Figure 66 shows the results of SDS-PAGE analysis of MacroCap Q purification of trypsin-digested dual-tagged precursor as described in Example 24. (Figure 66A) Shows 4-12% Bis-Tris SDS-PAGE analysis of protein samples loaded at 3 μg per lane. Gels were stained with Invitrogen SimplyBlue SafeStain. (Figure 66B) Shows 4-12% Bis-Tris SDS-PAGE analysis of protein samples loaded at 0.5 μg per lane. Gels were stained with Pierce Silver Stain Kit. (Figure 66C) Shows 4-12% Bis-Tris SDS-PAGE analysis of protein samples loaded at 0.5 μg per lane. Gels were stained with Pierce Silver Stain Kit. [Figure 67]

[00110] Figure 67 shows the results of a C18 RP-HPLC test for residual trypsin activity. (Figure 67A) Trace output from an analysis of the intact form of the synthetic [G2]GLP2 peptide. (Figure 67B) Trace output from an analysis of the synthetic [G2]GLP2 peptide digested with bovine trypsin. (Figure 67C) Trace output from an analysis of XTEN_AE869_Am1,C2 spiked with [G2]GLP2 and incubated overnight at 37°C, as described in Example 24. [Figure 68] This figure shows the preparation of a GLP2-XTEN conjugate from GLP2-Cys peptide and 1x amino-XTEN, as described in Example 26. A 20 μg protein sample was loaded onto a Phenomenex Jupiter C18 5 μM 300A 4.6 mm x 150 mm column. Protein was eluted with a 5-50% gradient of acetonitrile in 0.1% trifluoroacetic acid and detected by absorbance at 214 nm (left panels A-C). A 100 μg protein sample was desalted using a NanoSep 3K Omega centrifugal device (Pall Corp.). Protein solutions in 50% acetonitrile, 0.5% formic acid were injected into a high-resolution mass spectrometer at a flow rate of 10 μl / min. ESI-MS spectra were acquired in the 800-1600 amu (atomic mass unit) range and reconstructed into zero-charge spectra using Bayesian protein reconstruction software (right panels A-C). (Figure 68A): The initial 1x amino-XTEN protein. (Figure 68B): The product of the reaction between 1x amino-XTEN and sulfo-SMCC crosslinker. (Figure 68C): Purified GLP2-XTEN conjugate after the reaction between GLP2-Cys and N-Mal-XTEN. [Figure 69]This figure shows the preparation of a GLP2-XTEN conjugate from GLP2-Mal peptide and 1x thiol-XTEN, as described in Example 27. A 20 μg protein sample was loaded onto a Phenomenex Jupiter C18 5 μM 300A 4.6 mm x 150 mm column. Protein was eluted with a 5-50% gradient of acetonitrile in 0.1% trifluoroacetic acid and detected by absorbance at 214 nm (left panels A and B). A 100 μg protein sample was desalted using a NanoSep 3K Omega centrifugal device (Pall Corp.). Protein solutions in 50% acetonitrile, 0.5% formic acid were injected into a high-resolution mass spectrometer at a flow rate of 10 μl / min. ESI-MS spectra were acquired within the 800-1600 amu range and reconstructed into zero-charge spectra using Bayesian protein reconstruction software (right panels A and B). (Figure 69A): The initial 1x thiol-XTEN protein. (Figure 69B): The product of the reaction between GLP2-Mal and 1x thiol-XTEN. [Figure 70]

[0071] Figure 70A shows the results of purification of GLP2-XTEN using preparative C4 RP-HPLC, as described in Example 27. (Figure 70A) The chromatographic profile of the preparative RP-HPLC is shown. The fraction from 56 to 62 minutes was collected and evaporated under vacuum. (Figure 70B) The analysis of purified GLP2-XTEN by C18 RP-HPLC is shown. [Figure 71] Figure 71A shows the results of conjugation of DBCO-Mal to 1x thiol-XTEN, as described in Example 28. (Figure 71A) C18 RP-HPLC analysis of the reaction mixture. A 20 μg protein sample was loaded onto a Phenomenex Jupiter C18 5 μM 300A 4.6 mm x 150 mm column. Protein was eluted with a 5-50% gradient of acetonitrile in 0.1% trifluoroacetic acid. (Figure 71B) HIC purification of DBCO-XTEN. (Figure 71C) C18 RP-HPLC analysis of HIC-purified DBCO-XTEN. [Figure 72]Figure 72A shows the results of an analytical assay of XTEN conjugated with cross-linked FITC, as described in Example 31. (Figure 72A) The gel was imaged with a UV light box, demonstrating the large apparent MW of the FITC-containing conjugated species, and also showed co-migration in the gel as detected by SEC at OD 214 (protein signal) and OD 495 (FITC signal) in the SEC column, indicating good labeling of the XTEN with minimal free dye contamination. The material by lane (left to right) after the MW standard is labeled FITC-CL-CBD-XTEN, labeled FITC-CL-XTEN, purified FITC-CL-XTEN, purified FITC-CL-XTEN, and purified FITC-CL-XTEN. The gel was imaged with a UV light box, showing the FITC apparent MW of the FITC-containing species. (Figure 72B) The results of the SEC analysis of FITC-conjugated XTEN show the overlap of the output of the material detected at OD 214 and OD 495, and also the large apparent molecular weight. [Figure 73] 1 shows the results of SEC analysis of peak elution fractions of conjugates of GFP crosslinked to XTEN and free GFP, as described in Example 32. Crosslinking was confirmed by co-migration of the OD214 protein signal and the OD395 GFP signal in the SEC column. [Figure 74] 1 shows the results of a pharmacokinetic assay of GFP-X-XTEN and FITC-X-XTEN tested in cynomolgus monkeys, as described in Example 33. [Figure 75]Figure 1 shows the pharmacokinetic profiles (plasma concentrations) in cynomolgus monkeys after single doses of different compositions of GFP conjugated to unstructured polypeptides of varying lengths were administered either subcutaneously or intravenously, as described in Example 33. The compositions were GFP-L288, GFP-L576, GFP-XTEN_AF576, GFP-Y576, and XTEN_AD836-GFP. Blood samples were analyzed at various time points after injection, and plasma GFP concentrations were measured by ELISA using a polyclonal antibody against GFP for capture and a biotinylated preparation of the same polyclonal antibody for detection. Results are presented as plasma concentration versus time (h) after administration and show a significant increase in half-life, particularly for XTEN_AD836-GFP, the composition with the longest XTEN sequence length. The construct with the shortest sequence length, GFP-L288, had the shortest half-life. [Figure 76] 1 shows an SDS-PAGE gel of samples from a stability study of a fusion protein of XTEN_AE864 fused to the N-terminus of GFP. As described in Example 55, GFP-XTEN was incubated in cynomolgus monkey plasma and rat kidney lysate for up to 7 days at 37°C. Additionally, GFP-XTEN administered to cynomolgus monkeys was also evaluated. Samples were extracted on days 0, 1, and 7 and analyzed by SDS-PAGE followed by detection using Western analysis and an antibody against GFP. [Figure 77] 1 shows the near-UV circular dichroism spectrum of Ex4-XTEN_AE864, performed as described in Example 56. [Figure 78] 1 shows the results of size exclusion chromatography analysis of glucagon-XTEN construct samples measured against protein standards of known molecular weight, with the graphical output as absorbance versus retention volume, as described in Example 58. The glucagon-XTEN constructs are 1) glucagon-Y288, 2) glucagon-Y-144, 3) glucagon-Y72, and 4) glucagon-Y36. The results are shown. [Figure 79]5 is a schematic diagram of the logic flow chart of algorithm SegScore (Example 59). In this diagram, the following legend applies: i, j - counters used in the control loop that goes through the entire sequence; HitCount - this variable is a counter that keeps track of how many times a subsequence encounters the same subsequence within a block; SubSeqX - this variable holds the subsequence that is being checked for redundancy; SubSeqY - this variable holds the subsequence that SubSeqX is being checked for; BlockLen - this variable holds the user-determined length of the block; SegLen - this variable holds the length of the segment. The program is hard-coded to generate scores for subsequences of lengths 3, 4, 5, 6, 7, 8, 9, and 10; Block - this variable holds the length of the string, BlockLen. This string consists of characters from the input XTEN sequence and is determined by the position of the i counter; and SubSeqList - this is a list that holds all of the generated subsequence scores. [Figure 80] This figure illustrates the application of the algorithm SegScore to a hypothetical XTEN of 11 amino acids to determine repetitiveness. An XTEN sequence of N amino acids is divided into N-S+1 subsequences of length S (where S=3 in this case). Pairwise comparisons of all subsequences are performed and the average number of identical sequences is calculated, resulting in a subsequence score of 1.89 in this case. [Figure 81] Results are provided for an assay measuring the fluorescent signal of RP11 clones (pSD0107-pSD0118) as described in Example 12. One positive control (pLCW970) and two negative controls (pBr322 and pLCW970 + 10 mM phosphate) were included. GFP expression levels were measured using samples from two to three shake flasks per construct. [Figure 82A-C]Screening results of libraries LCW1157-1159 are shown. (Figures 82A-C) Fluorescence histograms of LCW1157-1159 are provided, showing the number of colonies identified for each luminescence signal region, as described in Example 12. The mean fluorescence readings of the negative control (black arrow) and the positive pSD0116 (white arrow) are labeled in the figures. (Figures 82D-F) Correlation between fluorescence readings in the original test and retest of selected clones is provided. [Fig. 82D-F] Screening results of libraries LCW1157-1159 are shown. (Figures 82A-C) Fluorescence histograms of LCW1157-1159 are provided, showing the number of colonies identified for each luminescence signal region, as described in Example 12. The mean fluorescence readings of the negative control (black arrow) and the positive pSD0116 (white arrow) are labeled in the figures. (Figures 82D-F) Correlation between fluorescence readings in the original test and retest of selected clones is provided. [Figure 83] SDS-PAGE analysis of the top eight expression construct products and controls under non-reducing conditions, as described in Example 12, is shown. The desired full-length protein end product, RP11-XTEN-GFP, is indicated by an arrow; the higher band is the protein dimer. Lanes 1-8: Top eight expression constructs (highest to lowest expression levels, based on retest fluorescence readings): 1. LCW1159.004, 2. LCW1159.006, 3. LCW1158.004, 4. LCW1157.040, 5. LCW1158.003, 6. LCW1157.039, 7. LCW1157.025, 8. LCW1157.038. C1-C3: Controls: C1.pSD0114, C2.pSD0116, C3.pCW1146 (negative control). [Figure 84]SDS-PAGE evaluation of MacroCap SP capture efficiency of the top four expression construct products under non-reducing conditions, as described in Example 12, is shown. Lanes 1-4: Load, flow-through, wash, and elution of LCW1159.004. Lanes 5-8: Load, flow-through, wash, and elution of LCW1159.006. Lanes 9-12: Load, flow-through, wash, and elution of LCW1158.004. Lanes 13-16: Load, flow-through, wash, and elution of LCW1157.040. Lanes 17-20 1-4: Load, flow-through, wash, and elution of negative control. Unlabeled lanes are molecular weight standards. [Figure 85] A summary of library LCW1163 screening results is shown, with the top four expression products in the retest compared to the fluorescent signal of the control, as described in Example 12. Each sample had four replicates and is represented in the figure by four individual dots. [Figure 86]

