Barcoded xten polypeptides and compositions thereof, and methods of making and using the same

Abstract

The present invention relates to barcoded XTEN polypeptides and compositions thereof and methods of making and using the same, specifically disclosing polypeptides comprising an extended recombinant polypeptide (XTEN) comprising a plurality of overlapping sequence motifs and one or more barcode fragments that are releasable upon protease digestion and detectable from all other proteolytically releasable fragments. Certain embodiments of these polypeptides further comprise a biologically active polypeptide, with advantageous embodiments thereof comprising a proteolytic cleavage releasable segment capable of cleaving the linkage between the XTEN polypeptide and the biologically active polypeptide. Methods of making the polypeptides and methods of using the polypeptides are also disclosed.

Description

[0001] This application is a divisional application of the Chinese patent application filed on November 13, 2020, with patent application number 202080090841.X and the invention title "XTEN polypeptide with barcode and composition thereof and method of preparation and use thereof".

[0002] sequence list

[0003] This application contains a sequence list, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on November 6, 2020, is named 20-1761-WO_Sequence_Listing_ST25.txt and is 1494 bytes in size. Background Technology

[0004] Peptides can be produced in a manner that results in a mixture of peptides. Peptide mixtures often include full-length peptides along with size variants (e.g., truncated ones). The presence of variants that differ in size from the desired full-length product can affect the biological behavior of the peptide drug substance, potentially impacting its safety and / or efficacy. For example, protein-based prodrugs for cancer treatment can be engineered to have a tumor-targeting activation mechanism. More specifically, full-length therapeutic proteins can be produced and administered in an inactive (non-cytotoxic) prodrug form, which is converted into an active drug by preferentially removing a portion of the prodrug peptide at the intended biological site (e.g., tumor). Truncated variants of the full-length construct can lose their protective sequence and become cytotoxic (active), thereby “contaminating” the prodrug composition and producing a mixture with components that are not intentionally active outside the intended biological site. In some cases, such shorter variants can pose a greater risk of immunogenicity, exhibit less selective toxicity to tumor cells, or display a less desirable pharmacokinetic profile compared to the full-length protein (e.g., resulting in a narrower therapeutic window), or harmfully have unintended effects on the receptor outside the intended site (e.g., in healthy tissue). Consequently, the detection and quantification of protein structural changes can be important for evaluating the biological properties of biotherapeutic agents (e.g., clinical safety and pharmacological efficacy) and for developing new biotherapeutic agents (e.g., with increased efficacy and reduced side effects). Existing techniques and methods for identifying and quantifying the amount of “contaminated” truncated products may include one or more drawbacks, such as limited sensitivity, ease of use, efficiency, or effectiveness. Summary of the Invention

[0005] This document discloses polypeptides comprising extended recombinant polypeptides (XTENs), said extended recombinant polypeptides comprising a plurality of non-overlapping sequence motifs. In the XTEN polypeptide of the present invention, the plurality of non-overlapping sequence motifs comprises: a set of non-overlapping sequence motifs, each of which is repeated at least twice in the XTEN polypeptide; and a unique non-overlapping sequence motif that appears only once within the XTEN polypeptide; said polypeptide further comprises a first barcode fragment that can be released from the polypeptide upon digestion by a protease. In the embodiments described herein, the first barcode fragment is part of the XTEN, comprising at least a portion of the sequence motif that appears only once within the XTEN, and is different in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide upon complete digestion by a protease. Further, in the XTEN embodiments of the present invention provided herein, the barcode fragment does not include an N-terminal amino acid or a C-terminal amino acid of the polypeptide. As further disclosed herein, the XTEN polypeptide of the present invention is characterized by comprising a length of at least 150 amino acids, more specifically, a length of 150-3000 amino acids. The amino acids constituting the XTEN polypeptide of the present invention are characterized, wherein at least 90% of these residues are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P), and the XTEN polypeptide contains at least four of these amino acids (G, A, S, T, E, or P). Additionally, the XTEN polypeptide provided herein contains non-overlapping sequence motifs of 9 to 14 amino acids in length, and within each of these non-overlapping motifs, the sequences having G, A, S, T, E, or P amino acids are substantially randomized with respect to any other non-overlapping sequence motifs constituting the XTEN polypeptide.

[0006] In some embodiments, the barcode fragment does not include a glutamic acid immediately adjacent to another glutamic acid in XTEN. In some embodiments, the barcode fragment has a glutamic acid at its C-terminus. In some embodiments, the barcode fragment has an N-terminal amino acid immediately preceding a glutamic acid residue. In some embodiments, the glutamic acid residue preceding the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, the barcode fragment does not include glutamic acid residues at positions other than the C-terminus of the barcode fragment, unless the glutamic acid is immediately followed by proline. In some embodiments, the barcode fragment is positioned 10 to 150 amino acids away from the N-terminus or C-terminus of the polypeptide.

[0007] In some embodiments, the sequence motifs of this set of non-overlapping sequence motifs are identified herein by SEQ ID NO: 182-203 and 1715-1722. In some embodiments, the sequence motifs of this set of non-overlapping sequence motifs are identified herein by SEQ ID NO: 186-189. In some embodiments, the set of non-overlapping sequence motifs comprises at least two, at least three, or all four of the sequence motifs SEQ ID NO: 186-189.

[0008] In specific embodiments, the polypeptides provided herein comprise XTEN polypeptides as disclosed herein, wherein the barcode fragments do not include the N-terminal or C-terminal amino acids of the polypeptide; do not include glutamic acid immediately adjacent to another glutamic acid in XTEN; have glutamic acid at its C-terminus; have an N-terminal amino acid immediately preceding a glutamic acid residue; and are located 10 to 125 amino acids from the N-terminus or C-terminus of the polypeptide.

[0009] In some of these specific embodiments, the glutamate residue preceding the N-terminal amino acid is not immediately adjacent to another glutamate residue. In some of these specific embodiments, the barcode fragment does not include glutamate residues at positions other than the C-terminus of the barcode fragment, unless the glutamate is immediately followed by proline.

[0010] In some embodiments, the XTEN peptide provided herein comprises a plurality of non-overlapping sequence motifs, wherein each of the sequence motifs is repeated at least twice in the XTEN peptide and has a length of 9 to 14 amino acids. In some embodiments, the sequence motifs of this set of non-overlapping sequence motifs are identified herein by SEQ ID NO: 182-203 and 1715-1722. In some embodiments, the sequence motifs of this set of non-overlapping sequence motifs are identified herein by SEQ ID NO: 186-189. In some embodiments, the set of non-overlapping sequence motifs comprises at least two, at least three, or all four of the sequence motifs SEQ ID NO: 186-189. In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN peptide are combinations of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P), wherein the XTEN peptide comprises at least four of these amino acids (G, A, S, T, E, or P). In some embodiments, the XTEN is 150 to 3000 amino acids in length. In some embodiments, the XTEN is 150 to 1000 amino acids in length. In some embodiments, the polypeptide can be cleaved by a protease that cleaves at the C-terminal side of a glutamate residue, which is not subsequently proline. In some embodiments, the protease is a Glu-C protease.

[0011] In some embodiments of the XTEN peptides provided herein, the barcode fragment is located within 200, 150, 100, or 50 amino acids from the N-terminus of the peptide. In some embodiments, the barcode fragment is located between 10 and 200, 30 and 200, 40 and 150, or 50 and 100 amino acids from the N-terminus of the protein. In some embodiments, the barcode fragment is located within 200, 150, 100, or 50 amino acids from the C-terminus of the peptide. In some embodiments, the barcode fragment is located between 10 and 200, 30 and 200, 40 and 150, or 50 and 100 amino acids from the C-terminus of the protein. In some embodiments, the length of the barcode fragment is at least 4 amino acids. In some embodiments, the length of the barcode fragment is 4 to 20, 5 to 15, 6 to 12, or 7 to 10 amino acids. In some embodiments, the barcode fragment is identified herein by SEQ ID No: 8020-8030 (BAR001-BAR011).

[0012] In some embodiments, the polypeptide further comprises a second barcode fragment, wherein the second barcode fragment is part of XTEN and is different in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide after complete digestion by the protease. In some embodiments, the polypeptide further comprises a third barcode fragment, wherein the third barcode fragment is part of XTEN and is different in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide after complete digestion by the protease.

[0013] In some embodiments, XTEN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the sequences identified herein by SEQ ID NO: 8001-8019. In some embodiments, the length of XTEN is at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.

[0014] In some embodiments, the polypeptide further comprises a bioactive polypeptide linked to an XTEN polypeptide (BPXTEN). In some embodiments, the XTEN polypeptide is linked to the bioactive polypeptide at an amino or carboxyl terminus of the XTEN. In any configuration, the barcode fragment is located within a region of the XTEN that extends 5% to 50%, 7% to 40%, or 10% to 30% of the length of the XTEN, as measured from the amino or carboxyl terminus linked to the bioactive polypeptide.

[0015] In some embodiments, the BPXTEN polypeptide further includes one or more reference fragments that are releasable from the polypeptide upon digestion by a protease, each of the reference fragments comprising a portion of the bioactive polypeptide. In some embodiments, the one or more reference fragments are single reference fragments that are different in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon digestion by a protease. In some embodiments, the reference fragment comprises a peptide whose presence in the polypeptide mixture indicates its presence or integrity (i.e., the protein has not yet been degraded or cleaved by a protease).

[0016] In some embodiments, the BPXTEN peptide further comprises a first release region (RS1) located between the XTEN and the bioactive peptide. In some embodiments, RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the sequences identified herein by any of the sequences in Tables 4a-4h and 8a-8b. In some embodiments, the bioactive peptide is identified herein by any of the sequences or combinations thereof in Tables 4a-4h and 8a-8b.

[0017] In some embodiments, BPXTEN peptides advantageously have at least twice the terminal half-life compared to bioactive peptides not linked to any XTEN.

[0018] In some embodiments, BPXTEN peptides are advantageously less immunogenic than bioactive peptides not linked to any XTEN, wherein immunogenicity can be determined by measuring the production of IgG antibodies selectively bound to the bioactive peptide after administration of a comparable dose to a human or animal.

[0019] In some embodiments, the BPXTEN peptide exhibits an apparent molecular weight factor greater than about 6 under physiological conditions.

[0020] In some embodiments, the BPXTEN polypeptide further comprises a second XTEN polypeptide, wherein the second XTEN polypeptide comprises an amino acid sequence having the same characteristics as the first XTEN component described throughout the foregoing and present disclosure of these embodiments of BPXTEN, and wherein the first XTEN polypeptide is located at the N-terminus of the bioactive polypeptide, and the second XTEN polypeptide is located at the C-terminus of the bioactive polypeptide. In some embodiments, the second XTEN polypeptide comprises an amino acid sequence different from the amino acid sequence of the first XTEN constituting these embodiments of BPXTEN. In some embodiments, the amino acid sequence of the second XTEN polypeptide is longer than the amino acid sequence of the first XTEN polypeptide.

[0021] In some embodiments, the BPXTEN peptide further comprises a second release segment (RS2) located between the bioactive peptide and the second XTEN peptide. In some embodiments, the RS1 and RS2 sequences of the first XTEN peptide are identical. In some embodiments, the RS1 of the first XTEN peptide and the RS2 of the second XTEN peptide are each substrates for cleavage by multiple proteases at one, two, three, or more cleavage sites within each release segment sequence.

[0022] In some of these embodiments, the BPXTEN polypeptide includes a further barcode fragment that is part of the second XTEN polypeptide and is different in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide after complete digestion by the protease. In some of these embodiments, the further barcode fragment does not include the C-terminal amino acid of the polypeptide. In some of these embodiments, the further barcode fragment includes a glutamic acid residue at its C-terminus. In some of these embodiments, the further barcode fragment of the second XTEN polypeptide is located within 200, 150, 100, or 50 amino acids from the C-terminus of the second XTEN component of the BPXTEN polypeptide. In some of these embodiments, the further barcode fragment of the second XTEN polypeptide is located at a position between 10 and 200, 30 and 200, 40 and 150, or 50 and 100 amino acids from the C-terminus of the second XTEN component of the BPXTEN polypeptide. In some of these embodiments, the length of the further barcode fragment is 4 to 20, 5 to 15, 6 to 12, or 7 to 10 amino acids. In some of these embodiments, further barcode fragments are identified herein by SEQ ID No: 8020-8030 (BAR001-BAR011).

[0023] In some embodiments, the second XTEN polypeptide further comprises a set of barcode fragments, including further barcode fragments and at least one additional barcode fragment, wherein each barcode fragment in the set is different in sequence and molecular weight from all other peptide fragments that can be released from the BPXTEN polypeptide after complete digestion of the polypeptide by the protease. In some embodiments, the second XTEN polypeptide is identified by SEQ ID NO: 8001-8019. In some embodiments, the further barcode fragments do not include a glutamic acid residue immediately adjacent to another glutamic acid residue in the polypeptide.

[0024] In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the second XTEN polypeptide are combinations of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), wherein the XTEN polypeptide contains at least four of these amino acids (G, A, S, T, E, or P). In some embodiments, the total number of amino acids in the first XTEN polypeptide and the sum of the total number of amino acids in the second XTEN polypeptide are at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids. In some embodiments, the second XTEN polypeptide contains a plurality of non-overlapping sequence motifs, each of which is repeated at least twice in the second XTEN polypeptide sequence and has a length of 9 to 14 amino acids.

[0025] In some embodiments, for the second XTEN polypeptide, the sequence motifs of a plurality of non-overlapping sequence motifs are identified herein by SEQ ID NO: 182-203 and 1715-1722. In some embodiments, the sequence motifs of a plurality of non-overlapping sequence motifs are identified herein by SEQ ID NO: 186-189. In some embodiments, for the second XTEN polypeptide, the plurality of non-overlapping sequence motifs comprise at least two, at least three, or all four of the following motifs: SEQ ID NO: 186-189. In some embodiments, the second XTEN polypeptide is 150 to 3000 amino acids in length. In some embodiments, the second XTEN polypeptide is 150 to 1000 amino acids in length. In some embodiments, the second XTEN polypeptide has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the sequences identified herein by SEQ ID NO: 8001-8019. In some embodiments, the length of the second XTEN polypeptide is at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.

[0026] In certain embodiments, the BPXTEN peptides provided herein comprise a first XTEN peptide containing a first RS sequence adjacent to, but not constituting, the C-terminus of the peptide, the first XTEN peptide being covalently linked to a tandemly covalently linked first and second bioactive peptides, wherein the second XTEN peptide is covalently linked to the C-terminus of the tandemly linked bioactive peptides, wherein the second XTEN peptide contains a second RS sequence adjacent to, but not constituting, the N-terminus of the second XTEN peptide, wherein the first RS sequence and the second RS sequence may be the same or different. In certain embodiments, the second XTEN peptide comprises an amino acid sequence longer than the amino acid sequence of the first XTEN peptide. In some embodiments, the first bioactive protein or the second bioactive protein, or both, comprise a specific binding protein, wherein in some embodiments, the specific binding protein specifically binds to an antigen or agonist expressed at a desired biological site. In certain embodiments, the desired biological site is a tumor, and the antigen is a tumor-specific antigen. In certain embodiments, the first bioactive peptide and the second bioactive peptide are different, including but not limited to having different specific binding affinities.

[0027] This document also discloses nucleic acids comprising polynucleotides encoding polypeptides such as any XTEN or BPXTEN polypeptide disclosed herein, or the reverse complement of said polynucleotides.

[0028] This document also discloses expression vectors comprising any polynucleotide sequence disclosed herein and a regulatory sequence operatively linked to the polynucleotide sequence, the regulatory sequence regulating the expression or other biological activities of the polynucleotide sequence.

[0029] This document discloses host cells comprising expression vectors as disclosed herein. In some embodiments, the host cell is a prokaryote. In some of these embodiments, the host cell is *Escherichia coli*. In some alternative embodiments, the host cell is a mammalian cell.

[0030] This document also discloses pharmaceutical compositions comprising polypeptides as disclosed herein and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions are formulated for administration to animals, and particularly humans, wherein said administration may be by any therapeutically effective route of administration. Pharmaceutical compositions disclosed herein can be prepared and used in any formulation known in the art and particularly suitable for the route of administration, site of administration, and intended effect on humans or animals.

[0031] This document discloses the use of polypeptides as disclosed herein, and in particular BPXTEN polypeptides, in the preparation of medicaments for treating diseases, conditions, or illnesses in humans or animals. In some embodiments, the disease, condition, or illness may be cancer.

[0032] This document discloses a method for treating diseases in humans or animals, as described above and throughout this disclosure, comprising administering one or more therapeutically effective doses of a pharmaceutical composition to a human or animal in need. In some embodiments, the pharmaceutical composition is administered to a human or animal as one or more therapeutically effective doses, said therapeutically effective doses being administered once daily, weekly, monthly, or annually on a clinically appropriate schedule and at a clinically appropriate dosage.

[0033] This document discloses mixtures comprising various peptides of varying lengths, particularly XTEN and BPXTEN peptides as disclosed herein, wherein the mixture comprises:

[0034] A first group of polypeptides, wherein each polypeptide in the first group of polypeptides comprises a barcode fragment, the barcode fragment being releaseable from the polypeptide by digestion with a protease, and having a sequence and molecular weight different from the sequence and molecular weight of all other fragments that can be released from the first group of polypeptides; and

[0035] A second group of peptides lacking the barcode fragments of the first group of peptides;

[0036] The first group of polypeptides and the second group of polypeptides each contain a reference fragment, which is common to both groups and is produced by digestion with a protease; and

[0037] The ratio of the first group of polypeptides to the polypeptides containing the reference fragment is greater than 0.7.

[0038] In some embodiments, the ratio of the first group of peptides to the peptide containing the reference fragment is greater than 0.8, 0.9, 0.95, or 0.98. In some embodiments, the reference fragment appears no more than once in each of the first and second group of peptides. In some embodiments, the protease is a protease that cleaves at the C-terminus of a glutamate residue. In some embodiments, barcode release from the peptide containing the first group of peptides is promoted by pepsin, elastase, thermophilic protease, or Glu-C protease. In some embodiments, barcode release is promoted by Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, peptides of various lengths include peptides containing at least one XTEN peptide as described herein.

[0039] In some embodiments, the first group of peptides comprises a full-length peptide, wherein the barcode fragment is a portion of the full-length peptide. In some embodiments, the full-length peptide is any peptide disclosed herein, and particularly XTEN and BPXTEN peptides. In some embodiments, the barcode fragment does not contain an N-terminal or C-terminal amino acid of the full-length peptide. In some embodiments, mixtures of peptides of various lengths differ from each other due to N-terminal truncation, C-terminal truncation, or both N-terminal and C-terminal truncation of the full-length peptide.

[0040] This document discloses a method for evaluating the relative amounts of a first group of peptides and a second group of peptides in a mixture comprising peptides of various lengths, particularly XTEN and BPXTEN peptides as disclosed herein, wherein each peptide in the first group shares a barcode fragment that appears once and only once in the peptide, and each peptide in the second group lacks a barcode fragment shared by the first group of peptides, wherein each peptide in both the first and second groups of peptides comprises a reference fragment, the method comprising:

[0041] The mixture is contacted with a protease to produce multiple protease-digested fragments derived from the cleavage of a first group of polypeptides and a second group of polypeptides, wherein the multiple protease-digested fragments comprise multiple reference fragments and multiple barcode fragments; and

[0042] The ratio of the amount of the barcode fragment to the amount of the reference fragment is determined to evaluate the relative amounts of the first group of peptides and the second group of peptides.

[0043] In some embodiments, the reference fragment appears no more than once in each of the first group of peptides and the second group of peptides.

[0044] In some embodiments, the protease cleaves polypeptides of various lengths at the C-terminal side of a glutamate residue, which is subsequently not a proline residue. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not a trypsin. In some embodiments, determining the ratio of the amount of barcode fragment to the amount of reference fragment includes quantifying the barcode fragment and reference fragment from the mixture after the polypeptide mixture has been contacted with the protease. In some embodiments, the barcode fragment and reference fragment are identified based on their respective masses. In some embodiments, the barcode fragment and reference fragment are identified by mass spectrometry. In some embodiments, the barcode fragment and reference fragment are identified by liquid chromatography-mass spectrometry (LC-MS). In some embodiments, determining the barcode fragment / reference fragment ratio includes isotopic labeling or stable isotope labeling. In some embodiments, determining the barcode fragment / reference fragment ratio includes one or a mixture of isotopically labeled reference fragments and isotopically labeled barcode fragments.

[0045] In some of these embodiments, polypeptides of various lengths comprise full-length polypeptides and truncated fragments thereof. In some of these embodiments, mixtures of polypeptides of various lengths differ from one another due to N-terminal truncation, C-terminal truncation, or both N-terminal and C-terminal truncation of the full-length polypeptide. In some of these embodiments, the ratio of barcode fragment to reference fragment is greater than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.98, or 0.99.

[0046] This document discloses a mixture comprising multiple peptides of various lengths, the mixture comprising a first group of peptides, wherein each peptide in the first group comprises a barcode fragment that can be released from the peptide by digestion with a protease and has a sequence and molecular weight different from that of all other fragments that can be released from the first group of peptides. The embodiments also include a second group of peptides lacking barcode fragments from the first group of peptides, wherein both the first and second groups of peptides each comprise a reference fragment common to both groups and can be released by digestion with a protease. In the embodiments, the number of reference fragments quantified in the peptide mixture after protease digestion is equal to the sum of the number of the first and second groups of peptides in the mixture, and the number of barcode fragments quantified in the peptide mixture after protease digestion is equal to the number of the first group of peptides in the mixture. In the embodiments, the first group of peptides comprises one reference fragment, and the ratio of the first group of peptides to peptides containing the reference fragment in the mixture is greater than 0.7.

[0047] In some embodiments, the mixture has a ratio of a first group of peptides to a peptide containing a reference fragment greater than 0.8, 0.9, or 0.95.

[0048] In one particular embodiment, the reference fragment appears no more than once in each of the first and second groups of polypeptides. In an alternative embodiment, the reference fragment appears twice in each of the first and second groups of polypeptides.

[0049] In some embodiments, the first group of polypeptides comprises a full-length polypeptide, wherein the barcode fragment is a portion of the full-length polypeptide.

[0050] In some embodiments, the full-length polypeptide includes the polypeptides disclosed herein.

[0051] In one particular embodiment, the mixture barcode fragment does not contain the N-terminal and C-terminal amino acids of the full-length polypeptide.

[0052] In some embodiments, the mixture contains polypeptides of various lengths, which differ from one another due to N-terminal truncation, C-terminal truncation, or both N-terminal and C-terminal truncation of the full-length polypeptide.

[0053] In some embodiments, the reference fragment appears no more than once in each of the first and second groups of peptides. In an alternative embodiment, the number of reference fragments in the first group of peptides may differ from the number of reference fragments in the second group of peptides, but the number of reference fragments in each peptide within each group must be the same.

[0054] In one particular embodiment, the reference fragments in the polypeptide mixture each have a sequence and molecular weight that differ from the sequences and molecular weights of all other fragments.

[0055] This document discloses a mixture comprising multiple peptides of various lengths, the mixture comprising a first group of peptides, wherein each peptide in the first group comprises

[0056] A barcode fragment, which can be released from the polypeptide by digestion with a protease, and has a sequence and molecular weight different from that of all other fragments that can be released from the first group of polypeptides. The mixture also contains a second group of polypeptides lacking the barcode fragment from the first group of polypeptides, wherein both the first and second group of polypeptides each contain a reference fragment common to both groups and can be released by digestion with a protease. The ratio of the first group of polypeptides to the polypeptides in the mixture is given by the following formula:

[0057] [Polypeptide containing barcode] / [(Polypeptide containing reference peptide) x N]

[0058] Where N is the number of times a reference peptide is released from each polypeptide in the mixture, and where the ratio of the first group of polypeptides containing a reference fragment to the polypeptides containing the reference fragment is greater than 0.7 when the first group of polypeptides contains a reference fragment.

[0059] In one particular embodiment, the ratio of the first group of peptides to the peptide containing the reference fragment is greater than 0.8, 0.9, or 0.95.

[0060] In some embodiments, the reference fragment appears no more than once in each of the first group of peptides and the second group of peptides.

[0061] In some embodiments, the reference fragment appears twice in each of the first group of peptides and the second group of peptides.

[0062] In one particular embodiment, the first group of polypeptides comprises a full-length polypeptide, wherein the barcode fragment is a portion of the full-length polypeptide.

[0063] In some embodiments, the full-length polypeptide includes the polypeptides disclosed herein. In one particular embodiment, the barcode fragment does not contain the N-terminal and C-terminal amino acids of the full-length polypeptide.

[0064] In some embodiments, mixtures of peptides of various lengths are different from each other due to N-terminal truncation, C-terminal truncation, or both N-terminal and C-terminal truncation of the full-length peptide.

[0065] In some embodiments, the reference fragment appears no more than once in each of the first and second groups of peptides. In further embodiments, the number of reference fragments in the first group of peptides may differ from the number of reference fragments in the second group of peptides, but the number of reference fragments in each peptide within each group must be the same. In some embodiments, the reference fragment in the peptides of the mixture has a sequence and molecular weight that differ from the sequence and molecular weight of all other fragments.

[0066] This document discloses a method for detecting the sequence integrity of a polypeptide comprising a first group of polypeptides in a mixture disclosed herein. The method includes the step of digesting the polypeptide mixture with a protease, the protease releasing barcode fragments and reference fragments from the first group of polypeptides and releasing reference fragments from the second group of polypeptides, and determining the ratio of barcode fragments from the first group of polypeptides to reference fragments from the first and second groups of polypeptides. In one particular embodiment, the sequence integrity of the polypeptide comprising the first group of polypeptides is detected by comparing the ratio of fragments to an expected ratio of fragments based on the number of barcode fragments and reference fragments in the polypeptide comprising the first and second groups of polypeptides.

[0067] The methods considered herein readily accommodate, for example, the qualitative and quantitative analysis of peptides containing barcode and / or reference fragments using LC / MS. In one particular embodiment, LC / MS is quantitative and detects isotopically distinguishable amounts of the barcode fragment, reference fragment, or both. In exemplary such methods, the mixture of peptides is doped with a known amount of a “standard material” to facilitate such analysis. For example, such a standard material is a standard material in the form of an isotopically labeled form comprising a mixture of said various lengths of peptides to be analyzed. This isotopically labeled standard can be added to the mixture as a complete sequence prior to digestion by the protease. Alternatively, the test sample of the mixture of peptides of various lengths and the isotopically labeled standard material are digested by the protease in separate reactions, and the protease-digested isotopically labeled standard material is added to the test sample prior to analysis by LC / MS. The method of the present invention also includes quantifying the amount of the barcode fragment, reference fragment, or both from the test sample by comparison with the quantification of the detected isotopically distinguishable amount of the barcode fragment, reference fragment, or both.

[0068] Upon review of this disclosure, those skilled in the art will recognize variations and modifications to these embodiments. The foregoing features and aspects can be implemented in any combination and sub-combination (including multiple dependent combinations and sub-combinations) of one or more other features described herein. The various features described or illustrated above, including any components thereof, can be combined or integrated in other embodiments. Furthermore, certain features may be omitted or not implemented.

[0069] Incorporate by reference

[0070] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference. Attached Figure Description

[0071] The various features of this disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of this disclosure can be obtained by referring to the following detailed description, along with the accompanying drawings, which illustrate illustrative embodiments utilizing the principles of the invention, in which:

[0072] Figure 1This describes a mixture of XTENylated protease-activated T cell engager (“XPAT”) peptides with various lengths. The full-length XPAT (top) comprises a 288-amino acid XTEN peptide at the N-terminus and an 864-amino acid XTEN peptide at the C-terminus. Various truncations can occur in one or both of the N-terminal and C-terminal XTEN peptides in the XPAT, for example, during fermentation, purification, or other steps in product preparation. While products with limited truncation (truncation close to the distal portion of the XTEN peptide from which it is linked) can function similarly to the full-length construct, severely truncation (truncation closer to the proximal portion of the XTEN peptide from which it is linked) can have significantly different pharmacological properties than their full-length counterparts. The presence of truncation poses a challenge to quantifying pharmacologically effective and ineffective variants in XPAT products. Figure 1 As shown in full-length XPAT, each XTEN peptide has a proximal and a distal end, wherein the proximal end is positioned closer to the bioactive peptide (e.g., T cell conjugates, cytokines, monoclonal antibodies (mAbs), antibody fragments, or other XTEN-modified proteins) than the distal end. Depending on the bonding orientation, the proximal or distal end of the XTEN peptide may correspond to the N-terminus or C-terminus of the XTEN peptide.