[0033] Figure 1 shows a summary of library LCW1160 screening results, as described in Example 12. Fluorescence histogram of LCW1157-1159 showing the number of colonies identified for each fluorescent signal region. The mean fluorescent readings of the negative control (black arrow), pSD0116 (white arrow), and LCW1159.004 (high-expressing candidate from screening LCW1157-1159, gray arrow) are labeled in the figure. [Figure 87] 4-12% SDS-PAGE / silver stain analysis of MacroCap Q fractions as described in Example 14. (Figure 87A): Batch 2, lane 1: molecular weight standard; lanes 2-5: MacroCap Q flow-through fractions 1-4, respectively; lanes 6-16: MacroCap Q elution fractions 1-11, respectively. (Figure 87B): 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. [Figure 88] 1 shows results from the analysis of intermediates and final products during the preparation of 1xDBCO, 3xLHRH-XTEN, as described in Example 34. [Figure 89]Figure 89 shows the results of analysis of reaction mixtures from the preparation of conjugates for 1x azide, 3x MMAE-XTEN analyzed by C18-RP-HPLC and mass spectrometry, as described in Example 35. (Figure 89A) Analysis of the initial 1x amino, 3x thiol-XTEN reaction product. (Figure 89B) Analysis of protein modification with MMAE-maleimide showing the mass increase corresponding to the modification of three cysteines with MMAE-Mal. (Figure 89C) Analysis of protein modification with azido-PEG4-NHS ester showing the mass increase corresponding to the single addition of an azido-PEG4 moiety. [Figure 90] Figure 90A shows an analysis of reaction products in the conjugation of 3xLHRH, 3xMMAE-XTEN, as described in Example 36. (Figure 90A): SDS-PAGE analysis of click conjugates. 0.5 μg of protein was loaded per lane on a 12% Bis-Tris NuPAGE minigel (Life Technologies). The gel was stained with a Pierce Silver Stain Kit (Thermo Scientific, catalog #24612). Lane 1, 1x azide, 3xMMAE-XTEN; lane 2, 1x DBCO, 3xLHRH-XTEN; lane 3, click chemistry reaction product. Conjugation product bands are indicated by arrows. (Figure 90B): C4 RP-HPLC analysis of click conjugation reactions and products—(1) 1x DBCO, 3xLHRH-XTEN; (2) 1x azide, 3xMMAE-XTEN; (3) click chemistry reaction product. [Figure 91]

[0093] Figure 91A shows a flow chart of the reactions during the preparation of 1xLHRH, 3xMMAE-XTEN conjugates as described in Example 37. (Figure 91A): Initial 1x amino, 3x thiol-XTEN; (Figure 91B): Protein modification with 2,2'-dipyridyl disulfide; (Figure 91C): Protein modification with DBCO-sulfo-NHS; (Figure 91D): Cysteine ​​deprotection with TCEP; (Figure 91E) Modification of three cysteines with MMAE-Mal; (91F): Conjugation of LHRH-azide to the N-terminal DBCO. [Figure 92]Figure 92A shows a flow chart of the reactions during the preparation of a 1xMal, 3xPTX-XTEN reactant conjugate, as described in Example 41. (Figure 92A): Initial 1x amino, 3x thiol-XTEN; (Figure 92B): Protein modification with PTX-Mal; (Figure 92C) Protein modification with sulfo-SMCC. [Figure 93]

[0093] Figure 93A shows analytical results of reaction mixtures from the preparation of iodoacetyl-XTEN, as described in Example 42. (Figure 93A): 1x amino-XTEN analyzed by C18-RP-HPLC before and after incubation with 10x excess SIA. (Figure 93B): ESI-MS analysis of 1x amino-XTEN modified with SIA. (Figure 93C): Sample analyzed by C18 RP-HPLC - bottom profile - HCKFWW peptide. Middle profile - IA-XTEN. Top profile - reaction of IA-XTEN with 5x excess HCKFWW peptide. [Figure 94]

[0049] Figures 94A-D show the results of screening libraries LCW1171, 1172, 1203, and 1204, as described in Example 14. (Figures 94A-D): Fluorescence histograms of LCW1171, 1172, 1203, and 1204, showing the number of colonies identified for each fluorescent signal region; mean fluorescence readings of the negative control (black arrow) and pSD0116 (white arrow) when screening LCW1171-1172 are labeled in Figures 94A and B, and mean fluorescence readings of the negative control (black arrow), pSD0116 (white arrow), and CBD control (gray arrow) when screening LCW1203-1204 are labeled in Figures 94C and D. [Figure 95] Figures 95A-C show the results of screening libraries LCW1208-1210 as described in Example 12. (Figures 95A-C): Fluorescence histograms of LCW1208-1210 showing the number of colonies identified for each fluorescent signal region, with the mean fluorescence readings of the negative control (black arrow) and CBD control (gray arrow) labeled in the figure. [Figure 96]Figure 96A shows the production of XTEN segments from precursors containing three repeated copies of XTEN of the same length and sequence. In Figure 96A, the XTEN precursor contains three identical copies of XTEN flanked by identical protease cleavage sites. In Figure 96B, the XTEN precursor further contains N- and C-terminal affinity purification tags to facilitate purification of the full-length precursor molecule. Following purification of the precursor, it is cleaved by a protease that acts on all of the incorporated cleavage sequences to release the tags from the XTEN, followed by purification to separate the individual units of XTEN, facilitating high-yield production of short- and medium-length XTEN from the long precursor molecule. [Figure 97] Figures 97A and 97B show different embodiments of trimeric branched XTEN-payload conjugates. All conjugates shown can be prepared from the same XTEN molecule via conjugation to its N-terminal amino group and functional groups located near the C-terminus (e.g., the thiol of cysteine). (Figures 97A and 97B) Conjugates with a single payload molecule are shown. Figure 97A uses a four-arm crosslinker with all of the XTEN conjugated in close proximity to the payload, resulting in significant shielding of payload interactions with other molecules. (Figure 97B) A configuration is shown in which the payload is conjugated to a single XTEN arm that is branched at the distal end of the configuration, resulting in reduced payload shielding compared to the configuration in Figure 97A. (Figure 97C) A conjugate with two payloads is shown, which can result in increased affinity or increased capacity. (Figures 97D and 97E) A configuration with three identical payloads is shown, further increasing capacity and / or affinity. (Figure 97F) A configuration with one payload A and two identical copies of payload B is shown for high-affinity binding or interaction. (Figure 97G) Shows a configuration with three different payloads, allowing for the inclusion of three different functions in a single XTEN conjugate. [Figure 98]A scheme for the synthesis of a conjugate between a branched XTEN and a single payload molecule is shown. First, the thiol group in the XTEN is blocked by reaction with iodoacetamide (alternatively, synthesis can begin with an XTEN lacking a thiol group). Next, a DBCO group is added to the α-amino group of the XTEN, which is then reacted with a tetrafunctional crosslinker containing one iodoacetyl group and three azide groups. The resulting XTEN is then reacted with a payload bearing a free thiol group, resulting in the final XTEN-payload conjugate. [Figure 99] Figure 1 shows a scheme for the synthesis of conjugates between branched XTEN and a single payload molecule. An intermediate is produced by reacting XTEN containing two azide functional groups and one NHS functional group with a trifunctional linker, followed by the addition of payload A to the thiol group via maleimide chemistry (the order of these two steps can be reversed). A second intermediate is produced by reacting XTEN with cysteine ​​bearing iodoacetamide to block the free thiol group, followed by the addition of DBCO to the α-amino group via NHS activation (the order of these two steps can be reversed). The two intermediate molecules are then conjugated using click chemistry to yield the final XTEN-payload conjugate. [Figure 100] A scheme for the synthesis of a conjugate containing one branched XTEN and two identical payload molecules is shown. An intermediate is produced by adding a DBCO group to the α-amino group of XTEN via NHS chemistry. A second intermediate is produced by blocking the free thiol group of XTEN with iodoacetamide, followed by the addition of a trifunctional crosslinker (two N-maleimide groups and one carboxyl group activated by NHS) to the α-amino group (the order of these two steps can be reversed). The two intermediates react to produce a branched conjugate, and then two Payload A molecules are added via click chemistry to produce the final XTEN-payload conjugate. [Figure 101]Figure 1 shows a scheme for the synthesis of a conjugate with one branched XTEN and three identical payload molecules. An intermediate is produced by adding a DBCO group to the α-amino group of the XTEN via NHS chemistry. Another intermediate is produced by conjugating payload A to the thiol group of the XTEN via an N-maleimide functional group. The three molecules are linked together via a trifunctional crosslinker containing three azide functional groups, resulting in the final XTEN-payload conjugate. [Figure 102] Figure 1 shows a scheme for the synthesis of a conjugate containing one branched XTEN and three identical payload molecules. The intermediate is produced by adding a DBCO group to the thiol group of XTEN A via N-maleimide chemistry. In the next step, Payload A is conjugated to the α-amino group of the XTEN intermediate via NHS chemistry. The three molecules are linked via a trifunctional crosslinker containing three azide functional groups, resulting in the final XTEN-payload conjugate. [Figure 103] Figure 1 shows a scheme for the synthesis of a conjugate with one branched XTEN, two payload A, and one payload B molecule per conjugate. An intermediate is produced by adding payload A to the thiol group of XTEN using an N-maleimide functional group, followed by the addition of a trifunctional crosslinker (two azide groups and one carboxyl group activated by NHS) to the α-amino group (the order of these two steps can be reversed). A second intermediate is produced by adding DBCO to the α-amino group of XTEN via NHS activation, followed by the addition of payload B to the free thiol group of XTEN using an N-maleimide group (the order of these two steps can be reversed). Two molecules of the second intermediate are reacted with one molecule of the first intermediate to form the final XTEN-payload conjugate. [Figure 104]A scheme for the synthesis of a conjugate with one branched XTEN and three different payloads is shown. An intermediate is produced by adding payload A to a thiol group on the XTEN using an N-maleimide functional group, followed by the addition of a trifunctional crosslinker (one azide group, one N-maleimide group, and one carboxyl group activated by NHS) to the α-amino group (the order of these two steps can be reversed). A second intermediate is produced by adding payload B to the α-amino group of the XTEN via NHS chemistry. A third intermediate is produced by adding DBCO to the α-amino group of the XTEN via NHS activation, followed by the addition of payload C to the free thiol group of the XTEN using an N-maleimide group (the order of these two steps can be reversed). The three intermediates react with each other to form the final XTEN-payload conjugate. [Figure 105] Figure 1 shows a scheme for the synthesis of conjugates with dimeric or tetrameric branched XTEN and payload A molecules. An intermediate is produced by adding DBCO to the thiol group of XTEN using an N-maleimide functional group, followed by adding payload A to the amino group of XTEN using NHS (the order of these two steps can be reversed). The intermediate is then multimerized by the addition of an azide crosslinker. The use of a divalent crosslinker results in a dimeric configuration, while the use of a tetravalent crosslinker results in a tetrameric final product. [Figure 106]Figure 1 shows a scheme for the synthesis of a conjugate with one branched XTEN and three different payloads. An intermediate is produced by adding payload A to the thiol group of XTEN using an N-maleimide functional group, followed by the addition of a trifunctional crosslinker (one azide group, one N-maleimide group, and one carboxyl group activated by NHS) to the α-amino group (the order of these two steps can be reversed). A second intermediate is produced by adding payload B to the free thiol group of XTEN via the N-maleimide functional group. A third intermediate is produced by adding DBCO to the α-amino group of XTEN via NHS activation, followed by the addition of payload C to the free thiol group using an N-maleimide group (the order of these two steps can be reversed). The three intermediates react with each other to form the final XTEN-payload conjugate. [Figure 107] Figure 107 shows the results of analysis of reaction mixtures from the preparation of conjugates for 1xDBCO, 3xFA(γ)-XTEN analyzed by C18-RP-HPLC and mass spectrometry, as described in Example 38. (Figure 107A) Analysis of the initial 1x amino, 3x thiol-XTEN reaction. (Figure 107B) Analysis of protein modification with folate-γ-maleimide showing the mass increase corresponding to the modification of three cysteines with FA(γ)-Mal. (Figure 107C) Analysis of protein modification with DBCO-sulfo-NHS ester showing the mass increase corresponding to the single addition of a DBCO moiety. [Figure 108] 1 shows C4 RP-HPLC analysis of click conjugation reactions and products 3xFA(γ), 3xMMAE-XTEN as described in Example 39. (1) 1xDBCO, 3xFA(γ)-XTEN; (2) 1xAzide, 3xMMAE-XTEN; (3) Product of click chemistry reaction. [Figure 109]Figure 109A shows an analysis of the final 3xFA(γ), 3xMMAE-XTEN product purified by preparative RP-HPLC as described in Example 39. (Figure 109A) Size exclusion chromatography analysis (Phenomenex BioSep-SEC-s4000 600x7.80mm column, 50mM sodium phosphate pH 6.5, 300mM NaCl buffer, flow rate 0.5ml / min, isocratic elution 70min). (Figure 109B) RP-HPLC analysis (Phenomenex Jupiter C18 5µM 300A 150x4.60mm column, Buffer A: 0.1% TFA in HO, Buffer B: 0.1% TFA in CAN, flow rate 1ml / min, gradient 5% to 50% B in 45min). Figure 3C shows the ESI-MS analysis (QSTAR-XL, calculated MW 85,085.4 Da, experimental MW 85,091 Da). [Figure 110] 1 shows the results of GPCR Ca 2+ mobilization activity of recombinant GLP2-2G-XTEN (filled squares) and conjugate GLP2-2G-XTEN (filled circles), performed as described in Example 62. [Figure 111] 1 shows the in vitro human plasma stability results of recombinant GLP2-2G-XTEN (filled squares) and conjugate GLP2-2G-XTEN (filled triangles) at various time points at 37° C., performed as described in Example 63. [Figure 112] 1 shows the results of pharmacokinetic profiles of recombinant GLP2-2G-XTEN (filled squares) and conjugate GLP2-2G-XTEN (filled triangles) in rats performed as described in Example 64. [Figure 113] (Figure 113A) SEC-HPLC analysis of the reaction products between tris-[2-maleimidoethyl]amine and 1x amino, 1x thiol-XTEN432. Figure 113A - Conjugate mixture: Peak 1 - trimer XTEN, Peak 2 - dimer XTEN, Peak 3 - unreacted monomeric XTEN; Figure 113B - Linear XTEN_1296 control; Figure 113C - Linear XTEN_864 control; Figure 113D - Linear XTEN_432 control. [Figure 114](Figure 114A) C18 RP-HPLC analysis of DBCO-sulfo-NHS conjugate to 1x amino-XTEN_288, as described in Example 65. Unreacted XTEN eluted at 19 minutes. 1x DBCO-XTEN_288 eluted at 27 minutes. The DBCO-sulfo-NHS reagent and its hydrolysis product eluted at 41.5 minutes and 38.5 minutes, respectively. (Figure 114B) C18 RP-HPLC analysis of azido-PEG4-NHS ester conjugate to tris(2-aminoethyl)amine. 3x azido-PEG4-TAEA was identified by MALDI-TOF MS and ESI-MS as the product with a MW of 966 Da. [Figure 115] 1 shows SEC-HPLC analysis of the reaction products between 3xAzide-PEG4-TAEA and 1xDBCO-XTEN_288, as described in Example 66: (Trace A) conjugate mixture: Peak 1—trimeric XTEN, Peak 2—dimeric XTEN, Peak 3—unreacted monomeric XTEN, Peak 4—low molecular weight compounds; (Trace B) linear XTEN_864 control; (Trace C) linear XTEN576 control; (Trace D) linear XTEN_288 control. [Figure 116] 1 shows the results of a cell killing assay demonstrating the selective cytotoxicity of 3xFA(γ), 3xMMAE-XTEN on KB cells, as described in Example 69. Inhibitory dose-response curves are shown for free MMAE (filled circles), 3xMMAE-XTEN (filled inverted triangles), and 3xFA(γ), 3xMMAE-XTEN groups in the presence (filled triangles) and absence (filled squares) of folate competitors on KB cells. [Figure 117A]The structures of XTEN-payload conjugates 3xFA(γ) and 3xMMAE-XTEN are shown. (Figure 117A) Two XTENs are joined by reaction of the azide 1-azido-3,6,9,12-tetraoxapentadecan-15-oic acid, N-hydroxysuccinimide ester, and the alkyne 6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoic acid, N-hydroxysuccinimide (or N-hydroxysulfonsuccinimide) ester. (Figure 117B) The X residue of Cys is modified with folate-γ-aminopentyl-maleimide. (Figure 117C) The Z residue of Cys is modified with maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethylauristatin E. [Figure 117B] The structures of XTEN-payload conjugates 3xFA(γ) and 3xMMAE-XTEN are shown. (Figure 117A) Two XTENs are joined by reaction of the azide 1-azido-3,6,9,12-tetraoxapentadecan-15-oic acid, N-hydroxysuccinimide ester, and the alkyne 6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoic acid, N-hydroxysuccinimide (or N-hydroxysulfonsuccinimide) ester. (Figure 117B) The X residue of Cys is modified with folate-γ-aminopentyl-maleimide. (Figure 117C) The Z residue of Cys is modified with maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethylauristatin E. [Figure 117C]The structures of XTEN-payload conjugates 3xFA(γ) and 3xMMAE-XTEN are shown. (Figure 117A) Two XTENs are joined by reaction of the azide 1-azido-3,6,9,12-tetraoxapentadecan-15-oic acid, N-hydroxysuccinimide ester, and the alkyne 6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoic acid, N-hydroxysuccinimide (or N-hydroxysulfonsuccinimide) ester. (Figure 117B) The X residue of Cys is modified with folate-γ-aminopentyl-maleimide. (Figure 117C) The Z residue of Cys is modified with maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethylauristatin E. DETAILED DESCRIPTION OF THE INVENTION