[0073] Figure 2A mixture of XPAT peptides with barcoded XTEN peptides of various lengths was depicted. In the full-length XPAT (top), the 288-amino acid-long N-terminal XTEN peptide contains three cleavably fused barcode sequences, “NA”, “NB”, and “NC” (from distal to proximal), and the 864-amino acid-long C-terminal XTEN peptide contains three cleavably fused barcode sequences, “CC”, “CB”, and “CA” (from proximal to distal). Each barcode is positioned to indicate the pharmacologically relevant length of the corresponding XTEN peptide. For example, a minor N-terminal truncated product of XPAT lacking barcode “NA” but having the more proximal barcodes “NB” and “NC” can exhibit substantially the same pharmacological properties as the full-length construct. In contrast, a major N-terminal truncated product of XPAT lacking, for example, all three barcodes at the N-terminus can be identifiable as different from the full-length construct in terms of pharmacological activity. A unique protease-cleavable sequence was identified from the bioactive polypeptide of XPAT (here, the tandem scFv containing the active portion of the T-cell conjugate). Because it is present in all length variants of XPAT (including full-length XPAT, minor truncated, and major truncated), the unique protease-cleavable sequence can be used as a reference for quantifying the amount of various truncated products relative to the total amount of bioactive protein.

[0074] Figure 3 This illustrates a potential design for a barcoded XTEN peptide by inserting a barcode-generating sequence into a generic (or conventional) XTEN peptide. An exemplary generic (or conventional) XTEN peptide (top) contains a non-overlapping 12-mer motif in the sequence “BCDABDCDABDCBDCDABDCB”, where the sequence motifs “A”, “B”, “C”, and “D” appear 3, 6, 5, and 7 times, respectively. Glu-C protease digestion of the exemplary generic XTEN peptide (top) does not produce a unique peptide other than the two ends (“NT” and “CT”). Inserting a barcode-generating sequence “X” (e.g., a unique 12-mer) into the XTEN peptide results in a unique proteolytically cleavable sequence (or barcode sequence) that does not appear anywhere else in the XTEN peptide. The barcode-generating sequence “X” can be positioned such that the resulting barcode marks the pharmacologically relevant length of the XTEN peptide. For example, an XTEN peptide lacking a barcode can be functionally different from a corresponding XTEN peptide with a barcode. Those skilled in the art will understand that the barcode generation sequence (“X”) can be the barcode sequence itself. Alternatively, the barcode generation sequence (“X”) can be different from the resulting barcode sequence. For example, the barcode sequence can overlap with and thus contain portions of a preceding or following 12-mer motif.

[0075] Figures 4A-4BThe quantification of the truncation level of the N-terminal XTEN peptide is shown. Figure 4A It has been confirmed that a barcoded XTEN peptide (bottom figure) can be constructed by replacing the sequence motif (e.g., the third sequence motif, "D") in a generic XTEN peptide (top figure) with a barcode-generating motif "X"; and in this example, the barcode-generating motif ("X") is itself a uniquely proteolytically cleavable barcode sequence. Figure 4A As shown in the image below, the barcodes are positioned such that all severely truncated forms of the XTEN peptide lack barcodes, while all finitely truncated forms of the XTEN peptide contain barcodes. Figure 4B The relative abundance of various cleavage products in two different mixtures of XPAT is shown. In one mixture, barcodes were present in 99% of the constructs containing bioactive proteins. In the other mixture, 13% of the constructs lacked barcodes. Figures 4A-4B The use of barcoded XTEN peptides to distinguish two peptide mixtures with substantially similar average molecular weights but distinctly different pharmacological activities is illustrated.

[0076] Figure 5A This paper illustrates the analytical size exclusion chromatography (SEC) method for XPAT protein and the detection of the full-length protein and its truncated derivatives. The synthetic protein + truncated fraction includes fragments as large as the full-length synthetic protein.

[0077] Figure 5B The abundance of barcode peptides in XPAT formulations, as detected by mass spectrometry, is shown. Each measurement is the XIC area of ​​the N-barcode SGPGSTPAE (SEQ ID No. 8029) and the C-barcode GSAPGTE (SEQ ID No. 8023), normalized to the 400 nM peak of their respective heavy isotope-labeled synthetic peptide.

[0078] The patent or application documents contain at least one color drawing. A copy of the color drawing disclosed in this patent or patent application will be provided by the Patent Office upon request and payment of the necessary fees. Detailed Implementation

[0079] the term

[0080] As used herein, unless otherwise stated, the following terms have their respective meanings.

[0081] As used in the specification and claims, the singular forms “an,” “a,” and “the” include the plural referents unless the context clearly indicates otherwise. For example, the term “cell” includes a variety of cells, including mixtures thereof.

[0082] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to amino acid polymers of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also covers amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation such as conjugation with a labeled component.

[0083] As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including but not limited to glycine and both its D or L optical isomers, as well as amino acid analogs and peptides. Standard single-letter or three-letter codes are used to designate amino acids.

[0084] "Host cell" includes an individual cell or cell culture that can be, or has been, a recipient of a human or animal vector. Host cells include the offspring of a single host cell. Due to naturally occurring or genetically modified variations, the offspring are not necessarily completely identical to the original parent cell (in terms of morphology or the genome of total DNA complement).

[0085] A "chimeric" protein contains at least one polypeptide comprising a region in its sequence located at a different position than that found in nature. These regions may normally be present in separate proteins and are bound together in a fusion polypeptide; or they may normally be present in the same protein but are arranged in a new arrangement in the fusion polypeptide. The protein may be described as a "conjugated," "linked," "fused," or "fusion" protein; these terms are used interchangeably herein and refer to the linking of two or more polypeptide sequences together by any means, including chemical conjugation or recombination. For example, chimeric proteins can be produced by chemical synthesis or by generating and translating polynucleotides in which peptide regions are encoded in a desired relationship.

[0086] The terms "polynucleotide," "nucleic acid," "nucleotide," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, which are deoxyribonucleotides or ribonucleotides or analogues thereof. Polynucleotides can have any three-dimensional structure and can perform any known or yet-to-be-discovered or developed function. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure can be conferred before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components.

[0087] The term "polynucleotide complement" refers to a polynucleotide molecule that has a complementary base sequence and reverse orientation compared to a reference sequence, in which it can hybridize with the reference sequence with perfect fidelity.

[0088] As used herein, a polynucleotide having “homology” or “homologous” is a polynucleotide that hybridizes to those sequences under strict conditions as defined herein and has at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity with those sequences.

[0089] When applied to polynucleotide sequences, the terms "percentage identity" and "% identity" refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a normalization algorithm. Such algorithms can insert gaps in the sequences being compared in a normalized and reproducible manner to optimize the alignment between the two sequences and thus enable a more meaningful comparison. Percentage identity can be measured, for example, over the entire length of the defined polynucleotide sequence as defined by a specific SEQ ID number, or over a shorter length, such as over the length of a fragment taken from a larger defined polynucleotide sequence, for example, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210, or at least 450 consecutive residues. Such lengths are merely exemplary, and it should be understood that any fragment length supported by the sequences shown herein in tables, figures, or sequence listings can be used to describe lengths on which percentage identity can be measured.

[0090] The "percentage (%) amino acid sequence identity" of the polypeptide sequence identified herein is defined as the percentage of amino acid residues in the query sequence that are identical to amino acid residues in a second reference polypeptide sequence or a portion thereof, after alignment and, if necessary, the introduction of vacancies to achieve maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percentage amino acid sequence identity can be performed in various ways within the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the sequences being compared. Percentage identity can be measured over the entire length of the defined polypeptide sequence, for example, as defined by a specific SEQ ID number, or over a shorter length, such as the length of a fragment taken from a larger defined polypeptide sequence, such as a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues. Such lengths are merely exemplary, and it should be understood that any segment length supported by the sequences shown herein in tables, figures, or sequence lists can be used to describe lengths on which percentage identity can be measured.

[0091] As used herein, “reproducibility” of an XTEN polypeptide amino acid sequence refers to trimer reproducibility and can be measured by a computer program or algorithm or by other means known in the art. Trimer reproducibility of an XTEN polypeptide amino acid sequence can be evaluated by determining the number of occurrences of overlapping trimer sequences within the polypeptide. For example, a polypeptide having 200 amino acid residues has 198 overlapping 3-amino acid sequences (trimers), but the number of unique trimer sequences depends on the amount of reproducibility within the sequence. A score reflecting the degree of trimer reproducibility throughout the polypeptide sequence (hereinafter, “subsequence score”) can be generated. In the context of this invention, “subsequence score” means the sum of the occurrences of each unique trimer construct across 200 consecutive amino acids of the polypeptide divided by the absolute number of unique trimer subsequences within the 200-amino acid sequence. Examples of such subsequence scores derived from the first 200 amino acids of repeating and non-reproducing polypeptides are presented in Example 73 of International Patent Application Publication No. WO 2010 / 091122 A1, which is incorporated herein by reference in its entirety. In some embodiments, the present invention provides BPXTEN polypeptides, each comprising at least one XTEN polypeptide, wherein the amino acid sequence of the XTEN polypeptide may have a subsequence score of less than 16, or less than 14, or less than 12, or more preferably less than 10.

[0092] As used herein, the term “substantially non-repeating XTEN polypeptide amino acid sequence” refers to an XTEN polypeptide in which there are few or no instances of four consecutive amino acids of the same amino acid type in the XTEN polypeptide amino acid sequence, and wherein the XTEN polypeptide amino acid sequence has a subsequence score of 12, or 10 or lower (as defined in the preceding paragraph), or does not exhibit a pattern of sequence motifs constituting the polypeptide sequence in order from the N-terminus to the C-terminus.

[0093] As described herein, the term “non-overlapping sequence motif” includes completely non-overlapping sequence motifs as well as partially non-overlapping sequence motifs, provided that the partially non-overlapping sequence motifs are not completely overlapping.

[0094] A “vector” is a nucleic acid molecule that preferably replicates itself in a suitable host, transferring an inserted nucleic acid molecule into and / or between host cells. This term includes vectors that primarily function to insert DNA or RNA into cells, replication vectors that primarily function to replicate DNA or RNA, and expression vectors that function to transcribe and / or translate DNA or RNA. It also includes vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. An “expression system” generally means a suitable host cell containing an expression vector that can function to produce the desired expression product.

[0095] As used in this article, the term "t" 1 / 2 "This means that the calculation is ln(2) / K" el The terminal half-life of K. el The terminal elimination rate constant is calculated through linear regression of the terminal linear portion of the logarithmic concentration versus time curve. Half-life generally refers to the time required for half the amount of applied substance deposited in a living organism to be metabolized or eliminated by normal biological processes. The term "t" is used in this context. 1 / 2 The terms “terminal half-life”, “elimination half-life”, and “cyclic half-life” can be used interchangeably in this article.

[0096] The terms “antigen,” “target antigen,” or “immunogen” are used interchangeably in this document to refer to an antibody fragment or a therapeutic agent based on the antibody fragment that binds to or has a structure or binding determinant specific to it.

[0097] As used herein, the term "payload" refers to a protein or peptide sequence having biological or therapeutic activity; a pair of pharmacophores for small molecules. Examples of payloads include, but are not limited to, cytokines, enzymes, hormones, and blood factors and growth factors. Payloads may also contain genetically fused or chemically conjugated portions, such as chemotherapeutic agents, antiviral compounds, toxins, or contrast agents. These conjugated portions may be linked to the remainder of the polypeptide via a linker, which may be cleavable or non-cleavable.

[0098] As used herein, the terms “treatment” or “treating,” “relief,” and “improvement” are used interchangeably and refer to methods for achieving or obtaining beneficial or desired results, including but not limited to therapeutic and / or preventive benefits. “Therapeutic benefit” means the eradication or improvement of an underlying condition being treated. Additionally, a therapeutic benefit is achieved by eradicating or improving one or more physiological symptoms associated with an underlying disease condition, wherein improvement is observed in a person or animal, although the person or animal may still suffer from the underlying condition. For preventive benefits, the composition may be administered to a person or animal at risk of developing a specific disease condition, or to a person or animal reporting one or more physiological symptoms of a disease, even if a diagnosis of the disease cannot yet be made.

[0099] As used herein, "therapeutic effect" refers to a physiological effect, including but not limited to the cure, mitigation, improvement, or prevention of disease conditions in humans or other animals caused by the fusion polypeptide of the present invention, or otherwise enhancing the physical or mental health of humans or animals, rather than the ability to induce the production of antibodies against antigenic epitopes possessed by biologically active proteins. In particular, the determination of a therapeutically effective amount is entirely within the capabilities of those skilled in the art, as provided in the detailed disclosure herein.

[0100] As used herein, the terms "therapeuticly effective amount" and "therapeuticly effective dose" refer to an amount of a bioactive protein, alone or as part of a fusion protein composition, that, when administered to a human or animal at a single dose or repeated doses, is capable of having any detectable beneficial effect on any symptom, aspect, measurement parameter, or characteristic of a disease state or condition. Such an effect need not be absolutely beneficial. A disease state can refer to a symptom or disease.

[0101] As used herein, the term “therapeutic effective dosing regimen” refers to a schedule of continuous administration of a bioactive protein, alone or as part of a fusion protein composition, wherein the dose is administered in a therapeutically effective amount to result in a sustained beneficial effect on any symptom, aspect, measurement parameter, or characteristic of a disease state or condition.

[0102] Fusion Peptides

[0103] This document discloses polypeptides comprising one or more extended recombinant polypeptides (XTENs or XTENs) (described more fully below) that may be fused or otherwise conjugated to another polypeptide, particularly a bioactive polypeptide, wherein the embodiments described herein are referred to as BPXTENs.

[0104] In some embodiments, the polypeptide comprises a first XTEN polypeptide (e.g., those described hereinafter in the “Extended Recombinant Polypeptides (XTEN)” section or elsewhere herein). In some embodiments, the polypeptide also comprises a second XTEN polypeptide (e.g., those described hereinafter in the “Extended Recombinant Polypeptides (XTEN)” section or elsewhere herein). In some embodiments, the polypeptide comprises an XTEN polypeptide (“N-terminal XTEN”) at or near its N-terminus. In some embodiments, the polypeptide comprises an XTEN polypeptide (“C-terminal XTEN”) at or near its C-terminus. In some embodiments, the polypeptide comprises both an N-terminal XTEN polypeptide and a C-terminal XTEN polypeptide. In some embodiments, the first XTEN polypeptide is an N-terminal XTEN polypeptide, and the second XTEN polypeptide is a C-terminal XTEN polypeptide.

[0105] The polypeptide may also contain a bioactive polypeptide (“BP”) linked to an XTEN polypeptide, thereby forming an XTEN-containing fusion polypeptide referred to herein as the “BPXTEN” polypeptide.

[0106] The XTEN peptide may comprise one or more barcode fragments (configured for release) that can be released from the XTEN peptide after the fusion peptide (or BPXTEN) has been digested by a protease (as described more fully below). In some embodiments, each barcode fragment is different in sequence and molecular weight from all other peptide fragments (including all other barcode fragments, if present) that can be released from the peptide after complete digestion by the protease.

[0107] (Fusion) polypeptides may include, for example, one or more reference fragments (as described more fully below) that can be released from the polypeptide after protease digestion, the protease digestion releasing barcode fragments from the polypeptide. In some embodiments, each reference fragment may be a single reference fragment that is different in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide after protease digestion.

[0108] Extended recombinant peptide (XTEN)

[0109] Chain length and amino acid composition

[0110] In some embodiments, the XTEN polypeptide comprises at least 150 amino acids. In some embodiments, the XTEN polypeptide has a length of 150 to 3,000 amino acids, or a length of 150 to 1,000 amino acids, or a length of at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids. In some embodiments, at least 90% of the amino acid residues of the XTEN polypeptide are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN polypeptide are selected from G, A, S, T, E, or P. In some embodiments, the XTEN polypeptide comprises at least four different types of G, A, S, T, E, or P amino acids. In some embodiments, the XTEN polypeptide is characterized in that it comprises at least 150 amino acids; at least 90% of the amino acid residues of the XTEN polypeptide are G, A, S, T, E, or P, and it comprises at least four different types of amino acids selected from G, A, S, T, E, and P, with any other non-overlapping sequence motifs constituting the XTEN polypeptide being substantially randomized. In some embodiments, the fusion polypeptide containing XTEN (e.g., a fusion polypeptide comprising a bioactive polypeptide conjugated thereto) comprises a first XTEN polypeptide and a second XTEN polypeptide. In some embodiments, the sum of the total number of amino acids in the first XTEN and the total number of amino acids in the second XTEN polypeptide is at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids.

[0111] Non-overlapping sequence motifs

[0112] In some embodiments, the XTEN peptides provided herein comprise or are formed by a plurality of non-overlapping sequence motifs. In some embodiments, at least one non-overlapping sequence motif is reproducible (or repeated at least twice in the XTEN), and at least one other non-overlapping sequence motif is non-reproducible (or found only once in the XTEN). In some embodiments, the plurality of non-overlapping sequence motifs comprise a set of (reproducible) non-overlapping sequence motifs, each of which is repeated at least twice in the XTEN; and a non-overlapping (non-reproducible) sequence motif that appears (or is found) only once in the XTEN. In some embodiments, each non-overlapping sequence motif is 9 to 14 (or 10 to 14, or 11 to 13) amino acids in length. In some embodiments, each non-overlapping sequence motif is 12 amino acids in length. In some embodiments, the plurality of non-overlapping sequence motifs comprise a set of non-overlapping (reproducible) sequence motifs, each of which is repeated at least twice in the XTEN; and is 9 to 14 amino acids in length. In some embodiments, the set of (reproduced) non-overlapping sequence motifs comprises the 12-mer sequence motifs identified herein by SEQ ID NOs: 182-203 and 1715-1722 in Table 1. In some embodiments, the set of (reproduced) non-overlapping sequence motifs comprises the 12-mer sequence motifs identified herein by SEQ ID NOs: 186-189 in Table 1. In some embodiments, the set of (reproduced) non-overlapping sequence motifs comprises at least two, at least three, or all four of the 12-mer sequence motifs of SEQ ID NOs: 186-189 in Table 1.

[0113] Table 1. Exemplary 12-merchant sequence motifs used to construct XTEN

[0114] Motif family* SEQ ID NO amino acid sequence AD 182 GESPGGSSGSES AD 183 GSEGSSGPGESS AD 184 GSSESGSSEGGP AD 185 GSGGEPSESGSS AE, AM 186 GSPAGSPTSTEE AE, AM, AQ 187 GSEPATSGSETP AE, AM, AQ 188 GTSESATPESGP AE, AM, AQ 189 GTSTEPSEGSAP AF, AM 190 GSTSESPSGTAP AF, AM 191 GTSTPESGSASP AF, AM 192 GTSPSGESSTAP AF, AM 193 GSTSSTAESPGP AG, AM 194 GTPGSGTASSSP AG, AM 195 GSSTPSGATGSP AG, AM 196 GSSPSASTGTGP AG, AM 197 GASPGTSSTGSP AQ 198 GEPAGSPTSTSE AQ 199 GTGEPSSTPASE AQ 200 GSGPSTESAPTE AQ 201 GSETPSGPSETA AQ 202 GPSETSTSEPGA AQ 203 GSPSEPTEGTSA BC 1715 GSGASEPTSTEP BC 1716 GSEPATSGTEPS BC 1717 GTSEPSTSEPGA BC 1718 GTSTEPSEPGSA BD 1719 GSTAGSETSTEA BD 1720 GSETATSGSETA BD 1721 GTSESATSESGA BD 1722 GTSTEASEGSAS

[0115] * indicates an individual motif sequence, which, when used together in various permutations, results in a "family sequence".

[0116] Barcode fragment

[0117] In some embodiments, the polypeptides provided herein comprise a barcode fragment (e.g., a first, second, or third barcode fragment of an XTEN polypeptide) that can be released from the polypeptide upon digestion by a protease. In some embodiments, the barcode fragment is part of an XTEN that includes at least a portion of a (non-reproducible, non-overlapping) sequence motif that appears (or is found) only once within the XTEN; and is different in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide upon complete digestion by a protease. Those skilled in the art will understand that the term "barcode fragment" (or "barcode" or "barcode sequence") can refer to a portion of an XTEN identified herein that is cleavably fused within the polypeptide, or to a peptide fragment obtained from the polypeptide.

[0118] In some embodiments, the barcode fragment does not include an N-terminal or C-terminal amino acid of the XTEN peptide. As described more fully below or anywhere herein, in some embodiments, the barcode fragment is releasable (configured to be releasable) after Glu-C digestion of the fusion peptide. In some embodiments, the barcode fragment does not include a glutamate immediately adjacent to another glutamate in the XTEN peptide. In some embodiments, the barcode fragment has a glutamate at its C-terminus. Those skilled in the art will understand that when cleavably fused within the XTEN peptide, the C-terminus of the barcode fragment may refer to the “last” (or C-terminal) amino acid residue within the barcode fragment, even if other “non-barcode” amino acid residues are located at the C-terminus of the barcode fragment within the same XTEN peptide. In some embodiments, the barcode fragment has an N-terminal amino acid immediately preceding a glutamate residue. In some embodiments, the glutamate residue preceding the N-terminal amino acid is not immediately adjacent to another glutamate residue. In some embodiments, the barcode fragment does not include a glutamate residue at a position other than the C-terminus of the barcode fragment, unless the glutamate is immediately followed by proline. In some embodiments, the barcode fragment is located 10 to 150, or 10 to 125 amino acids from the N-terminus or C-terminus of the polypeptide. In some embodiments, the barcode fragment is located within or at the location of the N-terminus of the polypeptide, or at a location within a range of 300, 280, 260, 250, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40, 36, 30, 24, 20, 12, or 10 amino acids from or at the location of the N-terminus, or within any of the foregoing. In some embodiments, the barcode fragment is located within 200, 150, 100, or 50 amino acids from the N-terminus of the polypeptide. In some embodiments, the barcode fragment is located between 10 and 200, 30 and 200, 40 and 150, or 50 and 100 amino acids from the N-terminus of the polypeptide. In some embodiments, the barcode fragment is located within 300, 280, 260, 250, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40, 36, 30, 24, 20, 12, or 10 amino acids from the C-terminus of the polypeptide, or within any of the foregoing. In some embodiments, the barcode fragment is located within 200, 150, 100, or 50 amino acids from the C-terminus of the polypeptide. In some embodiments, the barcode fragment is located between 10 and 200, 30 and 200, 40 and 150, or 50 and 100 amino acids from the C-terminus of the polypeptide.In some embodiments, the barcode fragment does not include the N-terminal or C-terminal amino acid of the peptide; does not include glutamic acid immediately adjacent to another glutamic acid in the XTEN; has glutamic acid at its C-terminus; has an N-terminal amino acid immediately preceding a glutamic acid residue; and (v) is positioned 10 to 150, or 10 to 125 amino acids away from the N-terminus or C-terminus of the peptide. In some embodiments, the glutamic acid residue preceding the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, the barcode fragment does not include glutamic acid residues at positions other than the C-terminus of the barcode fragment, unless the glutamic acid is immediately followed by proline. In some embodiments, for a barcoded XTEN peptide fused with a bioactive peptide, at least one barcode fragment (or at least two, or three barcode fragments) contained in the barcoded XTEN is positioned at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids away from the bioactive peptide. In some embodiments, the length of the barcode segment is at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids. In some embodiments, the length of the barcode segment is at least 4 amino acids. In some embodiments, the length of the barcode segment is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids, or within any of the aforementioned values. In some embodiments, the length of the barcode segment is 4 to 20, 5 to 15, 6 to 12, or 7 to 10 amino acids. In some embodiments, the barcode segment is selected from SEQ ID NO: 8020-8030 (BAR001-BAR011) in Table 2.

[0119] Table 2. Exemplary barcode fragments that can be released after Glu-C digestion

[0120] amino acid sequence SEQ ID NO: SPATSGSTPE BAR001 8020 GSAPATSE BAR002 8021 GSAPGTATE BAR003 8022 GSAPGTE BAR004 8023 PATSGPTE BAR005 8024 SASPE BAR006 8025 PATSGSTE BAR007 8026 GSAPGTSAE BAR008 8027 SATSGSE BAR009 8028 SGPGSTPAE BAR010 8029 SGSE BAR011 8030

[0121] In some embodiments, the barcoded XTEN polypeptide contains only one barcode fragment. In some embodiments, the barcoded XTEN polypeptide contains a set of barcode fragments, including a first barcode fragment, such as those described above or anywhere else herein. In these embodiments, each member of this set of barcode sequences may be distinguished from all other barcode sequences based on amino acid sequence or molecular weight (wherein these methods for distinguishing different barcode sequences will be relevant). In some embodiments, the set of barcode fragments includes a second barcode fragment (or further barcode fragments), such as those described above or anywhere else herein. In some embodiments, the set of barcode fragments includes a third barcode fragment, such as those described above or anywhere else herein. The set of barcode fragments fused within the N-terminal XTEN polypeptide may be referred to as an N-terminal group barcode (“N-terminal group”). The set of barcode fragments fused within the C-terminal XTEN polypeptide may be referred to as a C-terminal group barcode (“C-terminal group”). In some embodiments, the N-terminal group includes a first barcode fragment and a second barcode fragment. In some embodiments, the N-terminal group further comprises a third barcode fragment. In some embodiments, the C-terminal group comprises a first barcode fragment and a second barcode fragment. In some embodiments, the C-terminal group further comprises a third barcode fragment. In some embodiments, the second barcode fragment is located at the N-terminus of the first barcode fragment in the same group. In some embodiments, the second barcode fragment is located at the C-terminus of the first barcode fragment in the same group. In some embodiments, the third barcode fragment is located at the N-terminus of both the first and second barcode fragments. In some embodiments, the third barcode fragment is located at the C-terminus of both the first and second barcode fragments. In some embodiments, the third barcode fragment is located between the first and second barcode fragments. In some embodiments, the polypeptide comprises a set of barcode fragments including a first barcode fragment, a further (second) barcode fragment, and at least one additional barcode fragment, wherein each barcode fragment in the set is part of a second XTEN polypeptide and is different in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide after complete digestion by the protease.

[0122] Example of XTEN with barcode

[0123] Table 3a shows 13 exemplary barcoded XTEN amino acid sequences containing one barcode (e.g., SEQ ID NO: 8002-8003, 8005-8009, and 8013), two barcodes (e.g., SEQ ID NO: 8001, 8004, 8010, and 8012), or three barcodes (e.g., SEQ ID NO: 8011). Of these 13 exemplary barcoded XTEN peptides, six (SEQ ID NO: 8001-8003, 8008-8009, and 8011) can be fused to the C-terminus of a bioactive protein, and seven (SEQ ID NO: 8004-8007, 8010, and 8012-8013) can be fused to the N-terminus of a bioactive protein. In some embodiments, the XTEN polypeptide has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with sequences selected from SEQ ID NO:8001-8019 in Table 3a.

[0124] Table 3a. Exemplary XTEN with barcodes

[0125] SEQ ID NO. XTEN type Barcode # amino acid sequence AA's total # 8001 C-terminal XTEN 2 <![CDATA[PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGftabTSESATPESGPGSE PATSGPTE SGSEPATSGSETPGSPAGSPTSTEEGTTEPSE GSAPGTE STPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA]]> 864 8002 C-terminal XTEN 1 <![CDATA[PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PATSGPTE SGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGESEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA]]> 864 8003 C-terminal XTEN 1 <![CDATA[PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTE STPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA]]> 864 8004 N-terminal XTEN 2 <![CDATA[ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSTPA E SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSSATPESGPGE SPATSGSTPE GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP]]> 288 8005 N-terminal XTEN 1 <![CDATA[ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGE SPATSGSTPE GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP]]> 288 8006 N-terminal XTEN 1 <![CDATA[ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSTPA E SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGEEPATSGSTPEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP]]> 288 8007 N-terminal XTEN 1 <![CDATA[ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSTPA E SGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP]]> 288 8008 C-terminal XTEN 1 <![CDATA[PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTE STPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG]]> 864 8009 C-terminal XTEN 1 <![CDATA[PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTE STPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG]]> 576 8010 N-terminal XTEN 2 <![CDATA[SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE SGPGSTPAE SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE PATSGSTE ]]> 1152 8011 C-terminal XTEN 3 <![CDATA[SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSE PATSGSTE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTTEPSE GSAPGTATE SPEGSAPGTSESATPESGPGTSTEPSE GSAPGTSAE SATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTESAS]]> 1152 8012 N-terminal XTEN 2 <![CDATA[GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE GSAPATSE SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSE SASPE ]]> 864 8013 N-terminal XTEN 1 <![CDATA[GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE SATSGSE ]]> 864 8014 N-terminal XTEN (with His tag) 1 <![CDATA[SPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSTPAES GSE TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP]]> 292 8015 C endXTEN 1 <![CDATA[PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTE STPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA]]> 582 8016 C endXTEN 1 <![CDATA[TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE BETTING TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA]]> 576 8017 C endXTEN 1 <![CDATA[GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE SASPE SGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP]]> 576 8018 C endXTEN 1 <![CDATA[GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE PATSGS TEA TGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSATPESGPGSEPATS]]> 576 8019 C endXTEN 1 <![CDATA[EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE SASPE SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSEAPGSAPGTSESATPESGPGTSESAT]]> 576

[0126] In some embodiments, a barcoded XTEN peptide can be obtained by preparing one or more mutations in a generic XTEN peptide, such as any peptide listed in Table 3b, according to one or more of the following criteria: minimizing sequence changes in the XTEN peptide, minimizing changes in the amino acid composition of the XTEN peptide, substantially maintaining the net charge of the XTEN peptide, substantially maintaining (or improving) the low immunogenicity of the XTEN peptide, and substantially maintaining (or improving) the pharmacokinetic properties of the XTEN peptide. In some embodiments, the amino acid sequence of the XTEN peptide has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any of the SEQ ID NOs: 676-734 listed in Table 3b. In some embodiments, an XTEN sequence having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) but less than 100% sequence identity with any of the SEQ ID NOs: 676-734 listed in Table 3b is obtained by one or more mutations (e.g., fewer than 10, fewer than 8, fewer than 6, fewer than 5, fewer than 4, fewer than 3, or fewer than 2 mutations) from the corresponding sequences in Table 3b. In some embodiments, one or more mutations comprise the deletion, insertion, substitution, or replacement of a glutamate residue, or any combination thereof. In some embodiments, wherein the amino acid sequence of the XTEN peptide differs from any of the SEQ ID NOs: 676-734 listed in Table 3b, but has at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity with it, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the XTEN peptide amino acid sequence and the corresponding sequence in Table 3b involves the deletion, insertion, substitution, or replacement of a glutamate residue, or any combination thereof. In some such embodiments, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the XTEN peptide amino acid sequence and the corresponding sequence in Table 3b involves the substitution, or replacement of a glutamate residue, or both. As used herein, the term “substitution of the first amino acid” refers to the replacement of a second amino acid residue with a first amino acid residue, resulting in the second amino acid residue appearing at the substitution position in the obtained sequence. For example, “substitution of glutamate” refers to replacing a non-glutamate residue (e.g., serine (S)) with a glutamate (E) residue. As used herein, the term “substitution of the first amino acid” refers to replacing a first amino acid residue with a second amino acid residue, resulting in the first amino acid residue appearing at the substitution position in the obtained sequence. For example, “substitution of glutamate” refers to replacing a glutamate residue with a non-glutamate residue (e.g., serine (S)).