[0061] Before describing embodiments of the present invention, it is to be understood that such embodiments are provided by way of example only. and various alternatives to the embodiments of the invention described herein include: It will be understood that numerous variations may be employed in the practice of the present invention. Forms, modifications, and substitutions may be envisaged without departing from the invention.

[0062] Unless otherwise defined, all technical and scientific terms used herein are understood to be within the meaning of the present invention. The terms "a," "b," and "c" have the meaning broadly understood by those skilled in the art to which they pertain. Methods and materials similar or equivalent may be used in the practice or testing of the present invention. Suitable methods and materials that can be used are described below. The patent specification, including definitions, prevails. Further, the materials, methods, and examples are merely illustrative. and are not intended to be limiting. Those skilled in the art will recognize that numerous variations, modifications, and variations are possible. and substitutions will be envisioned without departing from the invention. definition In the context of this application, the following terms shall have the meanings ascribed to them unless otherwise specified: Has.

[0063] As used throughout this specification and claims, "a," "an," and "the" The term "at least one" is used interchangeably with "at least one" and "at least one" except where an upper limit is specifically stated thereafter. "at least the first," "one or more," or "plural" of referenced components or steps Therefore, the term "payload" as used herein means When used, it means "at least the first payload," but includes multiple payloads. The operating limits and parameters of the combination, as well as the amounts of any single agent, are set forth in light of this disclosure. It will be known to those skilled in the art.

[0064] The terms "polypeptide," "peptide," and "protein" are used herein to refer to In this context, the terms "polymer" and "polymer of amino acids" are used interchangeably to refer to polymers of amino acids of any length. It may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also refers to modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component, Contains.

[0065] As used herein, the term "amino acid" refers to naturally occurring and / or Refers to either unnatural or synthetic amino acids, both D and L optical isomers, and amino acid analogs, and peptidomimetics. Amino acids are designated using one-letter or three-letter codes.

[0066] A "pharmacologically active" agent is any substance or compound that is desired to be delivered to a subject. Any drug, compound, or composition (e.g., therapeutic, diagnostic, or drug delivery agent) of the mixture Several, often useful, methods have been proposed and can be demonstrated in vivo or in vitro. Such drugs provide or are expected to provide some pharmacological effect. These include peptides, proteins, carbohydrates, nucleic acids, nucleosides, oligonucleotides, and small molecule synthetic compounds, or analogs thereof.

[0067] The term "natural L-amino acids" refers to 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), L of glutamic acid (E), aspartic acid (D), serine (S), and threonine (T) It refers to optical isomeric forms.

[0068] The term "non-naturally occurring" when applied to a sequence and as used herein When used, it refers to the wild-type or naturally occurring sequence counterpart found in mammals ( counterpart), is not complementary, or has a high degree of homology For example, a non-naturally occurring polypeptide or polynucleotide sequence. The polypeptide or fragment, when properly aligned, has a 99% repeat sequence as compared to the native sequence. % or less, 98%, 95%, 90%, 80%, 70%, 60%, 50%, or even more The following amino acid sequence identities may be shared:

[0069] The terms "hydrophilic" and "hydrophobic" refer to the degree of affinity a substance has for water. Hydrophilic substances have a strong affinity for water and are soluble in, miscible with, or readily absorb water. or tends to be wetted by water, whereas hydrophobic materials lack an affinity for water and They tend to repel and not absorb water, and are not soluble or miscible with water, or They tend not to be wetted by water. Amino acids are characterized based on their hydrophobicity. Many scales have been developed. One example is Levitt, M, et al. The scale developed by I., J Mol Biol (1976) 104:59 Hopp, TP, et al., Proc Natl Acad Sci USA ( 1981) 78:3824. Examples of "hydrophilic amino acids" are arginine, Lysine, threonine, alanine, asparagine, and glutamine. The hydrophilic amino acids, aspartate, glutamate, and serine, and glycolipids Examples of "hydrophobic amino acids" are tryptophan, tyrosine, and phenylalanine. , methionine, leucine, isoleucine, and valine.

[0070] "Fragment" when applied to a biologically active protein is intended to mean a therapeutic and / or or truncations of biologically active proteins that retain at least a portion of the biological activity. "Variant" when applied to a biologically active protein is a biologically retain at least a portion of the therapeutic and / or biological activity of the active protein, A protein that has sequence homology to a naturally occurring biologically active protein, e.g. , the mutant protein has at least 70% or more of a nucleotide sequence similar to that of the reference biologically active protein. %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% As used herein, "biologically active" refers to a group of genes that share the same amino acid sequence identity as the target gene. The term "specific protein variant" refers to a variant of a gene encoding the protein, e.g., by site-directed mutagenesis, They may be intentionally modified by gene synthesis or accidentally modified through mutations, Contains proteins that retain activity.

[0071] The term "sequence variant" refers to a variant of a sequence that has been modified by one or more amino acid insertions, deletions, or substitutions. It refers to polypeptides that are modified compared to their native or original sequence. It may be located at either or both ends of the protein and / or internal to the amino acid sequence. Non-limiting examples include sequences of biologically active payload proteins. Another non-limiting example is the insertion of an XTEN sequence within an XTEN with a different amino acid. In deletion mutants, the polypeptides described herein are substituted with amino acids. Thus, deletion mutants are those in which one or more amino acid residues in the payload polypeptide are removed. It includes all fragments of the peptide sequence. In substitution variants, one of the polypeptides These amino acid residues are removed and replaced with alternative residues. is conservative in nature and conservative substitutions of this type are well known in the art.

[0072] The term "moiety" refers to a component of a larger composition or to a functional group of a drug molecule. It can be incorporated into larger compositions such as targeting peptides conjugated to groups or larger polypeptides. It is intended to be included.

[0073] As used herein, "terminal XTEN" refers to a compound in which the payload is a peptide or When it is a polypeptide, it is fused to the N-terminus or C-terminus of the payload, or It refers to the XTEN sequence contained therein.

[0074] The term "XTEN release site" refers to a site that can be recognized and cleaved by a protease and that is an XTEN release site. resulting in the release of XTEN or a portion of XTEN from the XTEN-payload polypeptide. As used herein, "XTEN" refers to the cleavage sequence in the XTEN-payload. "Mammalian proteases" are proteases typically found in mammalian body fluids, cells, or tissues. XTEN release sites are capable of binding to a variety of mammalian proteases ("XTEN release proteases"). ases), e.g., trypsin, FXIa, FXIIa, kallikrein , FVIIIa, FVIIIa, FXa, FIIa (thrombin), elastase-2, MMP-12, MMP-13, MMP-17, MMP-20, or any of the The protein can be engineered to be cleaved by a protease that recognizes a defined cleavage site. Other equivalent proteases (endogenous or exogenous) that can be used. The cleavage site can be adjusted and adapted to the protease being utilized.

[0075] The term "in" refers to a first polypeptide bound to a second polypeptide. When the first or second polypeptide is N-terminally linked to the second or first polypeptide, a bond connecting the C-terminus of the peptide, and the sequence of the first polypeptide to the sequence of the second polypeptide For example, XTEN may be attached "within" a payload polypeptide. When XTEN is attached to the N-terminus, C-terminus, or It can be inserted between any two amino acids.

[0076] "Activity" refers to the activity of the XTEN-payload compositions provided herein. When used, it is measured by in vitro, ex vivo, or in vivo assays. Whether measured by receptor binding, antagonist activity, or clinical efficacy, activity, agonist activity, cellular or physiological response, or any other activity generally known in the art. This refers to an action or effect, including but not limited to, an effect on a payload.