[0127] Table 3b. Exemplary generic XTENs for conversion into barcode-enabled XTENs

[0128] SEQ ID NO XTEN name amino acid sequence 676 AE144 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGESEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPATSGSETPGTSEPSATPESGPGSEPATSGSETPGTSEPSAT 677 AE144_1A SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGGTSTESETPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG 678 AE144_2A TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG 679 AE144_2B TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG 680 AE144_3A SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG 681 AE144_3B SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG 682 AE144_4A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSATPESGPGTSTEPSEGSAPG 683 AE144_4B TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSATPESGPGTSTEPSEGSAPG 684 AE144_5A TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG 685 AE144_6B TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSEPSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG 686 AE288_1 GTSESATPESGPGSEPATSGSETPGTSEPATPESGPGSEPATSGSETPGTSEPATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSATPESGPGSEPATSGSETPGTSEPATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSEPATPESGPGTSEPATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSETPGTSEPATPESGPGTSTEPSEGSAP 687 AE288_2 GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSAPGTSTEPSEGAP 688 AE576 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP 689 AE624 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSTGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPGTSTEPSEGSAPGST PGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSETAPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGSPAGSPTSTEEGTSETAPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSAP 690 AE864 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSTPEGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSEPATSGPGSEPATSGSETPGTSEPATSGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSEPATSGPGSEPATSGSETPGTSEPATSGPGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGSEPATSGSETPGTSEPATSGPGSPAGSPTSTEEGSPAGSPTSTEEGSTAPSGPGSEPATSGPGSEPATSGSETPG 691 AE865 GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGTSESATPESGPGSTE 692 AE866 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGSTEPSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTEP 693 AE1152 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP 694 AE144A STEPSEGSAPGSPAGSPTSTEEGTSPEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS 695 AE144B SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSPTSTEEGTSATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTPSTEGSAPG 696 AE180A TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS 697 AE216A PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT 698 AE252A ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS 699 AE288A TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPTSTEEGTSATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTESTEPSESATPESGPGTESATPESGPGTSESATPESGPGTSESATPESGPGTESATPESGPGTESATPESGPGTESATPESGPGTESATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTESTEPSESATPSGPGTESATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATS 700 AE324A PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATS 701 AE360A PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGTSTEPSEGSAPGTSESATPESGPGSETSGSETPGTSESATPESGPGSETSGSETPGTSESATPESGPGTSTEPSEGSAPGSPTSTEEGSATPESGPGSETSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSETSGSETPGSEPAT 702 AE396A PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGPTSTEEGTSTEPSEGSAPGTSTEPS 703 AE432A EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGPGSEPATSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSAPGTSESATPESGPGSEPATSGPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSPTSTEEGSPAGSPTSTEEGSPAGSAPGTSESATPESGPGSEPATS 704 AE468A EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSEPSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSPTSTEEGSPAGSPTSTEEGTSEPSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG 705 AE504A EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP 706 AE540A TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP 707 AE576A TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAP 708 AE612A GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSETAPESGPGSEPATSGSETPGTSTESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATS 709 AE648A PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTEPSESATPESGPGSTEPSESATPESGPGSTEPSESATPSGPGSTEPSESATPSGPGSTEPSESATPES TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSEPATS 710 AE684A EGSAPGSPAGSPTSTEEGTSTEPSEGAPGTSTEPSEGAPGTSESATPESGPGTSTEPSEGAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGAPGTSTEPSEGAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSTEPSEGAPGTSTE TSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPTSTEEGSATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG 711 AE720A TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGAPGTSTEPSEGAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGAP TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSAPGTSTESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSAPGTSTESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPG 712 AE756A TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGAPGTSTEPSEGAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGAP PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG 713 AE792A EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSETAPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSETAPESGPGTSTEPSEGSAP PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG 714 AE828A PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSEGSAPGTSTEPS TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGSTEPATSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSPTSTEEGSPAGSPTSTEEGTSTEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG 715 AE869 GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGSEPATSGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGSATPESGPGSEPATS 716 AE144_R1 SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTESASR 717 AE288_R1 SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSATPESGPGSEPATSGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS 718 AE432_R1 SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP 719 AE576_R1 SAGSPTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAP 720 AE864_R1 SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGPESGSETSGSETESASR 721 AE712 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSAPGTSESATPESGPGSEPATSGSETP 722 AE864_R2 GSPGAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSAP EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGPESGSETSGSETESASR 723 AE288_3 SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGPGSEPATSGSETPGSEPATSGSETPGSEPATSPGSETPGSEPATSPG 724 AE284 GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGAPGSPAGSPTSTEEGTSATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGAPGTSTEPSEGAPGSEPATSGSETPGTSESATPESGPGTSTEGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEGAPGTSTEPSEG 725 AE292 SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGGTSTEGSEPASAPGTSTEPSEGSAPGGSAP 726 AE864_2 AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG 727 AE867 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGSTEPSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTEP 728 AE867_2 SPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSETSGSETPGSEPATSGSETPGSEPATSGSETPGSEPATSGPGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGSEPATSGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGPGSPAGSPTSTEEGSPAGSPTSTEEGSTAPESGPGSEPATSGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGSTAPESGPGSEPATS 729 AE868 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGSTEPSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTEP 730 AE144_7A GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA 731 AE292 SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGSEPATSGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGSEPATSGGTSTEGSEPASAPGTSTEPSEGSAPGGSAP 732 AE293 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSTESTEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGSATPESGPGTSTEPSEGSAPGAAEPEA 733 AE300 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP 734 AE584 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGAAEPEA

[0129] In some embodiments, in order to construct the sequence of the barcoded XTEN peptide, amino acid mutations are performed on the XTEN peptides of medium length among those in Table 3b and the XTEN peptides of longer length than those in Table 3b, such as those XTEN peptides in which one or more 12-mer motifs of Table 1 are added to the N-terminus or C-terminus of the universal XTEN in Table 3b.

[0130] Further examples of the generic XTEN polypeptide amino acid sequences that can be used in accordance with this disclosure are disclosed in U.S. Patent Publications 2010 / 0239554 A1, 2010 / 0323956 A1, 2011 / 0046060 A1, 2011 / 0046061 A1, 2011 / 0077199 A1 or 2011 / 0172146 A1, or International Patent Publications WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, WO 2011028344 A2, WO2014 / 011819 A2 or WO In 2015 / 023891, the disclosures of the aforementioned patents are each expressly incorporated herein by reference.

[0131] In some embodiments, a barcoded XTEN peptide fused within the polypeptide chain adjacent to the N-terminus of the polypeptide chain (“N-terminal XTEN”) may be attached to a His tag containing multiple His residues, including six to eight His residues at the N-terminus, to facilitate purification of the fusion peptide. In some embodiments, a barcoded XTEN peptide fused within the polypeptide chain at the C-terminus (“C-terminal XTEN peptide”) may contain or be attached to a sequence EPEA at the C-terminus to facilitate purification of the fusion peptide. In some embodiments, the fusion peptide comprises both an N-terminal barcoded XTEN peptide and a C-terminal barcoded XTEN peptide, wherein the N-terminal barcoded XTEN is attached to a His tag comprising a plurality of poly(His) residues, including six to eight His residues at the N-terminus; and wherein the C-terminal barcoded XTEN peptide is attached to a sequence EPEA at the C-terminus, thereby facilitating the purification of the fusion peptide by chromatographic methods known in the art, for example to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% purity, including but not limited to IMAC chromatography, C-tagXL affinity matrix, and other such methods, including but not limited to those described in the example sections below.

[0132] Protease digestion

[0133] As described above or anywhere else herein, the barcode fragment can be cleavably fused into the XTEN polypeptide and can be released from the XTEN polypeptide (configured for release) after digestion by a protease. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease cleaves at the C-terminal side of a glutamate residue, which is subsequently not a proline. Those skilled in the art will understand that barcoded XTEN polypeptides (XTEN polypeptides containing the barcode fragment therein) are designed to achieve high efficiency, precision, and accuracy of protease digestion. For example, those skilled in the art will understand that adjacent Glu-Glu (EE) residues in the XTEN sequence can result in various cleavage patterns after Glu-C digestion. Accordingly, when the Glu-C protease is used for barcode release, the barcoded XTEN polypeptide or barcode fragment may not contain any Glu-Glu (EE) sequence. Those skilled in the art will also understand that if present in the fusion polypeptide, the dipeptide Glu-Pro (EP) sequence may be uncleavable by the Glu-C protease during the barcode release process.

[0134] BPXTEN structural configuration

[0135] In some embodiments, the BPXTEN fusion protein comprises a single BP polypeptide and a single XTEN polypeptide. Such BPXTEN proteins may have at least the following conformations, each listed in N-terminal to C-terminal orientation: BP-XTEN; XTEN-BP; BP-S-XTEN; and XTEN-S-BP, where “S” is a spacer sequence as described below.

[0136] In some embodiments, the BPXTEN protein comprises a C-terminal XTEN polypeptide and optionally a spacer region sequence (S) between the XTEN polypeptide and the BP polypeptide. Such a BPXTEN protein can be represented by Formula I (described as N-terminus to C-terminus):

[0137] (BP)-(S) x -(XTEN)(I)

[0138] Wherein BP is a bioactive protein as described below; S is a spacer sequence having 1 to 50 amino acid residues, which may optionally include a BP release segment (as described more fully below); x is 0 or 1; and XTEN may be any XTEN polypeptide described herein.

[0139] In some embodiments, the BPXTEN protein comprises an N-terminal XTEN polypeptide and optionally a spacer region sequence (S) between the XTEN polypeptide and the BP protein. Such a BPXTEN protein can be represented by Formula II (described as N-terminus to C-terminus):

[0140] (XTEN)-(S) x -(BP)(II),

[0141] Wherein BP is a bioactive protein as described below; S is a spacer sequence having 1 to 50 amino acid residues, which may optionally include a BP release segment (as described more fully below); x is 0 or 1; and XTEN may be any XTEN polypeptide as described herein.

[0142] In some embodiments, the BPXTEN protein comprises both an N-terminal XTEN polypeptide and a C-terminal XTEN polypeptide. Such BPXTEN proteins (e.g., Figure 1-2 XPAT in the equation can be represented by Equation III:

[0143] (XTEN)-(S) y -(BP)-(S) z -(XTEN)(III)

[0144] Wherein BP is a bioactive protein as described below; S is a spacer sequence having 1 to about 50 amino acid residues, which may optionally include a BP release segment (as described more fully below); y is 0 or 1; z is 0 or 1; and XTEN may be any XTEN polypeptide as described herein.

[0145] Bioactive peptides

[0146] Bioactive proteins (BPs) that can be fused to one or more XTEN polypeptides (as described herein), particularly those disclosed below, are contained in the sequences identified herein by means of Tables 4a-4h and 6a-6f, together with their corresponding nucleic acid and amino acid sequences, which are well known in the art. Descriptions and sequences of these BPs are available in public databases such as Chemical Abstracts Services Databases (e.g., CAS Registry), GenBank, The Universal Protein Resource (UniProt), and subscription-based databases such as GenSeq (e.g., Derwent). The polynucleotide sequence encoding the BP can be a wild-type polynucleotide sequence encoding a native BP (e.g., full-length or mature), or in some cases, a variant of a wild-type polynucleotide sequence (e.g., a polynucleotide encoding a wild-type, bioactive protein) whose nucleotide sequence has been optimized, for example, for expression in a particular species; or a polynucleotide encoding a variant of a wild-type protein, such as a site-directed mutant or allelic variant. Using methods known in the art and / or in conjunction with the guidance and methods provided herein, the BPXTEN constructs considered in this invention can be generated using wild-type or common cDNA sequences or codon-optimized variants of BP, entirely within the capabilities of those skilled in the art.

[0147] The BP used in BPXTEN proteins disclosed herein (e.g., fusion peptides comprising at least one BP and at least one XTEN peptide) can comprise any protein having a biological, therapeutic, preventive, or diagnostic purpose or function, or, when administered to humans or animals, may be used to mediate biological activity or to prevent or improve a disease, symptom, or condition. Particularly advantageous are BPs for which increased pharmacokinetic parameters, increased solubility, increased stability, activity masking, or some other enhanced pharmaceutical property are sought, or for those BPs for which increased terminal half-life would improve efficacy, safety, or result in reduced dosing frequency and / or improved patient compliance. Thus, various objectives can be considered in preparing BPXTEN fusion protein compositions, including improving the therapeutic efficacy of the biologically active compound compared to BPs not linked to an XTEN peptide, by, for example, increasing in vivo exposure or the length of time BPXTEN remains within the therapeutic window when administered to humans or animals.

[0148] BP can be a natural full-length protein, or it can be a fragment or sequence variant of a bioactive protein that retains at least a portion of the biological activity of the natural protein.

[0149] In one embodiment, the BP incorporated into a human or animal composition may be a recombinant polypeptide having a sequence corresponding to a protein found in nature. In another embodiment, the BP may be a sequence variant, fragment, homolog, or mimic of the natural sequence, retaining at least a portion of the biological activity of the natural BP. In a non-limiting example, the BP may be a sequence showing at least about 80% sequence identity with a protein sequence selected from Tables 4a-4h, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In a further non-limiting example, BP can be a bispecific sequence comprising a first binding domain and a second binding domain, wherein the first binding domain has a specific binding affinity for tumor-specific markers or antigens of target cells, and shows at least about 80% sequence identity with the paired VL and VH sequences of the anti-CD3 antibodies identified in Table 6f, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 6%, 97%, 98%, 99%, or 100% sequence identity; and wherein the second binding domain, which has specific binding affinity for effector cells, shows at least about 80% sequence identity with the paired VL and VH sequences of the anti-target cell antibodies identified in Table 6a, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one embodiment, the BPXTEN fusion protein may comprise a single BP protein linked to an XTEN peptide. In another embodiment, the BPXTEN protein may comprise a first BP and a second molecule of the same BP, resulting in a fusion protein comprising two BPs (e.g., two glucagon molecules or two hGH molecules) linked to one or more XTEN peptides.

[0150] Generally, when used in vivo or utilized in in vitro assays, a BP exhibits binding specificity to a given target (or a given number of targets) or another desired biological property. For example, a BP can be an agonist, receptor, ligand, antagonist, enzyme, antibody (e.g., monospecific or bispecific), or hormone. Of particular interest are BPs intended for or known to be applicable to diseases or conditions, wherein the natural BP has a relatively short terminal half-life, and enhancement of its pharmacokinetic parameters (which may optionally be released from the fusion protein by cleavage of the spacer region sequence) would allow for less frequent dosing or enhanced pharmacological effects. Also of interest are BPs having a narrow therapeutic window between the minimum effective dose or blood concentration (Cmin) and the maximum tolerated dose or blood concentration (Cmax). In this case, the linking of a BP to a fusion protein containing a selected XTEN peptide sequence can lead to improvements in these properties compared to a BP not linked to one or more XTEN peptides, making it more suitable for use as a therapeutic or prophylactic agent.

[0151] Glucose-regulated peptides

[0152] Endocrine and obesity-related diseases or conditions have reached epidemic proportions in most developed countries and represent a huge and ever-increasing healthcare burden, encompassing a wide variety of conditions affecting the body's organs, tissues, and circulatory systems. Of particular concern are endocrine and obesity-related diseases and conditions, primarily diabetes, one of the leading causes of death in the United States.

[0153] Most metabolic processes in glucose homeostasis and insulin response are regulated by multiple peptides and hormones, and many of these peptides and hormones, as well as their analogues, have been found to be effective in the treatment of metabolic diseases and conditions. Many of these peptides tend to be highly homologous to each other, even when they have opposing biological functions. Peptides that increase glucose include the peptide hormone glucagon, while those that decrease glucose include exendin-4, glucagon-like peptide-1, and amylin. However, even when intensified with the use of small molecule drugs, the use of therapeutic peptides and / or hormones has achieved limited success in the management of these diseases and conditions. In particular, dosage optimization is important for drugs and biologics used to treat metabolic diseases, especially those with narrow therapeutic windows. Hormones in general and peptides involved in glucose homeostasis often have narrow therapeutic windows. This narrow therapeutic window, coupled with the fact that such hormones and peptides typically have short half-lives (requiring frequent dosing to achieve clinical benefit), leads to difficulties in the management of these patients. While chemical modifications to therapeutic proteins, such as PEGylation, can alter their in vivo clearance and subsequent serum half-life, this requires additional manufacturing steps and results in heterogeneous end products. Furthermore, unacceptable side effects from prolonged administration have been reported. Alternatively, genetic modifications via the fusion of the Fc domain with the therapeutic protein or peptide can increase the size of the therapeutic protein, reduce clearance by the kidneys, and promote lysosomal recycling via the FcRn receptor. Unfortunately, the Fc domain cannot fold efficiently during recombinant expression and tends to form insoluble precipitates called inclusion bodies. These inclusion bodies must be dissolved, and the functional protein must be reactivated; this is a time-consuming, inefficient, and costly process.

[0154] Therefore, one aspect of the present invention is to incorporate peptides relating to glucose homeostasis, insulin resistance, and obesity (collectively, “glucose-regulating peptides”) into a BPXTEN fusion protein to produce compositions effective in the treatment of glucose, insulin, and obesity disorders, diseases, and related conditions. Suitable glucose-regulating peptides that can be linked to the XTEN polypeptides disclosed herein to produce BPXTEN proteins (which in particular include all bioactive peptides) increase glucose-dependent insulin secretion through pancreatic β-cells or enhance the action of insulin. Glucose-regulating peptides may also include bioactive peptides that stimulate transcription of the proinsulin gene in pancreatic β-cells. Furthermore, glucose-regulating peptides may also include bioactive peptides that slow gastric emptying time and reduce food intake. Glucose-regulating peptides may also include bioactive peptides that inhibit glucagon release from α-cells of Langerhans islands. Table 4a provides a non-limiting list of glucose-regulating peptide sequences that may be covered by the BPXTEN fusion protein of the present invention. The glucose-regulating peptide of the BPXTEN composition of the present invention disclosed herein may be a peptide that shows at least about 80% sequence identity with the amino acid sequence selected from Table 4a (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0155] Table 4a: Glucose-regulating peptides

[0156] Protein name (synonyms) SEQ ID NO amino acid sequence Adrenomedullin (ADM) 1 YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY Amylin, rat 2 KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY Amylin, human 3 KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY Calcitonin (hCT) 4 CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP Calcitonin, salmon 5 CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP Calcitonin gene-related peptide (h-CGRPα) 6 ACDTATCVTHRLAGLLSRSGGVVKNMVPTNVGSKAF Calcium-lowering gene-related peptide (h-CGRP β) 7 ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF Cholecystokinin (CCK) 8 MNSGVCLCVLMAVLAAGALTQPVPPADPAGSGLQRAEEAPRRQLRVSQRTDGESRAHLGALLARYIQQARKAPSGRMSIVKNLQNLDPSHRISDRDYMGWMDFGRRSAEEYEYPS CCK-33 9 KAPSGRMSIVKNLQNLDPSHRISDRDYMGWMDF CCK-8 10 DYMGWMDF Exendin-3 11 HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS Exendin-4 12 HGEGTFTSDLSKQMEEEAVR LFIEWLKNGGPSSGAPPPS FGF-19 13 MRSGCVVVHVWILAGLWLAVAGRPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCARGQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAFEEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK FGF-21 14 MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS Gastrin 15 QLGPQGPPHLVADPSKKQGPWLEEEEEAYGWMDF Gastrin-17 16 DPSKKQGPWLEEEEEAYGWMDF Gastric inhibitory polypeptide (GIP) 17 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ Ghrelin 18 GSSFLSPEHQRVQQRKESKKPPAKLQPR Glucagon 19 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT Glucagon-like peptide-1 (hGLP-1) (GLP-1; 1-37) 20 HDEFERHAEGTFTSDVSSTLEGQAALEFIAWLVKGRG GLP-1 (7-36), human 21 HAEGTFTSDVSSYLEGQAALEFIAWLVKGR GLP-1 (7-37), human 22 HAEGTFTSDVSSTLEGQAALEFIAWLVKGRG GLP-1, Frog 23 HAEGTYTNDVTEYLEEKAAKEFIEWLIKGKPKKIRYS Glucagon-like peptide-2 (GLP-2), human 24 HADGSFSDEMNTILDNLAARDFINWLIETKITD GLP-2, Frog 25 HAEGTFTNDMTNYLEEKAAKEFVGWLIKGRP-OH IGF-1 26 GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA IGF-2 27 AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE INGAP peptide (insulin-associated islet regeneration protein) 28 EESQKKLPSSRITCPQGSVAYGSYCYSLILIPQTWSNAELSCQMHFSGHLAFLLSTGEITFVSSLVKNSLTAYQYIWIGLHDPSHGTLPNGSGWKWSSSNVLTFYNWERNPSIAADRGYCAVLSQKSGFQKWRDFNCENELPYICKFKV AFP-6 (Pituitary mesotheliin) 29 TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY Leptin, human 30 VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC Neurotransmitter (U-8) in pigs 31 YFLFRPRN Neurotransmitter (U-9) 32 GYFLFRPRN Neurotransmitter (U25) (human) 33 FRVDEEFQSPFASQSRGYFLFRPRN Neurotransmitter (U25) in pigs 34 FKVDEEFQGPIVSQNRRYFLFRPRN Neurotransmitter S, human 35 ILQRGSGTAAVDFTKKDHTATWGRPFFLFRPRN Neurotransmitter U, rat 36 YKVNEYQGPVAPSGGFFLFRPRN Oxygen oxidase (OXM) 37 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA Peptide YY (PYY) 38 YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY Pramlinpeptide 39 KCNTATCATNRLANFLVHSSNNFGPILPPTNVGSNTY-NH2 Urocorticin (Ucn-1) 40 DNPSLSIDLTFHLLRTLLELARTQSQRERAEQNRIIFDSV Urocorticin (Ucn-2) 41 IVLSLDVPIGLLQILLEQARARAAREQATTNARILARVGHC Urocorticin (Ucn-3) 42 FTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI

[0157] "Adrenal medullaris" or "ADM" refers to human adrenal medullaris peptide hormone and species and sequence variants of ADM that possess at least a portion of the biological activity of mature ADM. ADM is generated from a 185-amino acid prohormone through sequential enzymatic cleavage and amidation, resulting in a 52-amino acid bioactive peptide with a measured plasma half-life of 22 minutes. The ADM-containing fusion protein of the present invention can be used specifically in diabetes for stimulating insulin secretion from pancreatic islet cells for glucose regulation, or in humans or animals with persistent hypotension. The complete genomic basis of human AM has been reported (Ishimitsu et al., 1994, Biochem. Biophys. Res. Commun 203:631-639), and analogues of the ADM peptide have been cloned, as described in U.S. Patent No. 6,320,022.

[0158] "Amylin" refers to human peptide hormones called amylin, pramlinopeptide, and species variations of amylin possessing at least a portion of the biological activity of mature amylin, as described in U.S. Patent No. 5,234,906. Amylin is a 37-amino acid polypeptide hormone co-secreted by pancreatic β-cells with insulin in response to nutrient intake (Koda et al., 1992, Lancet 339:1179-1180), and has been reported to regulate several key pathways of carbohydrate metabolism, including glucose incorporation into glycogen. The amylin-containing fusion protein of the present invention can complement the action of insulin, which regulates the rate of glucose loss from circulation and its uptake by peripheral tissues. Amylin analogues have been cloned, as described in U.S. Patent Nos. 5,686,411 and 7,271,238.

[0159] Amylin mimics that retain biological activity can be produced. For example, pramlinin has the sequence KCNTATCATNRLANFLVHSSNNFGPILPPTNVGSNTY (SEQ ID NO: 43), wherein an amino acid from the rat amylin sequence replaces an amino acid in the human amylin sequence. In one embodiment, the invention contemplates a fusion protein comprising an amylin mimic containing the sequence KCNTATCATX1RLANFLVHSSNNFGX2ILX2X2TNVGSNTY (SEQ ID NO: 44), wherein X1 is independently N or Q and X2 is independently S, P, or G. In one embodiment, an amylin mimic incorporated into BPXTEN may have the sequence KCNTATCATNRLANFLVHSSNNFGGILGGTNVGSNTY (SEQ ID NO: 45). In another embodiment, wherein the amylin mimic is used at the C-terminus of BPXTEN, the mimic may have the sequence KCNTATCATNRLANFLVHSSNNFGGILGGTNVGSNTY(NH2) (SEQ ID NO: 46).

[0160] "Calcitonin" (CT) refers to human calcitonin protein and species and sequence variants of CT with at least a portion of the biological activity of the mature CT, including salmon calcitonin ("sCT"). CT is a 32-amino acid peptide cleaved from a larger prothyroid hormone, which appears to function in the nervous and vascular systems, but has also been reported as a potent hormonal mediator of the satiety reflex (reviewed in Becker, JCEM, 89(4): 1512-1525 (2004) and Sexton, Current Medicinal Chemistry 6: 1067-1093 (1999)). The calcitonin-containing fusion protein of the present invention can be used specifically for the treatment of osteoporosis and as a therapy for Paget's disease. Synthetic calcitonin peptides have been produced as described in U.S. Patent Nos. 5,175,146 and 5,364,840.

[0161] "Calcitonin gene-related peptide" or "CGRP" refers to human CGRP peptide and species and sequence variants of CGRP possessing at least a portion of the biological activity of mature CGRP, which is a member of the calcitonin family of peptides and exists in humans in two forms: α-CGRP (a 37-amino acid peptide) and β-CGRP. CGRP shares 43-46% sequence identity with human amylin. The CGRP-containing fusion protein of the present invention can be specifically used to reduce the incidence of diabetes-related diseases, improve hyperglycemia and insulin deficiency, inhibit lymphocyte infiltration into the islets of Langerhans, and protect β-cells from autoimmune destruction. Methods for preparing synthetic and recombinant CGRP are described in U.S. Patent No. 5,374,618.

[0162] "Cholecystokinin" or "CCK" refers to human CCK peptides and species and sequence variants of those possessing at least a portion of the biological activity of mature CCK. CCK-58 is the mature sequence, while the CCK-33 amino acid sequence, first identified in humans, is the major circulating form of this peptide. The CCK family also includes an in vivo C-terminal fragment of 8 amino acids ("CCK-8"), the pentapeptide gastrin or CCK-5 of the C-terminal peptide CCK (29-33), and the CCK-4 of the C-terminal tetrapeptide CCK (30-33). CCK is a peptide hormone of the gastrointestinal system responsible for stimulating the digestion of fats and proteins. The fusion protein of the present invention containing CCK-33 and CCK-8 can be specifically used to reduce the increase in circulating glucose and enhance the increase in circulating insulin after dietary intake. Analogs of CCK-8 have been prepared as described in U.S. Patent No. 5,631,230.

[0163] "Exendin-3" refers to a glucose-regulating peptide isolated from the beaded venom lizard (Heloderma horridum) and its sequence variants possessing at least a portion of the biological activity of mature exendin-3. Exendin-3 amides are specific exendin receptor antagonists that mediate increases in pancreatic cAMP and the release of insulin and amylase. The exendin-3-containing fusion protein of this invention can be specifically used to treat diabetes and insulin resistance. The sequence and methods for its determination are described in U.S. Patent 5,424,286.

[0164] "Exendin-4" refers to the glucose-regulating peptide found in the saliva of the American venomous lizard (Heloderma suspectum) and its species and sequence variants, including the native 39-amino acid sequence HGEGTFTSDLSKQMEEEAVRLFIEYLKNGGPSSGAPPPS (SEQ ID NO: 47) and homologous sequences and peptide mimics and their variants; such as native sequences from primates and non-natural sequences possessing at least a portion of the biological activity of mature exendin-4. Exendin-4 is an incretin polypeptide hormone that lowers blood glucose, promotes insulin secretion, slows gastric emptying, and improves satiety, providing significant improvement in postprandial hyperglycemia. Table 4b shows sequences from a wide variety of species, while Table 4c shows a list of synthetic GLP-1 analogs; all of these are considered for use in the BPXTEN protein described herein.