[0077] As used herein, the term "ELISA" refers to the method described herein. or as otherwise known in the art. Refers to adsorption assay.

[0078] A "host cell" can be a recipient for a subject vector such as those described herein. A host cell includes an individual cell or cell culture that has received or been a recipient. This includes the progeny of the cell, which may not necessarily be the product of natural, accidental, or deliberate mutation. It is not necessarily completely identical (morphologically or genomically for the entire DNA complement) to the original parent cell. Host cells include cells transformed in vivo with vectors of the invention. .

[0079] "Isolated" is used to describe the various polypeptides disclosed herein. When a substance is identified and separated and / or recovered from components of its natural environment, Contaminant components of its natural environment typically affect the diagnostic properties of the polypeptide. Materials that interfere with the diagnostic or therapeutic use of enzymes, hormones, and other proteinaceous or As will be apparent to those skilled in the art, non-naturally occurring polysaccharides may also be present. oligonucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof A compound does not require "isolation" to distinguish it from its naturally occurring counterpart. Additionally, "enriched," "isolated," or "diluted" polynucleotides, peptides, A peptide, polypeptide, protein, antibody, or fragment thereof is a peptide or polypeptide that is a polypeptide of a type that is a naturally occurring peptide or polypeptide. The molecular concentration or number per volume is generally determined by the Generally, they are produced by recombinant means and expressed in host cells. The expressed polypeptide is considered to be "isolated."

[0080] An "isolated" nucleic acid is one that has been identified and that is not part of the natural sequence of the nucleic acid that normally encodes a polypeptide. Separated from at least one contaminant nucleic acid molecule with which it is associated in the source. For example, an isolated nucleic acid molecule encoding a polypeptide is a nucleic acid molecule encoding a polypeptide in the form in which it is found in nature. or otherwise present in the context of the isolated polypeptide. is distinguishable from a nucleic acid molecule that encodes a specific polypeptide as it exists in natural cells. However, a nucleic acid molecule encoding an isolated polypeptide is generally The nucleic acid molecule encoding the polypeptide contained in the cell expressing the peptide includes, for example, The nucleic acid molecule may be present in a chromosomal or extrachromosomal location that is distinct from that of natural cells.

[0081] A "chimeric" protein has at least one position in the sequence that differs from that found in nature. The fusion polypeptide comprises at least one fusion polypeptide comprising two regions. These regions are typically , may be present in separate proteins and carried together in a fusion polypeptide, or They may normally be present in the same protein, but may be arranged in a new sequence in the fusion polypeptide. Chimeric proteins can be prepared, for example, by chemical synthesis or by combining peptide regions. by creating and translating a polynucleotide encoding the desired obtain.

[0082] "Fused" and "fusion" are used interchangeably herein and refer to the synthesis of two or more peptides. The term "ligand" refers to the joining of peptide or polypeptide sequences together by recombinant means.

[0083] "Operably linked" means that the DNA sequences being linked are contiguous and in reading phase. "In-frame fusion" means that the original ORF is two to form a long continuous ORF in a manner that maintains the correct reading frame. refers to the junction of the open reading frame (ORF) or An agent is said to be engineered into a coding sequence for a polypeptide if it affects the transcription of the polypeptide sequence. Therefore, the resulting recombinant fusion protein is Two or more segments corresponding to the encoded polypeptide (these segments are typically It is a single protein containing a nucleotide sequence (which is not normally so joined in nature).

[0084] "Cross-linked," "conjugated," "bonded," "attached to," and "attached to" are Used interchangeably herein, they refer to the covalent joining of two different molecules by a chemical reaction. Crosslinking can occur in one or more chemical reactions, as described more fully below.

[0085] As used herein, the term "conjugation partner" refers to a group of molecules in a conjugation reaction. The term "complex" refers to individual components that can be or are joined together.

[0086] The term "conjugate" refers to the heterogeneous structure formed as a result of covalently linking conjugation partners together. A seed molecule, e.g., a biologically active payload, or an antigen, covalently attached to the XTEN molecule. Refers to an XTEN molecule or crosslinker covalently attached to a reactive XTEN.

[0087] "Crosslinker" and "linker" are used interchangeably and in their broadest context. refers to a chemical entity used to covalently bond two or more entities. For example, crosslinking An agent may contain two, three, four, or more X, as those entities are defined herein. XTEN is conjugated or a payload is conjugated to XTEN. The reaction products of zero-length, homo- or heterobifunctional, and multifunctional crosslinker compounds, two crosslinkers cross-linking agents include, but are not limited to, the reaction products of cross-linking chemical reactants. It will be understood by those skilled in the art that the term "covalently linked reaction product" may refer to the covalently linked reaction product remaining after crosslinking of the The crosslinker may contain one or more unreacted molecules that can react with another entity. The reactants may also include:

[0088] In the context of a polypeptide, a "linear sequence" or "sequence" refers to sequences that are adjacent to each other in the sequence. The amino acid sequence of the corresponding residues is consecutive in the primary structure of the polypeptide, and is arranged in the carboxyl-terminal direction. A "subsequence" is the order of amino acids in a polypeptide. is the linear sequence of the portion of the polypeptide that is known to contain it in both directions.

[0089] "Heterologous" means derived from a genotypically distinct entity from the rest of the entity being compared. For example, it can be removed from its native coding sequence and engineered into a coding sequence other than the native sequence. The glycine-rich sequence linked to the polynucleotide is a heterologous glycine-rich sequence. When applied to a polynucleotide or polypeptide, the term "polynucleotide" refers to that polynucleotide or polypeptide. This means that the genotype of the genotyped genotype is different from that of the rest of the genotyped ... do.

[0090] The terms "polynucleotide," "nucleic acid," and "oligonucleotide" are synonymous. They are used to synthesize polymeric forms of nucleotides of any length, deoxyribonucleic acid (dT). Polynucleotides refer to either nucleotides or ribonucleotides, or their analogs. A protease may have any three-dimensional structure and may perform any function, known or unknown. Examples of polynucleotides include, but are not limited to: coding or non-coding sequences of a gene or gene fragment. gene regions, loci(s) defined from linkage analysis, exons, introns, message mRNA, transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers Polynucleotides are composed of methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be The sequence of nucleotides may be added before or after assembly of the nucleotides. A polynucleotide can be further interrupted after polymerization, such as by conjugation with a labeling component. It can be further modified.

[0091] The term "polynucleotide complement" refers to a polynucleotide that hybridizes with perfect fidelity to a reference sequence. The complementary base sequence and reverse orientation are compared to the reference sequence so that the 1 shows a polynucleotide molecule having

[0092] "Recombinant," when applied to a polynucleotide, means that the polynucleotide is A variety of recombination steps may include sequencing, restriction, and / or ligation steps. The recombinant protein may be the product of various combinations and other procedures that result in the expression of the recombinant protein in a host cell. This means that

[0093] The terms "gene" and "gene fragment" are used interchangeably herein. They are capable of encoding specific proteins after being transcribed and translated. A polynucleotide containing at least one open reading frame is referred to as a polynucleotide containing at least one open reading frame. A gene or gene fragment is a polynucleotide that contains the entire coding region or any portion thereof. As long as it contains at least one open reading frame capable of covering the fragment. A "fusion gene" is a gene that contains at least two genes linked together. It is a gene consisting of two heterologous polynucleotides.

[0094] "Homology" or "homologous" or "sequence identity" refers to the difference between two or more polynucleotide sequences. Refers to the sequence similarity or compatibility between sequences or between two or more polypeptide sequences. Use a program such as Fit to find sequence identity, similarity, or sequence identity between two different amino acid sequences. When determining identity or homology, default settings may be used, or alternatively, identity, similarity, Or to optimize the homology score, use blosum45 or blosum80 etc. An appropriate scoring matrix can be selected. Preferably, the homologous polynucleotides A nucleotide is a nucleotide that hybridizes under stringent conditions, as defined herein. and compared to those sequences, it is at least 70%, preferably at least 80% %, more preferably at least 90%, more preferably at least 95%, more preferably have 97%, more preferably 98%, and even more preferably 99% sequence identity. The homologous polypeptides are preferably at least 70%, preferably at least 8%. 0%, even more preferably at least 90%, even more preferably at least 95% They share a sequence identity of 99%.

[0095] "Ligation," as applied to polynucleic acids, refers to the joining of two nucleic acid fragments or genes. Refers to the process of creating phosphodiester bonds between genes and joining them together. In order to ligate the A fragments or genes together, the ends of the DNA must be compatible with each other. In some cases, the ends are directly compatible after endonuclease digestion. However, first, the crossed ends, which are generally produced after endonuclease digestion, are converted to blunt ends. It may be necessary to convert the ends to make them suitable for ligation.

[0096] "Stringent conditions" or "stringent hybridization conditions" The term "antigenic" refers to a polynucleotide that is more likely to be present than other sequences (e.g., less likely to be present in the background). "Contains reference to conditions under which a target sequence hybridizes to a detectably high (at least 2-fold) degree to its target sequence." Generally, the stringency of hybridization is determined, in part, by the length of the wash steps. Expressed with reference to the temperature and salt concentration at which it is carried out. Typically, stringent conditions is a salt concentration less than about 1.5M sodium ions, typically at pH 7.0-8.3. The sodium ion concentration is about 0.01 to 1.0 M (or other salts), and the temperature is For short polynucleotides (e.g., about 10-50 nucleotides), at least about 30°C, and for long polynucleotides (e.g., greater than 50 nucleotides) The temperature is at least about 60°C, and for example, "stringent conditions" are 50% formaldehyde, Hybridization at 37°C in amide, 1M NaCl, 1% SDS, and 0 May include three washes in 1x SSC / 1% SDS at 60-65°C for 15 minutes each Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. The SC concentration can vary from about 0.1 to 2xSSC, and SDS is present at about 0.1%. Such wash temperatures typically provide a wash temperature that is suitable for a particular sequence at a defined ionic strength and pH. The Tm is selected to be approximately 5°C to 20°C below the thermal melting point. hybridize to a perfectly matched probe (under defined ionic strength and pH) The equation for calculating the Tm and conditions for nucleic acid hybridization is Knowledge, Sambrook, J. et al., "Molecular Cloning g:A Laboratory Manual,″3 rd Edition, Cold Spring Harbor Laboratory Press, 2001 Typically, a blocking reagent is used to prevent non-specific hybridization. Such blocking agents can be used to block, for example, shear and denaturing proteins. Contains salmon sperm DNA at approximately 100-200 μg / ml. Organic solvents such as amides are used in certain situations, such as RNA:DNA hybridization. Useful variations on these wash conditions will be readily apparent to one of skill in the art. It would be.

[0097] The terms "percent identity," "percent sequence identity," and "% identity" are used interchangeably. The term, when applied to polynucleotide sequences, is used to align sequences using standardized algorithms. It refers to the percentage of residue matches between at least two polynucleotide sequences identified by a sequence identifier. Such algorithms are standardized and reproducible for optimizing alignments between two sequences. In this way, gaps can be inserted into the sequences being compared, allowing for a more meaningful comparison of the two sequences. The percent identity is calculated over the entire length of the defined polynucleotide sequence. It may be measured over a longer distance, or over a shorter length, e.g., a larger defined a fragment taken from the polynucleotide sequence, e.g., at least 45, at least 60, at least 90, at least 120, at least 150, at least 210, or or over the length of a fragment of at least 450 contiguous residues. Such lengths are merely exemplary and may not be used in any tables, figures, or sequence listings herein. Any fragment length supported by the sequence shown can be used to calculate percent identity. It is understood that the length over which the sequence identity can be measured may also be described. The algorithm compares two optimally aligned sequences over a comparison window and identifies matched positions. (identical residues occur in both polypeptide sequences) and matched positions. Dividing the number of positions by the total number of positions in the comparison window (i.e., window size); The result is calculated by multiplying by 100 to give the percentage of sequence identity. When sequences of different lengths are compared, the shortest sequence defines the length of the comparison window. Conservative substitutions are not considered when calculating sequence identity.