[0165] Fibroblast growth factor 21, or "FGF-21", refers to the human protein encoded by the FGF-21 gene, or species and sequence variants thereof possessing at least a portion of the biological activity of mature FGF-21. FGF-21 stimulates glucose uptake in adipocytes, but not in other cell types; this effect is additive with respect to insulin activity. The FGF-21-containing fusion protein of the present invention can be specifically used to treat diabetes, including by inducing increased energy expenditure, fat utilization, and lipid excretion. FGF-21 has been cloned, as disclosed in U.S. Patent No. 6,716,626.

[0166] "Fibroblast growth factor 19" or "FGF-19" means the human protein encoded by the FGF-19 gene, or a species and sequence variant thereof having at least a portion of the biological activity of mature FGF-19. FGF-19 is a protein member of the fibroblast growth factor (FGF) family. FGF-19 increases hepatic expression of the leptin receptor, metabolic rate, stimulates glucose uptake in adipocytes, and leads to weight loss in obese mouse models (Fu et al., 2004, Endocrinology 145:2504-2603). The FGF-19-containing fusion protein of the present invention can be specifically used to increase metabolic rate and reverse diet and leptin-deficient diabetes. FGF-19 has been cloned and expressed as described in U.S. Patent Application No. 20020042367.

[0167] "Gastrin" refers to the human gastrin peptide, its truncated form, and species and sequence variants possessing at least a portion of the biological activity of mature gastrin. Gastrin is primarily found in three forms: gastrin-34 ("large gastrin"); gastrin-17 ("small gastrin"); and gastrin-14 ("small gastrin"), and shares sequence homology with CCK. The gastrin-containing fusion protein of the present invention can be specifically used to treat obesity and diabetes for glucose regulation. Gastrin has been synthesized as described in U.S. Patent No. 5,843,446.

[0168] "Ghrelin" refers to a human hormone that induces satiety, or species and sequence variants thereof, including natural and processed sequences of 27 or 28 amino acids and homologous sequences. Ghrelin levels increase before meals and decrease after meals, and can lead to increased food intake and increased fat mass through its action at the hypothalamic level. The ghrelin-containing fusion proteins of the present invention can be used particularly as agonists; for example, selectively stimulating the motility of the GI tract in gastrointestinal motility disorders, accelerating gastric emptying, or stimulating the release of growth hormone. For example, ghrelin analogs with sequence substitutions or truncated variants as described in U.S. Patent No. 7,385,026 can be used particularly as fusion couples with XTEN peptides as antagonists for improving glucose homeostasis, treating insulin resistance, and treating obesity. The isolation and characterization of ghrelin have been reported (Kojima et al., 1999, Nature. 402:656-660), and synthetic analogs have been prepared by peptide synthesis as described in U.S. Patent No. 6,967,237.

[0169] "Glucagon" refers to human glucagon glucose-regulating peptide, or species and sequence variants thereof, including the natural 29-amino acid sequence and homologous sequences; for example, natural sequence variants from primates; and non-natural sequence variants having at least a portion of the biological activity of mature glucagon. As used herein, the term "glucagon" also includes peptide mimics of glucagon. The glucagon-containing fusion protein of the present invention can be particularly used to increase blood glucose levels in individuals with existing liver glycogen reserves and to maintain glucose homeostasis in diabetic patients. Glucagon has been cloned, as disclosed in U.S. Patent No. 4,826,763.

[0170] "GLP-1" refers to human glucagon-like peptide-1 and its sequence variants possessing at least a portion of the biological activity of mature GLP-1. The term "GLP-1" includes human GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36) amide. GLP-1 stimulates insulin secretion, but only during periods of hyperglycemia. The safety of GLP-1 compared to insulin is enhanced by this property and the observation that the amount of insulin secreted is proportional to the magnitude of hyperglycemia. The biological half-life of GLP-1(7-37)OH is only 3 to 5 minutes (US Patent No. 5,118,666). The GLP-1-containing fusion protein of the present invention can be specifically used to treat diabetes and insulin resistance for glucose regulation. GLP-1 has been cloned and derivatives prepared as described in US Patent No. 5,118,666. Non-limiting examples of GLP-1 sequences from a wide variety of species are shown in Table 4b, while Table 4c shows sequences of many synthetic GLP-1 analogs; all of these are considered for use in the BPXTEN compositions described herein.

[0171] Table 4b: Representative naturally occurring GLP-1 homologs as BP candidates

[0172] Gene name SEQ ID NO amino acid sequence GLP-1 {Frog} 48 HAEGTYTNDVTEYLEEKAAKEFIEWLIKGKPKKIRYS GLP-1a {African clawed frog (Xenopus laevis)} 49 HAEGTFTSDVTQQLDEKAAKEFIDWLINGGPSKEIIS GLP-1b {African clawed frog} 50 HAEGTYTNDVTEYLEEKAAKEFIIEWLIKGKPK GLP-1c {African Xenopus Lagoon} 51 HAEGTFTNDMTNYLEEKAAKEFVGWLIKGRPK Gastric inhibitory polypeptide {Mus musculus} 52 HAEGTFISDYSIAMDKIRQQDFVNWLL Glucose-dependent insulinotropic peptide {Equus caballus} 53 HAEGTFISDYSIAMDKIRQQDFVNWLL Glucagon-like peptide {Petromyzon marinus} 54 hadgtftndmtsyldakaardfvswlarsdks Glucagon-like peptide {Anguilla rostrata} 55 Haegtytsdvssylqdqaakefvswlktgr Glucagon-like peptide {Anguilla anguilla} 56 haegtytsdvssylqdqaakefvswlktgr Glucagon-like peptide {Hydrolagus colliei} 57 hadgiytsdvasltdylkskrfveslsnynkrqndrrm Glucagon-like peptide {Amia calva} 58 yadapyisdvysylqdqvakkwlksgqdrre GLUC_ICTPU / 38-65 59 HADGTYTSDVSSYLQEQAAKDFITWLKS GLUCL_ANGRO / 1-28 60 HAEGTYTSDVSSYLQDQAAKEFVSWLKT GLUC_BOVIN / 98-125 61 HAEGTFTSDVSSYLEGQAAKEFIAWLVK GLUC1_LOPAM / 91-118 62 HADGTFTSDVSSYLKDQAIKDFVDRLKA GLUCL_HYDCO / 1-28 63 HADGIYTSDVASLTDYLKSKRFVESLSN GLUC_CAVPO / 53-80 64 HSQGTFTSDYSKYLDSRRAQQFLKWLLN GLUC_CHIBR / 1-28 65 HSQGTFTSDYSKHLDSRYAQEFVQWLMN GLUC1_LOPAM / 53-80 66 HSEGTFSNDYSKYLEDRKAQEFVRWLMN GLUC_HYDCO / 1-28 67 HTDGIFSSDYSKYLDNRRTKDFVQWLLS GLUC_CALMI / 1-28 68 HSEGTFSSDYSKYLDSRRAKDFVQWLMS GIP_BOVIN / 1-28 69 YAEGTFISDYSIAMDKIRQQDFVNWLLA VIP_MELGA / 89-116 70 HADGIFTTVYSHLLAKLAVKRYLHSLIR PACA_CHICK / 131-158 71 HIDGIFTDSYSRYRKQMAVKKYLAAVLG VIP_CAVPO / 45-72 72 HSDALFTDTYTRLRKQMAMKKYLNSVLN VIP_DIDMA / 1-28 73 HSDAVFTDSYTRLLKQMAMRKYLDSILN EXE1_HELSU / 1-28 74 HSDATFTAEYSKLLAKLALQKYLESILG SLIB_CAPHI / 1-28 75 YADAIFTNSYRKVLGQLSARKLLQDIMN SLIB_RAT / 31-58 76 HADAIFTSSYRRILGQLYARKLLHEIMN SLIB_MOUSE / 31-58 77 HVDAIFTTNYRKLLSQLYARKVIQDIMN PACA_HUMAN / 83-110 78 VAHGILNEAYRKVLDQLSAGKHLQSLVA PACA_SHEEP / 83-110 79 VAHGILDKAYRKVLDQLSARRYLQTLMA PACA_ONCNE / 82-109 80 HADGMFNKAYRKALGQLSARKYLHSLMA GLUC_BOVIN / 146-173 81 HADGSFSDEMNTVLDSLATRDFINWLLQ SECR_CANFA / 1-27 82 HSDGTFTSELSRLRESARLQRLLQGLV SECR_CHICK / 1-27 83 HSDGLFTSEYSKMRGNAQVQKFIQNLM EXE3_HELHO / 48-75 84 HSDGTFTSDLSKQMEEEAVRLFIEWLKN

[0173] Table 4c: Representative GLP-1 synthetic analogs

[0174] SEQ ID NO Amino acid sequence 85 HAEGTFTSDVSSYLEGQAAREFIAWLVKGRG 86 HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG 87 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGKG 88 HAEGTFTSDVSSYLEGQAAREFIAWLVRGKG 89 HAEGTFTSDVSSYLEGQAAREFIAWLVRGKGR 90 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRK 91 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRRK 92 HAEGTFTSDVSSYLEGQAAREFIAWLVKGKG 93 HAEGTFTSDVSSYLEGQAAKEFIAWLVRGKG 94 HAEGTFTSDVSSYLEGQAAREFIAWLVKGRGRK 95 HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRGRRK 96 HAEGTFTSDVSSYLEGQAAREFIAWLVRGKGRK 97 HAEGTFTSDVSSYLEGQAAREFIAWLVRGKGRRK 98 HGEGTFTSDVSSYLEGQAAREFIAWLVKGRG 99 HGEGTFTSDVSSYLEGQAAKEFIAWLVRGRG 100 HGEGTFTSDVSSYLEGQAAKEFIAWLVKGKG 101 HGEGTFTSDVSSYLEGQAAREFIAWLVRGKG 102 HGEGTFTSDVSSYLEGQAAREFIAWLVRGRGRK 103 HGEGTFTSDVSSYLEGQAAREFIAWLVRGRGRRK 104 HGEGTFTSDVSSYLEGQAAREFIAWLVKGKG 105 HGEGTFTSDVSSYLEGQAAKEFIAWLVRGKG 106 HGEGTFTSDVSSYLEGQAAREFIAWLVKGRGRK 107 HGEGTFTSDVSSYLEGQAAKEFIAWLVRGRGRRK 108 HGEGTFTSDVSSYLEGQAAREFIAWLVRGKGRK 109 HGEGTFTSDVSSYLEGQAAREFIAWLVRGKGRRK 110 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGK 111 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRK 112 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRRK 113 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREK 114 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFK 115 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPK 116 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPEK 117 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPEEK 118 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGK 119 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRK 120 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRRK 121 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREK 122 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFK 123 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPK 124 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPEK 125 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPEEK 126 DEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRK 127 DEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRRK 128 DEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREK 129 DEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFK 130 DEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPK 131 DEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPEK 132 DEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPEEK 133 EFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGK 134 EFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRK 135 EFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRRK 136 EFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREK 137 EFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFK 138 EFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPK 139 EFERHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFPEK 140 EFERHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFPEEK 141 FERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGK 142 FERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRKGRK 143 FERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRRK 144 FERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREK 145 FERHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFK 146 FERHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFPK 147 FERHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFPEK 148 FERHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFPEEK 149 ERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGK 150 ERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRKGRK 151 ERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRRK 152 ERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREK 153 ERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFK 154 ERHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPK 155 ERHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFPEK 156 ERHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFPEEK 157 RHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGK 158 RHAEGTFTSDVSSYLEGQAAREFIAWLVRGRKGRK 159 RHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRRK 160 RHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREK 161 RHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFK 162 RHAEGTFTSDVSSYLEGQAAREFIAWLVRGRGRREFPK 163 RHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFPEK 164 RHAEGTFTSDVSSYLEGQKAAREFIAWLVRGRGRREFPEEK 165 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVKGRGK 166 HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK 167 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGKGK 168 HAEGTFTSDVSSYLEGQAAREFIAWLVKGRGK 169 HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRGK 170 HAEGTFTSDVSSYLEGQAAREFIAWLVRGKGK 171 HAEGTFTSDVSSYLEGQAAREFIAWLVRGRGK 172 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVKGRGRK 173 HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVRGRGRK 174 HDEFERHAEGTFTSDVSSYLEGQAAREFIAWLVRGKGRK 175 HAEGTFTSDVSSYLEGQAAREFIAWLVKGRGRK 176 HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRGRK 177 HAEGTFTSDVSSYLEGQAAREFIAWLVRGKGRK 178 HGEGTFTSDVSSYLEGQAAREFIAWLVKGRGK 179 HGEGTFTSDVSSYLEGQAAREFIAWLVRGKGK

[0175] The natural GLP sequence can be described by several sequence motifs presented below. The letters in parentheses represent the amino acids that are acceptable at each sequence position: {HVY} {AGISTV} {DEHQ} {AG} {ILMPSTV} {FLY}{DINST} {ADEKNST} {ADENSTV} {LMVY} {ANRSTY} {EHIKNQRST} {AHILMQVY} {LMRT}{ADEGKQS} {ADEGKNQSY} {AEIKLMQR} {AKQRSVY} {{AILMQSTV} {GKQR} {DEKLQR}{FHLVWY} {ILV} {ADEGHIKNQRST} {ADEGNRSTW} {GILVW} {AIKLMQSV} {ADGIKNQRST}{GKRSY} (SEQ ID NO: 9399). In addition, synthetic analogues of GLP-1 can be used as fusion partners of XTEN peptides to produce BPXTEN proteins with bioactivity that can be used to treat glucose-related diseases.

[0176] “GLP-2” refers to human glucagon-like peptide-2 and its sequence variants that possess at least a portion of the biological activity of mature GLP-2. More specifically, GLP-2 is a 33-amino acid peptide co-secreted by enteroendocrine cells in the small and large intestines, together with GLP-1.

[0177] "Insulin-like growth factor 1" or "IGF-1" refers to the human IGF-1 protein and species and sequence variants possessing at least a portion of the biological activity of mature IGF-1. IGF-1 consists of 70 amino acids and is primarily produced by the liver as an endocrine hormone, as well as in target tissues via paracrine / autocrine pathways. The IGF-1-containing fusion protein of this invention can be specifically used to treat diabetes and insulin resistance for glucose regulation. IGF-1 has been cloned and expressed in *E. coli* and yeast, as described in U.S. Patent No. 5,324,639.