[0098] "Percent (%) sequence identity" refers to a sequence identity to a polypeptide sequence identified herein. Align the sequences, if necessary, to achieve the maximum percent sequence identity. After introducing the gap, the amino acid residues of the second reference polypeptide sequence or a portion thereof are It is defined as the percentage of amino acid residues in the query sequence that are identical, and any homology Substitutions are not considered as part of the sequence identity, thereby resulting in optimal alignment. Alignment for the purposes of determining percent identity of sequences can be performed by a variety of methods within the skill of the art. By using methods such as BLAST, BLAST-2, ALIGN, or Megalign, Use publicly available computer software such as (DNASTAR) software Those skilled in the art will be able to determine appropriate parameters for measuring alignment. and any necessary adjustments to achieve optimal alignment over the entire length of the sequences being compared. The percent identity is calculated over the entire length of the defined polypeptide sequence. It may be measured over a longer distance, or over a shorter length, e.g., a larger defined fragments taken from the polypeptide sequence, e.g., at least 15, at least 2 0, at least 30, at least 40, at least 50, at least 70, or less The length of such fragments can be measured over a length of at least 150 consecutive residues. Lengths are merely examples and are not intended to be limiting unless otherwise specified by the sequences shown in the tables, figures or sequence listings herein. Percent identity is measured using any fragment length supported by the column. It is understood that the length that can be achieved may be described.

[0099] "Repetitive" as used in the context of polynucleotide sequences refers to, for example, sequences of a given length It refers to the degree of internal homology in sequences, such as the frequency of identical nucleotide sequences. For example, it can be determined by analyzing the frequency of identical sequences.

[0100] A "vector" is a nucleic acid molecule that is preferably self-replicating in a suitable host; Transferring inserted nucleic acid molecules into and / or between cells. This term primarily refers to A vector that functions for the insertion of DNA or RNA into a cell, primarily for the replication of DNA or RNA. Replication of the vector, and transcription and / or translation of DNA or RNA. Expression vectors that function for the purpose of the invention include vectors that provide more than one of the above functions. An "expression vector" is a vector that, when introduced into an appropriate host cell, expresses a polypeptide(s). ) is a polynucleotide that can be transcribed and translated into a desired expression system. It implies a suitable host cell comprising an expression vector that can function to produce a product.

[0101] "Serum degradation resistance," when applied to a polypeptide, refers to the resistance of the polypeptide to blood or other substances. This refers to the ability of a protein to withstand degradation in the presence of other components, typically those in serum or plasma that do not require proteases. Resistance to serum degradation is achieved by converting the protein into human (or mouse, rat, or monkey, as appropriate) ) serum or plasma and typically for several days (e.g., 0.25, 0.5, 1, 2, 4, 8, 1 6 days), typically at about 37°C. The sample can be run on a Western blot assay and the protein can be identified by antibodies. The antibody can be directed against a tag in the protein. The size of the protein is identical to the size of the injected protein, and the protein appears as a single band on the Western In this exemplary method, 50% of the protein was degraded. is degraded, as determined by Western blot or equivalent technique. This is the serum degradation half-life or "serum half-life" of the protein.

[0102] "t 1 / 2 ", "half-life", "terminal half-life", "elimination half-life", and "circulating half-life" The terms " and " are used interchangeably herein and, as used herein, ln(2) / K el The terminal half-life is calculated as K elis the time curve is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration Half-life is typically the time it takes for half of the amount of administered substance deposited in the body to be eliminated by normal biological processes. This refers to the time required for a substance to be metabolized or eliminated by the body.

[0103] "Active clearance" refers to a mechanism by which proteins are removed from the circulation other than by filtration. refers to the cell, receptor-mediated removal of proteins from the circulation, metabolism, or Includes decomposition.

[0104] "Apparent molecular weight factor" and "apparent molecular weight" refer to the molecular weight of a particular amino acid or polypeptide. A related term that refers to a measure of the relative increase or decrease in apparent molecular weight exhibited by a peptide sequence. The molecular weight of the product was determined by size exclusion chromatography by comparison with globular protein standards. Determined using a SEC or similar method and measured in "apparent kD" units. The apparent molecular weight factor is the ratio between the apparent molecular weight and the actual molecular weight, is calculated based on the amino acid composition and adding the calculated molecular weight of each type of amino acid in the composition. by calculation or by estimation from comparison with molecular weight standards in an SDS electrophoresis gel. For representative proteins, the apparent molecular weight and apparent molecular weight factor are The determination of both children is described in the Examples.

[0105] The term "hydrodynamic radius" or "Stokes radius" refers to the mass of a body moving through a solution. The viscosity of the solution is measured by assuming that the The effective radius (R h (nm)). In an embodiment of the invention, the XTEN polypeptide The hydrodynamic radius measurements correlate with a more intuitive measure, the "apparent molecular weight factor." The "hydrodynamic radius" of a protein determines its diffusion rate in aqueous solution and in polymer gels. The hydrodynamic radius of a protein depends on its molecular weight, which affects its ability to move. It is determined by its structure, including its shape and compactness. Determine the hydrodynamic radius. Methods for doing so are well known in the art and are described, for example, in U.S. Pat. Size exclusion chromatography, as described in US Pat. Nos. 632 and 7,294,513, Most proteins are separated by the smallest hydrodynamic medium (SEC). It has a spherical structure, which is the most compact three-dimensional structure that can be obtained. Proteins adopt a random and open unstructured or "linear" structure, resulting in , which have a much larger hydrodynamic radius compared to typical globular proteins of similar molecular weight. do.

[0106] "Physiological conditions" refers to the set of conditions in a living host, as well as those conditions of a living subject. Refers to in vitro conditions, including temperature, salt concentration, and pH, that are mimicked in in vitro assays. Many physiologically relevant conditions have been established for the preparation of anaerobic buffers. The neutral pH ranges from about 6.5 to about 7.8, preferably from about 7.0 to about 7.5. The various physiological buffer solutions are listed in Sambrook et al. (2001). The physiologically relevant temperature is about 25°C to about 38°C, preferably about 35°C to about 37°C. The range is.

[0107] A "single atom residue of a payload" refers to a subject XTEN or XTEN-linker composition. After reaction with the XTEN, the atoms of the payload, typically sulfur, oxygen, or nitrogen, are chemically bonded to the XTEN. For example, the atom residue of the payload refers to the cis- or trans-atomic atom in the payload. Thethiol reactive sulfur residue, peptide or polypeptide or small molecule payload Nitrogen molecules, peptides, proteins or small molecules, or synthetic organic drugs with amino reactive groups of or a reactive carboxyl or aldehyde group.

[0108] A "reactive group" is a chemical structure that can be attached to a second reactive group. An example of a reactive group is an amino group. , carboxyl group, sulfhydryl group, hydroxyl group, aldehyde group, azide group Some reactive groups can be activated and subjected to a second reaction, either directly or through a cross-linker. As used herein, a reactive group is a group capable of facilitating conjugation with a chemical group. As long as they have the ability to participate in chemical reactions, XTEN, crosslinkers, azide / alkyne click chemistry It can be part of the reactant or the payload. Once reacted, the conjugate bond forms a or binding residues of crosslinkers or XTEN reactants.

[0109] "Controlled release," "sustained release," "depot preparation," and "sustained release" are used to describe polypeptides. The release period of the polypeptide of the present invention is compared to the release period when the drug is administered in the absence of the drug. The terms "anticoagulant" and "anticoagulant" are used interchangeably to refer to agents that can extend the time between injections. may have different release rates, resulting in different therapeutic doses.

[0110] The term "payload," as used herein, refers to a compound that, when administered to a subject, have biological, pharmacological, or therapeutic activity or beneficial effect, or Any protein, peptide sequence, small molecule, drug, or other substance that can be demonstrated in vitro. Payloads also include molecules that can be used for imaging or in vivo diagnostic purposes. Examples of payloads include cytokines, enzymes, hormones, blood clotting factors, and growth factors. compounds, chemotherapy drugs, antiviral compounds, toxins, anticancer drugs, radioactive compounds, and imaging agents, and target peptides, proteins, antibodies, antibody fragments, or receptors or ligands. Examples of suitable compounds include, but are not limited to, compounds used to bind to the ligand.

[0111] The terms "antigen," "target antigen," and "immunogen" refer to an antibody fragment or an antibody a structure or molecule to which a body fragment-based therapeutic agent binds or has specificity are used interchangeably herein to refer to binding determinants.

[0112] The term "antagonist" as used herein refers to an antagonist of any of the compounds disclosed herein. Partially or completely block, inhibit, or neutralize the biological activity of the naturally occurring polypeptide. The method for identifying antagonists of a polypeptide includes identifying any molecule that binds to a naturally occurring polypeptide. contacting the peptide with a candidate antagonist molecule and, typically, determining whether the peptide is a natural polypeptide; and measuring a detectable change in one or more associated biological activities. In the context of the present invention, antagonists may be proteins, nucleic acids, carbohydrates, antibodies, or or any other molecule that reduces the effect of a biologically active protein.

[0113] A "defined medium" is one in which the components of the medium are known, ensuring the survival and viability of cells in culture. refers to a medium that contains the nutrients and hormonal requirements necessary for growth and / or development. Traditionally, synthetic media The soil was formulated with the addition of nutrients and growth factors necessary for growth and / or survival. Typically, a synthetic medium contains at least one component from one or more of the following categories: Provides: a) all essential amino acids (usually the 20 amino acids plus the basic cysteine) b) a source of energy (usually in the form of carbohydrates such as glucose); c) vitamins and and / or other organic compounds required in low concentrations; d) free fatty acids; and e) trace elements. Trace elements (trace elements) are inorganic compounds or naturally occurring elements that are typically required in very low concentrations. Elements, usually in the micromolar range. It may optionally be supplemented with one or more ingredients from any of the categories: a) one b) mitogens; b) salts and buffers (e.g., calcium, magnesium, and c) nucleotides and bases (e.g., adenosine and thymidine, hypoxamine, d) Protein and tissue hydrolysates.

[0114] The term "agonist" is used in its broadest sense and refers to the naturally occurring polypeptides disclosed herein. Suitable agonist molecules include, inter alia, any molecule that mimics the biological activity of a peptide. Agonist antibodies or antibody fragments, such as naturally occurring polypeptides, peptides, small organic molecules, etc. Agonists of naturally occurring polypeptides include, but are not limited to, fragments, or variants of the amino acid sequence. A method for identifying an agonist involves contacting the native polypeptide with a candidate agonist molecule. and detectable in one or more biological activities normally associated with the native polypeptide. and measuring the change.

[0115] "Inhibition constant" or "Ki" are used interchangeably and refer to the dissociation constant of the enzyme-inhibitor complex, denotes the reciprocal of the binding affinity of the inhibitor to the enzyme.

[0116] As used herein, "treatment" or "treating" or "alleviating" or or "ameliorate" are used interchangeably and refer to administering a drug or biologic to achieve a therapeutic benefit. to produce a therapeutic effect, to cure or reduce the severity of an existing condition, or to provide a preventative benefit to achieve a desired outcome, prevent the occurrence of a condition, or reduce the likelihood or severity of the condition Therapeutic benefit means that the subject may still suffer from the underlying disease. However, the underlying condition being treated, or the underlying condition, such that improvement is observed in the subject, "Ablation or amelioration of one or more of the physiological symptoms associated with

[0117] "Therapeutic effect" or "therapeutic benefit," as used herein, refers to a physiological effect. refers to the alleviation, amelioration, or prevention of disease in humans or other animals, or the treatment of biological Other than the ability to induce the production of antibodies against antigenic epitopes possessed by the protein, Any physical or other adverse effects in humans or animals that may result from the administration of a polypeptide of the present invention. These include, but are not limited to, enhancing mental health. Therefore, the composition may be used to treat a particular disease, condition, or condition, even if no diagnosis of the disease has been made. or at risk of developing a condition (e.g., bleeding in a diagnosed hemophilia A subject) It may be administered to a subject or to a subject reporting one or more of the physiological symptoms of the disease.

[0118] The terms "therapeutically effective amount" and "therapeutically effective dose" are used herein. When used in combination with a drug or biological agent, either alone or as part of a polypeptide composition, refers to the amount of biologically active protein that, when administered to a subject in a single or repeated dose, is capable of inhibiting a pathological condition. or any detection of any symptom, aspect, measured parameter, or characteristic of a condition. Such effects need not be absolutely beneficial. Determination of a therapeutically effective amount is readily understood, especially in light of the detailed disclosure provided herein. It is within the capabilities of one skilled in the art.

[0119] The term "therapeutically effective dosage regimen" as used herein refers to a single or multiple or as part of a polypeptide composition. refers to a schedule for sequential administration of doses (i.e., 2 or 3), and these doses are , any symptom, aspect, measured parameter, or characteristic of a disease state or condition. The therapeutically effective amount is administered to provide a therapeutically beneficial effect. I). General techniques The practice of the present invention is within the skill of the art, unless otherwise indicated, in the art of immunology, biochemistry, immunology, and the like. , chemistry, molecular biology, microbiology, cell biology, genomics, and conventional techniques of recombinant DNA Sambrook, J. et al., "Molecular Cloning" ing:A Laboratory Manual,″3 rd edition,Col d Spring Harbor Laboratory Press, 2001, ″C current protocols in molecular biology″,F .M. Ausubel, et al. eds., 1987, the series ″M ethods in Enzymology, “Academic Press, San Diego, CA., ``PCR 2: a practical approach'', MJMacPherson,BDHames and GRTaylor eds., Oxford University Press, 1995, “Antib odies,a laboratory manual″Harlow,E.and L ane,D.eds.,Cold Spring Harbor Laboratory ,1988,″Goodman & Gilman's The Pharmacolo logical Basis of Therapeutics,″11 th Editio n, McGraw-Hill, 2005, and Freshney, R.I., "Cul ture of Animal Cells:A Manual of Basic T echnique,″4 th edition, John Wiley & Sons, Somerset, NJ, 2000 (the contents of which are incorporated herein by reference in their entirety). (incorporated into the

[0120] Host cells can be cultured in a variety of media, including Ham's F10 (Sigma), Mi nimal Essential Medium (MEM, Sigma), RPMI-1 640 (Sigma), and Dulbecco's Modified Eagle' Commercially available media such as DMEM (Sigma) are suitable for culturing eukaryotic cells. Additionally, animal cells lack serum but can tolerate hormones, growth factors, or specific cell types. in a defined medium supplemented with any other factors necessary for the survival and / or growth of the cell type. A defined medium that supports cell survival improves viability, morphology, metabolic capacity, and And potentially, a defined medium that promotes cell growth while maintaining the ability of cells to differentiate. Provides all chemicals necessary for proliferation or growth. Induces the survival and growth of mammalian cells. The general parameters governing in vitro are well established in the art. Physicochemical parameters that can be controlled in a cell culture system include, for example, pH, pO2, temperature, The nutritional requirements of cells are usually determined by a system developed to provide an optimal environment. Nutrients are provided in standard media formulations. Nutrients are divided into several categories: amino acids and their derivatives, carbohydrates, sugars, fatty acids, complex lipids, nucleic acid derivatives, and vitamins. Apart from nutrients to maintain cellular metabolism, most cells grow in serum-free media. To achieve this, the following groups are considered: steroids, prostaglandins, growth factors, pituitary hormones, and one or more hormones from at least one of the peptide hormones. (Sato,GH,et al.in ″Growth of Cells in Hormonally Defined Media″,Cold Spring Ha In addition to hormones, cells were also stimulated with in vitro For survival and growth in the circulating plasma, transferrin (a plasma iron transport protein), ceruloplasia Transport proteins such as sumin (copper transport protein) and high density lipoprotein (lipid carrier) The optimal set of hormones or transport proteins may be different for each cell type. Most of these hormones or transport proteins are either added exogenously or In rare cases, mutant cell lines have been identified that do not require a particular factor. , know about other factors necessary to maintain cell cultures without undue experimentation. It would be.

[0121] The growth medium for growing prokaryotic host cells is nutrient broth (liquid nutrient medium) or LB medium. Suitable media include synthetic and natural media. Generally, a medium contains a carbon source such as glucose necessary for bacterial growth, water, and The medium contains a source of amino acids and nitrogen, such as beef or yeast extract ( The medium may contain either natural or known amounts of amino acids (as in synthetic media). In some embodiments, the growth medium is LB broth, e.g., LB Miller broth or LB Lennox broth. LB broth is made from peptone (an enzymatic digestion product of casein), yeast, and In some embodiments, the extract contains an antibiotic. A selective medium is used in which the desired cells that are resistant to the antibiotic are selected. Only the sacs grow. II). XTEN Protein Polymers and Conjugate Compositions The present invention relates, in part, to a substantially homogeneous composition comprising an extended recombinant polypeptide (XTEN). In a first aspect, the present invention relates to a composition of XTEN that is substantially uniform in length. Such compositions include an XTEN-crosslinker intermediate and an XTEN-payload. It is useful as a reagent conjugate partner for creating compositions. The present invention provides a method for creating a substantially homogeneous XTEN composition. Also provided are methods for creating such substantially homogeneous XTEN compositions in high yield.

[0122] In a second aspect, the present invention provides XTEN, e.g., one or more payloads. XTEN that can be bound to a conjugation partner, resulting in a payload-XTEN conjugate is synthesized by the addition of Peyrothrombohedral hydroxybenzoates, either directly or via crosslinkers or azide / alkyne reactants. Specifically engineered to incorporate a defined number of reactive amino acids for binding to The present invention provides compounds with enhanced pharmaceutical properties, including enhanced pharmacokinetic and pharmacological properties. for use in creating conjugates with payload drugs of interest as compositions having Also provided are methods of forming such engineered XTEN polymers.

[0123] In another aspect, the present invention provides a method for the preparation of hydroxybenzoates, in monomeric and multimeric configurations, as reactive conjugate partners. , a substantially homogeneous XTEN polymer containing a defined number of crosslinkers or azide / alkyne reactants. The present invention provides crosslinkers or azide / alkyne reactions and methods for preparing such reactions. XTEN derivatives containing reactants are used as reactants in the conjugation of payload agents to produce the desired The present invention provides XTEN-payload conjugates that exhibit the physical, pharmaceutical, and pharmacological properties of vinegar.

[0124] In another aspect, the present invention provides compositions of XTEN-payloads, comprising one or more XTEN- TEN is a defined number of payloads, including a combination of different payloads, in one or more payloads. , chemically conjugated in either monomeric or multimeric configurations to enhance pharmaceutical and pharmacokinetic properties. and compositions having pharmacological properties. Such compositions, when administered to a subject, exert the pharmacological or biological effect of their payload. Due to their activity, they may have utility in the prevention, treatment, or amelioration of diseases or conditions. 1. XTEN: Extended Recombinant Polypeptides In one aspect, the present invention provides a method for producing a polymeric polymer comprising, either directly or via a crosslinker reactant, one or more and a conjugation partner to bind to the payload and result in an XTEN-payload complex. The present invention provides substantially homogeneous XTEN polypeptide compositions useful as therapeutic agents.

[0125] XTEN may have a low-order structure under physiological conditions, or may have a secondary or tertiary structure. XTEN is a polypeptide having a non-naturally occurring, substantially non-repetitive sequence. Typically, a nucleic acid sequence has from about 36 to about 3000 amino acids, most or all of which are The term "XTEN" refers to a small hydrophilic amino acid. , whole antibodies or antibody fragments (e.g., single chain antibodies and Fc fragments) specifically XTEN polypeptides are used as conjugation partners to fulfill various roles. They have many uses and, when attached to a payload, confer certain desirable properties. The XTEN-payload conjugates may have enhanced properties, e.g., the ability to bind to the XTEN without Enhanced pharmacokinetic, physiochemical, pharmacological, and It has pharmaceutical properties and its payload is known to be used in the art. This makes them useful in the treatment of certain conditions.

[0126] The unstructured character and physicochemical properties of XTEN are due in part to the presence of four to six hydrophilic amino acids. Total amino acid composition disproportionately limited to amino acids, amino acids in a quantifiable non-iterative design The linkage of the XTEN polypeptide and the length of the XTEN polypeptide are common to XTENs, but the natural polypeptide In advantageous features not common to polypeptides, the properties of XTEN disclosed herein are , which are not linked to a complete primary amino acid sequence and are evident from the various exemplary sequences in Table 2. As such, within a variable range of lengths, they have similar characteristics, many of which are described in the examples. Therefore, XTEN is more similar to a non-proteinaceous hydrophilic polymer than to a protein. The XTEN of the present invention have the following advantageous properties: conformational flexibility, low secondary structure Reduced or deleted, highly water soluble, highly protease resistant, low immunogenicity, mammalian Low binding to solvents, a defined degree of charge, and increased hydrodynamic (or stoke) These properties make them particularly useful as conjugation partners. It is similar to certain hydrophilic polymers (e.g., polyethylene glycol) that are used in

[0127] The XTEN component(s) of the subject conjugates, regardless of the extended length of the polymer, They are designed to behave like denatured peptide sequences under physiological conditions. is the state of peptides in solution, characterized by a large degree of conformational freedom of the peptide backbone. Most peptides and proteins are degraded in the presence of high concentrations of denaturing agents or At elevated temperatures, they adopt a denatured conformation. Denatured conformational peptides exhibit, for example, characteristic circular dichroism behavior. (CD) spectrum and long-range interactions as determined by NMR. "Degenerated structure" and "unstructured structure" are used herein to refer to In some embodiments, the present invention relates to a method for producing a protein that is primarily In another example, the XTEN sequence is provided as a denatured sequence lacking secondary structure. The EN sequence is substantially devoid of secondary structure under physiological conditions. When used in pulse, as measured or determined by the means described herein, This means that less than 50% of the XTEN amino acid residues in the XTEN sequence contribute to the secondary structure. "Substantially devoid," as used in this context, of the ability to at least about 100 of the XTEN amino acid residues of the XTEN sequence, as measured or determined by 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about This means that 97%, or at least about 99%, do not contribute to secondary structure.

[0128] A variety of methods and assays for determining the physicochemical properties of a subject XTEN are available. Such properties include secondary or tertiary structure, solubility, Protein aggregation, stability, absolute and apparent molecular weight, purity and homogeneity, solubility characteristics, fouling Methods for measuring such properties include, but are not limited to, dye content and water content. Methods include analytical centrifugation, EPR, HPLC-ion exchange, and HPLC-size exclusion. Chromatography (SEC), HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism method, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NM IR, Raman spectroscopy, refractometry, and UV / visible spectroscopy. for example, in circular dichroism spectroscopy in the "far ultraviolet" spectral region (190-250 nm). Secondary structural elements, such as α-helices and β-helices, can thus be measured spectroscopically. Each sheet gives rise to a characteristic shape and size of the CD spectrum, and these The same applies to deletion of structural elements. Secondary structure is described in U.S. Patent Application Publication No. 20030228309 As described in issue A1, the well-known Chou-Fasman algorithm (Chou, P .Y.,et al.(1974)Biochemistry,13:222-45) and the Garnier-Osguthorpe-Robson algorithm ("Garnier-Osguthorpe-Robson"). Garnier J, Gibrat JF, Robson B. (199 6),GOR method for predicting protein sec ondary structure from amino acid sequence e. Methods Enzymol 266:540-553) Predictions about polypeptide sequences can also be made through programs or algorithms. For a given sequence, these algorithms determine whether several secondary structures exist or not. For example, the formation of alpha helices or beta sheets can be predicted. The total number and / or percentage of residues in the sequence that form random coils (secondary The percentage of residues in the sequence that are predicted to result in a structural defect (lacking structure). Polypeptide sequences can be found on the world wide web, for example at fasta.bioch.virg inia.edu / fasta_www2 / fasta_www.cgi?rm=mis Chou-Fasman algorithm using sites on c1, and npsa-pb il.ibcp.fr / cgi-bin / npsa_automat.pl?page= The Gor algorithm in npsa_gor4.html (both as of September 5, 2012) (accessed on 2014). Additional methods are described in Arnau, et al., Prot Expr and Purif (2006) 48, 1-13. It has been disclosed.

[0129] In one embodiment, the XTEN sequence used in the subject conjugate is Chou-Fas α-helical percentages ranging from 0% to less than approximately 5%, as determined by the MAN algorithm In another embodiment, the XTEN sequence has a Chou-Fasman β-sheet percentages ranging from 0% to less than approximately 5%, as determined by the algorithm In one embodiment, the XTEN sequence of the conjugate has a Chou-Fasman α-helical percentages ranging from 0% to less than approximately 5%, as determined by the algorithm In one embodiment, the β-sheet percentage ranges from 0% to less than about 5%. wherein the XTEN sequence of the conjugate has an alpha helix percentage of less than about 2% and a helix percentage of less than about 2%. The XTEN sequence of the conjugate composition has a beta sheet percentage of less than 100%. It has a high degree of random coil percentage as determined by the algorithm. In some embodiments, the XTEN sequence is Preferably, at least about 80%, more preferably at least about 90%, more preferably at least 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%, and even more preferably is at least about 96%, more preferably at least about 97%, more preferably at least It has about 98%, and most preferably at least about 99% random coil. In this embodiment, the XTEN sequence of the conjugate composition is determined by the Chou-Fasman algorithm. an alpha helix percentage ranging from 0% to less than about 5%, and 0% ~Beta-sheet percentage in the range of less than about 5%, as determined by the GOR algorithm In another embodiment, the nucleic acid has at least about 90% random coil. The XTEN sequences of the disclosed compositions have an alpha helix percentage of less than about 2%, and % β-sheet percentage, as determined by the GOR algorithm In another embodiment, the XT of the composition The EN sequence is virtually devoid of secondary structure as determined by circular dichroism.

[0130] The criteria for selecting the XTEN to be attached to the payload used to form the conjugate composition are: Generally, the physicochemical and conformational structural attributes of XTEN are discussed, in turn, in relation to its use. This provides the composition with enhanced pharmaceutical, pharmacological, and pharmacokinetic properties. 1. Non-repetitive sequences In particular, the subject XTEN sequences included in the subject conjugate composition embodiments are substantially non-repetitive. Generally, the repeating amino acid sequences are found in collagen and leucine dimers. aggregate or form higher order structures, as exemplified by naturally occurring repeat sequences such as These repeating amino acids tend to form contacts that result in crystalline or pseudocrystalline structures. In contrast, the low tendency of non-repetitive sequences to aggregate may explain why they are otherwise Long sequences with relatively low frequency charged amino acids that may aggregate if the sequence is repeated The non-repetitive nature of a subject XTEN can be determined by one or more of the following characteristics: In one embodiment, the substantially non-repetitive XTE The N sequence has three adjacent amino acids in the sequence that are the same amino acid type, unless the amino acid is serine. In this case, no more than three adjacent amino acids are serine residues. In embodiments, substantially non-repetitive XTEN sequences, as described more fully below, 80-99% of the sequences consist of motifs of 9-14 amino acid residues, and these The motifs are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (GA), and glutamic acid (G). 3, 4, 5, or 6 amino acids selected from thiamin (E) and proline (P) Any two adjacent amino acid residues in any one motif are composed of The sequence does not repeat the sequence motif more than twice.

[0131] The degree of repetitiveness of a polypeptide or gene can be determined by computer programs or algorithms. can be measured by rhythm or by other means known in the art. In accordance with the present invention, the repetitiveness of a particular polypeptide, such as XTEN, can be calculated. The algorithms used are disclosed herein, and the sequences analyzed by the algorithms are Exemplary sequences are provided (see Examples below). In one embodiment, a polypeptide of a predetermined length The repeatability of the subsequence can be calculated according to the formula shown in Equation I (hereafter referred to as the "subsequence sequence"). core").

[0132] [ka]

[0133] The algorithm, called "SegScore," applies the aforementioned equation to XTEN, etc. It was developed to quantify the repetitiveness of polypeptides, where the sequence of a given amino acid length "n" is , determine the number of times ("count") that a unique subsequence of length "s" appears in the set length. , the subsequence to be analyzed for repetition by dividing it by the absolute number of subsequences in the sequence of that given length. Figure 79 shows the logic flow chart of the SegScore algorithm. 80 shows the subsequence scores for 11 amino acids and 3 amino acid residues. For a hypothetical XTEN with a sequence length of 200, we sketch how it is derived. The defined polypeptide length of 192 amino acid residues is determined by 192 overlapping 9-amino acid subsequences, and and 198 3-mer subsequences, but the subsequence score of any given polypeptide is the absolute number of unique subsequences, and each unique subsequence (meaning a different amino acid sequence) , depending on how often they appear in a sequence of a given length.

[0134] In the context of the present invention, a "subsequence score" refers to the sum of 200 of the cumulative XTEN polypeptides. The total occurrence of each unique 3-mer frame across consecutive amino acids of 200 amino acids This is the number divided by the absolute number of unique 3-mer subsequences in the sequence. An example of such a subsequence score derived from 200 consecutive amino acids of a polypeptide is In one embodiment, the present invention provides an XTEN- and the XTEN provides a payload of less than 12, more preferably less than 10, more preferably More preferably, it is less than 9, more preferably, it is less than 8, more preferably, it is less than 7, more preferably, it is less than 6, and and more preferably has a subsequence score of less than 5. In another embodiment, the present invention provides The present invention provides an XTEN-crosslinker conjugate comprising XTEN, wherein the XTEN is less than 10, more preferably or less than 9, more preferably less than 8, more preferably less than 7, more preferably less than 6, and more preferably has a subsequence score of less than 5. In another embodiment, the present invention provides and an XTEN-click chemistry conjugate comprising an XTEN, the XTEN comprising less than 10, More preferably less than 9, more preferably less than 8, more preferably less than 7, more preferably have a subsequence score of less than 6, and more preferably less than 5. wherein the invention provides an XTEN conjugate comprising at least two linked XTENs, Each individual XTEN may contain less than 10, or less than 9, or less than 8, or less than 7, or less than 6 In yet another embodiment, the subsequence score is less than 0.5, or less than 5, or less than or equal to 0. In accordance with the present invention, there is provided an XTEN conjugate composition comprising at least three conjugated XTENs. provided that each individual XTEN has less than 10, or less than 9, or less than 8, or less than 7, or have a subsequence score of less than 6, or less than 5, or even lower. In embodiments of the XTEN compositions, the XTEN composition may have 10 or fewer (i.e., 9, 8, 7, etc.) subsequence sequences. XTEN having a core are characterized as being substantially non-repetitive.

[0135] In one embodiment, the non-repetitive nature of the XTEN of the present invention is due to the fact that the XTEN is not an absolute primary sequence. XTEN and the resulting XTEN-Peiro along with the specific types of amino acids predominating therein. This confers one or more of the enhanced physicochemical and biological properties of the bond conjugate. These enhanced properties include the ability to deliver payloads that are not conjugated to XTEN or that contain repeat sequences. The resulting conjugate exhibits a higher affinity for the protein in the host cell compared to a payload conjugated to a protein with the Higher XTEN protein expression, greater genetic stability of the XTEN-encoding gene These include improved solubility, lower aggregation tendency, and enhanced pharmacokinetics. These enhanced properties result in more efficient manufacturing, better uniformity in the final product, lower Allow for product costs and / or are extremely high, sometimes exceeding 100mg / ml This facilitates the formulation of XTEN, including pharmaceutical preparations containing protein concentrations that are In one embodiment, the XTEN polypeptide sequence of the conjugate, when administered to a mammal, Designed to have a low degree of internal repetition to reduce or virtually eliminate immunogenicity It is mainly limited to only three amino acids: glycine, serine, and glutamate. Polypeptide sequences consisting of short repeated motifs are predicted to be present in these sequences. Despite the absence of cellular epitopes, it elicits relatively high antibody titers when administered to mammals. Protein aggregates, cross-linked immunogens, and repeated enzymes containing repeating carbohydrates can result. Immunogens bearing the pitope are highly immunogenic, e.g., B activators that induce B cell activation. This is due to the repetitive nature of the polypeptide, which has been shown to result in cross-linking of cell receptors. This can be caused by quality (Johansson, J., et al. (2007) V accine, 25:1676-82, Yankai, Z., et al. (2006) Biochem Biophys Res Commun,345:1365-71, H su,CT,et al.(2000)Cancer Res,60:3701-5 ), Bachmann MF, et al. Eur J Immunol. (1995) 25(12):3445-3451). 2. Exemplary Sequence Motifs The present invention uses conjugation partners that contain multiple units of shorter sequences, or motifs. The amino acid sequence of the motif is substantially non-repetitive. The properties are multimerized to form XTEN sequences, as shown in Figures 18-19. The use of a "building block" approach using libraries of sequence motifs has not been met. Although XTEN sequences can consist of multiple units of as few as four different species of sequence motifs, These motifs themselves generally consist of non-repetitive amino acid sequences, so the entire XTEN sequence is designed to make the sequence substantially non-repetitive.

[0136] In one embodiment, the XTEN is greater than about 36 to about 3000, or from about 100 to about 200 0, or a substantially non-repetitive sequence of about 144 to about 1000 amino acid residues, At least about 80%, or at least about 85%, or at least about 90% of the TEN sequence %, or at least about 95%, or at least about 97%, or about 99% to about 100% % consist of non-redundant sequence motifs, and each of these motifs consists of approximately 9-36 As used herein, "non-overlapping" means that the amino acid residues are distinct motifs. The motifs do not share amino acid residues, but rather are linked in a linear fashion to other motifs or amino acid residues. In other embodiments, at least about 80% of the XTEN sequence is fused to %, or at least about 85%, or at least about 90%, or at least about 95% or at least about 97%, or about 99% to about 100% from non-overlapping sequence motifs Each of these motifs has 9 to 14 amino acid residues. In embodiments, at least about 80%, or at least about 85%, or or at least about 90%, or at least about 95%, or at least about 97%, or Approximately 99% to 100% of the sequences consist of non-overlapping sequence motifs, and each of these motifs has 12 amino acid residues. In these embodiments, the sequence motif is substantially consist primarily (e.g., 90% or more) or exclusively of small hydrophilic amino acids, and the entire sequence It is preferable to make XTEN have unstructured flexible characteristics. Examples include arginine, lysine, threonine, alanine, asparagine, glutamine , aspartate, glutamate, serine, and glycine. In this case, the XTEN sequence is about 36 to about 3000, or about 100 to about 2000, or about 1 arranged in substantially non-repetitive sequences that are between 44 and approximately 1000 amino acid residues in length , glycine (G), alanine (A), serine (S), threonine (T), glutamate It mainly contains 4 to 6 amino acids selected from the group consisting of α, β-glucan ... In some embodiments, the XTEN sequence is selected from the group consisting of glycine (G), alanine (A), serine (S), and ribonucleotides. ), threonine (T), glutamate (E), or proline (P). In some embodiments, X is formed of 4, 5, or 6 amino acids selected from the group consisting of: TEN is about 36 to about 1000, or about 100 to about 2000, or about 400 to about 30 00 amino acid residues, at least about 80% of the sequence having non-overlapping sequence motifs Each motif has 9 to 36 amino acid residues. At least 90%, or at least 91%, or at least 92%, or less at least 93%, or at least 94%, or at least 95%, or at least 9 6%, or at least 97%, or 100% are glycine (G), alanine (A), Choose from serine (S), threonine (T), glutamate (E) and proline (P) It consists of 4 to 6 amino acids and contains one of the amino acid types of full-length XTEN. In another embodiment, at least about 90% of the XTEN sequences are , consisting of non-overlapping sequence motifs, each of the motifs having 9 to 36 amino acids; These motifs consist of glycine (G), alanine (A), serine (S), and threonine (T). ), glutamate (E), and proline (P). The content of any one amino acid type of full-length XTEN is 40%, or about 30%. , or 25%, or about 17%, or about 12%, or about 8%. In this configuration, at least about 90% of the XTEN sequence consists of non-overlapping sequence motifs. Each of these motifs consists of glycine (G), alanine (A), serine ( S), threonine (T), glutamate (E), and proline (P). It has 12 amino acids consisting of 4 to 6 amino acids, and is one of the amino acids of full-length XTEN. The amino acid content is 40%, or about 30%, or 25%, or about 17%, or In still other embodiments, the XTEN sequence is at least about 12%, or at most about 8%. 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% Approximately 100% consist of non-overlapping sequence motifs, each of which contains a glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and It has 12 amino acid residues consisting of ribonucleotides (RIs) and proline (P).

[0137] In some embodiments, the invention provides a polypeptide comprising from about 36 to about 1000 amino acid residues, or The amino acid sequence is composed of one, two, or three amino acid residues of approximately 100 to approximately 3,000 in total. an XTEN-payload comprising four or more substantially non-repetitive XTEN sequence(s); XTEN-crosslinker and XTEN-click chemistry conjugates are provided, and the sequences thereof are at least about 80%, or at least about 90%, or about 91%, or about 92%, or is about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or About 98%, or about 99% to about 100%, of the amino acid sequences are four or more selected from the amino acid sequences in Table 1. The whole sequence remains substantially non-repetitive. In some embodiments, the XTEN comprises non-overlapping sequence motifs and has approximately 8 sequences. 0%, or at least about 85%, 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%, or about 100% are selected from Table 1 A family sequence consisting of multiple units of non-overlapping sequences selected from a single motif family. Family, when applied to a motif, means that XTEN is a single motif from Table 1. Motifs selected from chief categories, i.e., AD, AE, AF, AG, AM, In other embodiments, the XTEN has: AQ, BC, or BD. Among the motif families in Table 1, selected to achieve the desired physicochemical characteristics A motif sequence comprising multiple units from two or more sequences, and having a net charge, as described more fully below. , repetitiveness, which can be conferred by hydrophilicity, lack of secondary structure, or amino acid composition of the motif In the above embodiments described in this paragraph, XTEN The motif or portion of the motif to be incorporated into the Selected and assembled, approximately 36, approximately 42, approximately 72, approximately 144, approximately 288, approximately 576, Approximately 864, approximately 1000, approximately 2000 to approximately 3000 amino acid residues, or any intermediate length XTEN families useful for incorporation into subject conjugates can be achieved. Non-limiting examples of Lee sequences are presented in Table 2. The specific sequences described for Table 2 are Although the sequence is described in Any AE sequence having 44 amino acid residues (e.g., AE144_1A, AE144 _2A, etc.) or generalized with respect to the AG144 sequence Reference is made, for example, to any AG sequence having 144 amino acid residues (e.g., AG144 _1, AG144_2, AG144_A, AG144_B, AG144_C, etc.) It is intended that

[0138] [Table 1]

[0139] [Table 2-1]

[0140]

Table 2-2

[0141]

Table 2-3

[0142]

Table 2-4

[0143]

Table 2-5

[0144]

Table 2-6

[0145]

Table 2-7

[0146]

Table 2-8

[0147]

Table 2-9

[0148]

Table 2-10

[0149]

Table 2-11

[0150]

Table 2-12

[0151]

Table 2-13

[0152]

Table 2-14

[0153]

Table 2-15

[0154]

Table 2-16

[0155]

Table 2-17

[0156]

Table 2-18

[0157]

Table 2-19

[0158]

Table 2-20

[0159]

Table 2-21

[0160] [Table 2-22]

[0161] [Table 2-23]

[0162] [Table 2-24]

[0163] [Table 2-25]

[0164] [Table 2-26]

[0165] [Table 2-27]

[0166] [Table 2-28]

[0167] [Table 2-29]

[0168] [Table 2-30]

[0169] XTEN contains glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and 4, 5, or 6 amino acids selected from proline (P). less than 100% of its amino acids consist of a sequence motif from Table 1, or Table 2, 3, and some experiments with less than 100% of the sequences consisting of 22-25 XTEN sequences. In embodiments, the other amino acid residues in the XTEN are any of the other 14 naturally occurring L-amino acids. wherein the XTEN sequence is selected from at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1111%, 112%, 113%, 11 4%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids Preferably, the amino acids are selected from hydrophilic amino acids so as to have a specific amino acid sequence. (G), alanine (A), serine (S), threonine (T), glutamate (E) and Short sequences of amino acids other than ribonucleotides (RIs) and proline (P) can be incorporated into XTEN to The desired properties, such as allowing the incorporation of a restriction site, can be achieved by the addition of a This may facilitate the incorporation of binding or cleavage sequences into the loading moiety. In one exemplary embodiment, the present invention provides a method for producing a hydroxybenzoate comprising the steps of: The present invention provides XTEN that incorporate 10, or 1-5 lysine residues, and the reactive lysines are As described herein, the crosslinker may be used to conjugate to a payload. In another embodiment more fully described, the XTEN may be present in an amount of 1 to about 20, or 1 to about 1 5, or 1 to about 10, or 1 to 5 cysteine ​​residues, reactive cysteines is utilized to conjugate to a crosslinker or payload as described herein. In another embodiment, the XTEN comprises from 1 to about 20 cysteine ​​and lysine residues. These lysines and cysteines are incorporated into different cross-linking groups as described herein. In another embodiment, the XTEN is utilized to bind to an agent or payload. Combination of 1, 2, 3, 4, 5, or more arginine residues not followed by a proline residue and combine to form XTEN segments, as described more fully herein below. Glycine (G), alanine (A), and methylamino acids provide a cleavage sequence that can be cleaved by trypsin. (A), serine (S), threonine (T), glutamate (E) and proline (P) XTEN amino acids that are not present may be scattered throughout the XTEN sequence or may be present within or outside of a sequence motif. located between them, or, for example, at or near the N- or C-terminus. The XTEN sequences are either concentrated in one or more short stretches of hydrophobic amino acids. The present invention provides XTENs utilized in conjugate constructs because they provide structure to polypeptides. The content of hydrophobic amino acids in the The hydrophobic amino acid content of the XTEN is 0.01%. These include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, and vari In addition, the following amino acids are included: methionine (to avoid oxidation) less than 5% of the amino acids (to avoid deamidation), asparagine, and glutamine (to avoid deamidation) or contains less than 4%, or less than 3%, or less than 2%, or less than 1% In other embodiments, the XTEN sequence can be designed to contain or not contain: Amino groups of methionine and tryptophan in the XTEN components used in the conjugate constructs The acid content is typically less than 5%, or less than 2%, and most preferably less than 1%. In other embodiments, the XTEN of the subject XTEN conjugates comprises 10% positively charged XTEN. less than about 7%, or less than about 5%, or about 2% of the amino acid residues are positively charged The total of methionine and tryptophan residues is less than 2%. Thus, the sum of asparagine and glutamine residues is less than 5% of the total XTEN sequence. 3. Cysteine ​​and Lysine Engineered XTEN Sequences In another aspect, the present invention provides a method for treating a malaria parasite comprising administering to a patient a defined number of incorporated cysteine ​​or lysine residues. XTEN with a cysteine-engineered group, and XTEN with a lysine-engineered group, respectively. The present invention provides XTEN, which is a nucleotide sequence that is capable of binding to the thiol group of cysteine ​​or the ε-alkali of lysine. The conjugation between the amino group and a reactive group on the payload or crosslinker that is conjugated to the XTEN backbone To allow for the synthesis of XTEN with a defined number of cysteine ​​and / or lysine residues, In one embodiment of the above, the XTEN of the present invention comprises about 1 to about 100 a lysine residue, 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, ... About 10 lysine residues, or about 1 to about 5 lysine residues, or 1 to about 3 lysine residues In another embodiment of the foregoing, the present invention has only a single lysine residue. The XTEN of the invention may contain from about 1 to about 100 cysteine ​​residues, or from about 1 to about 70 cysteine ​​residues. cysteine ​​residues, or about 1 to about 50 cysteine ​​groups, or about 1 to about 30 cysteine ​​residues a group, 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 In another embodiment of the above, the XT of the present invention has only a single cysteine ​​residue. EN has about 1 to about 10 lysine residues and about 1 to about 10 cysteine ​​residues The aforementioned lysine and / or cysteine-containing XTEN can be used to synthesize XTEN, any crosslinking Constructing a conjugate containing a bridge agent plus a payload useful in treating a condition in a subject The maximum number of molecules of payload agent attached to the XTEN moiety can be lysine, cysteine, or methyl. or reactive side groups (e.g., terminal amino or thiol) incorporated into XTEN The amino acid sequence is determined by the number of other amino acids that have the

[0170] In one embodiment, the present invention provides cysteine ​​engineered XTEN, nucleotides encoding one or more amino acids are replaced with a cysteine ​​amino acid; In another embodiment, the present invention provides a method for producing a cysteine-engineered XTEN gene. and providing engineered XTEN, a nucleotide sequence encoding one or more cysteine ​​amino acids. The cysteine-engineered XTEN gene is inserted into the XTEN coding gene. In another example, about 9 to about 10 nucleotides containing codons encoding one or more cysteines are formed. Oligonucleotides encoding one or more motifs of four amino acids are called XTEN motifs. The oligos are linked in frame to the full-length XTEN or other oligos encoding the cysteine ​​residues. One or more cysteines are inserted during the formation of the XTEN gene. In one embodiment of the above, a cysteine-encoding nucleotide is inserted into the XTEN sequence. The nucleotides are linked to codons that encode the amino acids used in XTEN, Use the method described in the literature (see Example 61 and Figures 40-41) or use a standard Cysteine-XT with cysteine(s) at defined positions by molecular biology techniques EN motifs can be formed, and these motifs are expressed by full-length cysteine-engineered X In such cases, for example, a single cis- The nucleotides encoding the tein are linked to DNA encoding a motif selected from Table 1. When added, the resulting motif has 13 amino acids but contains two cysteines. Incorporation of the nucleotide sequence results in a motif with 14 amino acids, etc. The in-motif can be formed de novo and contains the nucleotide encoding cysteine. nucleotides encoding one or more amino acid residues used in XTEN (e.g. For example, G, S, T, E, P, A) by joining at defined positions. The motifs encoding the cysteine ​​amino acids can be any number of cysteine ​​amino acids, as described herein and shown in FIG. As shown in Figures 1 to 8, other XTEN-encoding motif sequences were used to generate full-length XTENs. It is then assembled by annealing to the gene encoding the lysine engineered When XTEN is utilized to form the conjugates of the invention, the above techniques are This is done by using a codon that codes for lysine instead of lysine. , containing about 9-14 amino acid residues and containing one or more reactive amino acids, namely cysteine Alternatively, new XTEN motifs can be formed that may contain a single cysteine. The non-specific motifs suitable for use in engineered XTENs containing amines or lysines are Examples of limitations are as follows: GGSPAGSCTSP GASASCAPSTG TAEAAGCGTAEAA GPEPTCPAPSG However, the present invention is based on the Motifs of different lengths, such as those shown in Tables 5 and 11, are contemplated for embedding.

[0171] Genes encoding XTENs with one or more cysteine ​​and / or lysine motifs In some cases, the gene may be constructed from existing XTEN motifs or segments. Existing "building block" polynucleotides encoding both short and long XTENs Nucleotides (e.g., AE48, AE144, AE288, AE432, AE576, A E864, AM48, AM875, AE912, AG864), or cysteine ​​and and / or may be fused in frame with nucleotides encoding a lysine-containing motif. The nucleotides encoding the 36'mers in Examples 1 to 4 and Tables 22 to 25 are linked together. Alternatively, cysteine ​​and The nucleotides encoding the islets of Tables 4 and 5 are also suitable for use in the islets encoding the islets of Tables 4 and 5. Nucleotides are used to identify existing XTEN sequences (described more fully below and in the Examples). (as described below) can be used to construct engineered XTENs, which contain reactive cysteines. and / or lysines are placed at one or more designed positions in the sequence in a desired amount. Non-limiting examples of such engineered XTEN are provided in Table 3. Thus, in one embodiment In one embodiment, the present invention provides a sequence or fragment of a sequence selected from Table 3 when optimally aligned. fragments with at least about 80% sequence identity, or at least about 90%, or or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or or about 96%, or about 97%, or about 98%, or about 99% sequence identity However, the present invention provides an XTEN sequence that is the same as or identical to the cysteine ​​or lysine residues. Application of cysteine ​​or lysine engineered methods to create XTEN encompassing residues means to be constrained to the exact composition or range of composition identities of the preceding embodiments. As will be appreciated by those skilled in the art, the cysteine ​​or lysine incorporated into the XTEN The exact positions and numbers of residues may vary without departing from the invention as described. do.

[0172] [Table 3-1]

[0173] [Table 3-2]

[0174] [Table 3-3]

[0175] [Table 3-4]

[0176] [Table 3-5] ...

Claims

[Claim 1] A composition as described in the specification.

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