[0178] "Insulin-like growth factor 2" or "IGF-2" refers to the human IGF-2 protein and species and sequence variants that possess at least a portion of the biological activity of mature IGF-2. IGF-2 has been cloned, as described in Bell et al., 1985, Proc Natl AcadSci US A. 82:6450-4.

[0179] "Insulin-associated protein" (INGAP) or "pancreatic β-cell growth factor" refers to human INGAP peptide and species and sequence variants of INGAP possessing at least a portion of the biological activity of mature INGAP. The INGAP-containing fusion protein of this invention can be specifically used for the treatment or prevention of diabetes and insulin resistance. INGAP has been cloned and expressed, as described in R Rafaeloff et al., 1997, J Clin Invest. 99(9): 2100–2109.

[0180] "Pituitary middle lobe peptide" or "AFP-6" refers to human pituitary middle lobe peptide and species and sequence variants of it having at least a portion of the biological activity of mature pituitary middle lobe peptide. Treatment with pituitary middle lobe peptide results in a decrease in blood pressure in normal and hypertensive humans or animals, as well as inhibition of gastric emptying activity, and involves glucose homeostasis. The pituitary middle lobe peptide-containing fusion protein of the present invention can be specifically used to treat diabetes, insulin resistance, and obesity. The pituitary middle lobe peptide and variants have been cloned as described in U.S. Patent No. 6,965,013.

[0181] "Lepin" refers to naturally occurring leptin from any species, as well as the biologically active D-isoform, or fragments and sequence variants thereof. The leptin-containing fusion protein of this invention can be specifically used to treat diabetes, for glucose regulation, insulin resistance, and obesity. Leptin has been cloned as described in U.S. Patent No. 7,112,659, and leptin analogs and fragments have been cloned as described in U.S. Patent Nos. 5,521,283, 5,532,336, PCT / US96 / 22308, and PCT / US96 / 01471.

[0182] "Neurotransferase" refers to the neurotransferase family of peptides, including neurotransferase U and S peptides, and their sequence variants. The neurotransferase U family includes various truncated or spliced ​​variants, such as FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN (SEQ ID NO: 180). An example of the neurotransferase S family is human neurotransferase S having the sequence ILQRGSGTAAVDFTKKDHTATWGRPFFLFRPRN (SEQ ID NO: 181), particularly its amide form. The neurotransferase fusion protein of the present invention can be particularly used to treat obesity, diabetes, reduced food intake, and other related conditions and disorders as described herein.

[0183] "Gasitin" or "OXM" refers to human gastrin and species and sequence variants of it that possess at least a portion of the biological activity of mature OXM. OXM is a 37-amino acid peptide produced in the colon, containing the 29-amino acid sequence of glucagon, followed by an 8-amino acid carboxyl-terminal extension. The OXM-containing fusion protein of the present invention can be specifically used to treat diabetes for glucose regulation, insulin resistance, obesity, and can also be used as a weight loss therapy.

[0184] "PYY" refers to the human peptide YY polypeptide and species and sequence variants of it that possess at least a portion of the biological activity of mature PYY. The PPY-containing fusion protein of this invention can be specifically used to treat diabetes for glucose regulation, insulin resistance, and obesity. Analogs of PYY have been prepared as described in U.S. Patent Nos. 5,604,203, 5,574,010, and 7,166,575.

[0185] "Urocorticin" refers to human urocorticin peptide hormone and its sequence variants having at least a portion of the biological activity of mature urocorticin. Three human urocorticins exist: Ucn-1, Ucn-2, and Ucn-3. Further urocorticins and analogues are described in U.S. Patent No. 6,214,797. The BPXTEN protein containing urocorticin of the present invention can also be used specifically to treat or prevent conditions associated with stimulation of ACTH release, hypertension due to vasodilatory effects, inflammation mediated by factors other than ACTH elevation, high fever, appetite disorders, congestive heart failure, stress, anxiety, and psoriasis. Urocorticin-containing fusion proteins can also be combined with natriuretic peptide modules, amylin family and exendin family or GLP1 family modules to provide enhanced cardiovascular benefits, such as treating CHF by providing a beneficial vasodilatory effect.

[0186] Metabolic diseases and cardiovascular proteins

[0187] Metabolic and cardiovascular diseases represent a significant healthcare burden in most developed countries, with cardiovascular disease remaining the leading cause of death and disability in the United States and most European countries. Metabolic diseases and conditions encompass a wide range of conditions affecting the body's organs, tissues, and circulatory system.

[0188] Dyslipidemia is frequently observed in people with diabetes and in animals with cardiovascular disease; it is typically characterized by parameters such as elevated plasma triglycerides, low HDL (high-density lipoprotein) cholesterol, normal to elevated LDL (low-density lipoprotein) cholesterol, and increased levels of small, dense LDL particles in the blood. Dyslipidemia and hypertension are major contributors to increased morbidity of coronary events, kidney disease, and death in people and animals with metabolic diseases such as diabetes and cardiovascular disease.

[0189] Cardiovascular diseases can manifest as a wide range of conditions, symptoms, and changes in clinical parameters involving the heart, blood vessels, and organ systems throughout the body. These include aneurysms, angina pectoris, atherosclerosis, stroke, cerebrovascular disease, congestive heart failure, coronary artery disease, myocardial infarction, decreased cardiac output, peripheral vascular disease, hypertension, hypotension, and blood markers such as C-reactive protein, BNP, and enzymes such as CPK, LDH, SGPT, and SGOT.

[0190] Most metabolic processes and many cardiovascular parameters are regulated by multiple peptides and hormones (“metabolic proteins”), and many such peptides and hormones, as well as their analogues, have been found to be effective in the treatment of these diseases and conditions. However, even when enhanced with the use of small molecule drugs, the use of therapeutic peptides and / or hormones has achieved limited success in the management of these diseases and conditions. In particular, dosage optimization is important for drugs and biologics used to treat metabolic diseases, especially those with narrow therapeutic windows. Hormones in general and peptides involved in glucose homeostasis often have narrow therapeutic windows. The narrow therapeutic window, coupled with the fact that such hormones and peptides typically have short half-lives (requiring frequent dosing to achieve clinical benefit), leads to difficulties in the management of these patients. Therefore, there remains a need for therapeutic agents with increased efficacy and safety in the treatment of metabolic diseases.

[0191] Therefore, one aspect of the present invention is to incorporate bioactive metabolic proteins relating to or used in the treatment of metabolic and cardiovascular diseases and conditions into a BPXTEN fusion protein to produce a composition effective in the treatment of such diseases and conditions. Metabolic proteins may include any protein having a biological, therapeutic, or preventative purpose or function that can be used to prevent, treat, mediate, or improve metabolic or cardiovascular diseases, conditions, or conditions. Table 4d provides a non-limiting list of such sequences of metabolic BPs covered by the BPXTEN fusion protein of the present invention. The metabolic protein of the BPXTEN composition of the present invention may be a protein that shows at least about 80% sequence identity with the protein sequences selected from Table 4d, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0192] Table 4d: Bioactive proteins used in metabolic disorders and cardiology

[0193] Protein Name (synonyms) Sequence SEQ ID NO. Anti-CD3 See U.S. Patent Nos. 5,885,573 and 6,491,916 IL-1ra, human full length MEICRGLRSHLITLLLFLFHSETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE 1723 IL-1ra, canine METCRCPLSYLISFLLFLPHSETACRLGKRPCRMQAFRIWDVNQKTFYLRNNQLVAGYLQGSNTKLEEKLDVVPVEPHAVFLGIHGGKLCLACVKSGDETRLQLEAVNITDLSKNKDQDKRFTFILSDSGPTTSFESAACPGWFLCTALEADRPVSLTNRPEEAMMVTKFYFQKE 1724 IL-1ra, rabbit MRPSRSTRRHLISLLLFLFHSETACRPSGKRPCRMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNAKLEERIDVVPLEPQLLFLGIQRGKLCLSCVKSGDKMKLHLEAVNITDLGKNKEQDKRFTFIRSNSGPTTTFESASCPGWFLCTALEADQPVSLTNTPDDSIVVTKFYFQED 1725 IL-1ra, rat MEICRGPYSHLISLLLILLFRSESAGHIPAGKRPCKMQAFRIWDTNQKTFYLRNNQLIAGYLQGPNTKLEEKIDMVPIDFRNVFLGIHGGKLCLSCVKSGDDTKLQLEEVNITDLNKNKEEDKRFTFIRSETGPTTSFESLACPGWFLCTTLEADHPVSLTNTPKEPCTVTKFYFQED 1726 IL-1ra, mouse MEICWGPYSHLISLLLILLFHSEAACRPSGKRPCKMQAFRIWDTNQKTFYLRNNQLIAGYLQGPNIKLEEKIDMVPIDLHSVFLGIHGGKLCLSCAKSGDDIKLQLEEVNITDLSKNKEEDKRFTFIRSEKGPTTSFESAACPGWFLCTTLEADRPVSLTNTPEEPLIVTKFYFQEDQ 1727 Anakinra MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE 1728 α - Natriuretic Peptide (ANP) SLRRSSCFGGRMDRIGAQSGLGCNSFRY 1729 β - Natriuretic Peptide, human (BNP human) SPKMVQGSGGFGRKMDRISSSSGLGCKVLRRH 1730 Brain Natriuretic Peptide, rat; (BNP rat) NSKMAHSSSCFGQKIDRIGAVSRLGCDGLRLF 1731 C - type Natriuretic Peptide (CNP, pig) GLSKGCFGLKLDRIGSMSGLGC 1732 Fibroblast Growth Factor 2 (FGF - 2) PALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVAL KRTGQYKLGS KTGPGQKAIL FLPMSAKS 1733 TNF receptor (TNFR) LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD 1734

[0194] "Anti-CD3" refers to monoclonal antibodies, species, sequence variants, and fragments thereof targeting the T-cell surface protein CD3, including OKT3 (also known as moromuzumab) and humanized anti-CD3 monoclonal antibodies (hOKT31 (Ala-Ala)) (Herold et al., 2002, New England Journal of Medicine 346:1692-1698). The anti-CD3-containing fusion protein of the present invention can be specifically used to slow the onset of new-onset type 1 diabetes, including the use of anti-CD3 as a therapeutic effector and targeting moiety of a second therapeutic BP in BPXTEN compositions. Sequence of the variable region and the generation of anti-CD3 have been described in U.S. Patent Nos. 5,885,573 and 6,491,916.

[0195] "IL-1ra" refers to human IL-1 receptor antagonist proteins and species and sequence variants that possess at least a portion of the biological activity of mature IL-1ra, including the sequence variant anakinetin (Kineret®). Anakinetin is a non-glycosylated recombinant human IL-1ra, and differs from endogenous human IL-1ra by the addition of an N-terminal methionine residue. The commercial version of anakinetin is marketed as Kinet®. It binds to the IL-1 receptor with the same affinity as native IL-1ra and IL-1β, but does not result in receptor activation (signal transduction); the effect is attributed to the presence of only one receptor-binding motif on IL-1ra, compared to two such motifs on IL-1α and IL-1β. Anakinetin has 153 amino acids and a size of 17.3 kD, and a reported half-life of approximately 4–6 hours.

[0196] Increased IL-1 production has been reported in patients suffering from various microbial infectious diseases and other conditions. The IL-1ra fusion protein of the present invention can be specifically used to treat any of the aforementioned diseases and conditions. IL-1ra has been cloned as described in U.S. Patent Nos. 5,075,222 and 6,858,409.

[0197] "Natriuretic peptide" refers to atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP or B-type natriuretic peptide), and C-type natriuretic peptide (CNP); both human and non-human species and sequence variants possessing at least a portion of the biological activity of their mature paired natriuretic peptides. Sequences of useful forms of natriuretic peptides are disclosed in U.S. Patent Publication 20010027181. Examples of ANPs include human ANP (Kangawa et al., 1984, BBRC 118:131) or ANPs from various species, including porcine and rat ANPs (Kangawa et al., 1984, BBRC 121:585). Sequence analysis revealed that the original BNP precursor consisted of 134 residues and was cleaved into a 108-amino acid precursor. A 32-amino acid cleavage from the C-terminus of the BNP precursor resulted in human BNP (77-108), which is the circulating physiologically active form. Human BNP, consisting of 32 amino acids, is involved in the formation of disulfide bonds (Sudoh et al., 1989, BBRC 159:1420) and is cited in US patents 5,114,923, 5,674,710, 5,674,710, and 5,948,761. BPXTENs containing one or more natriuretic functions can be used to treat hypertension, induce diuresis, induce natriuresis, diuresis or relaxation, vasodilation or relaxation, natriuretic peptide receptor (e.g., NPR-A) binding, inhibition of aldosterone secretion from the adrenal glands, cardiovascular diseases and conditions, reduction, cessation, or reversal of cardiac remodeling following cardiac events or due to congestive heart failure, kidney diseases and conditions; treatment or prevention of ischemic stroke, and treatment of asthma.

[0198] "Heparin-binding growth factor 2" or "FGF-2" refers to the human FGF-2 protein and species and sequence variants of it that have at least a portion of the biological activity of their mature counterparts. FGF-2 has been cloned, as described in Burgess, WH and Maciag, T., Ann. Rev. Biochem., 58:575-606 (1989); Coulier, F. et al., 1994, Prog. Growth Factor Res. 5:1; and PCT Publication WO 87 / 01728.

[0199] "TNF receptor" refers to the human receptor for TNF and its biological receptor species and sequence variants that possess at least a portion of the activity of the mature TNFR. The X-ray crystal structure of the complex formed by the extracellular domain of the human p55 TNF receptor and TNFβ has been determined (Banner et al., 1993 Cell 73:431, which is incorporated herein by reference).

[0200] clotting factors

[0201] In hemophilia, blood clotting is disrupted due to the lack of certain plasma clotting factors. Human factor IX (FIX) is the proenzyme of a serine protease, which is an important component of the endogenous pathway of the coagulation cascade. Factor VIIa (FVIIa) protein has been found to be useful for treating bleeding episodes in patients with hemophilia A or B who have inhibitors against FVIII or FIX and in patients with acquired hemophilia, as well as for preventing bleeding during surgical interventions or invasive procedures in patients with hemophilia A or B who have inhibitors against FVIII or FIX. Therefore, there remains a need for a combination of factor IX and factor VIIa with an extended half-life and retained activity, when administered as part of a prophylactic and / or therapeutic regimen for hemophilia B, and for formulations that reduce side effects and can be administered via both intravenous and subcutaneous routes.

[0202] Coagulation factors included in the BPXTEN of this invention may include proteins having biological, therapeutic, or preventative purposes or functions that can be used to prevent, treat, mediate, or improve blood clotting disorders, diseases, or defects. Suitable coagulation proteins include bioactive polypeptides involved in the coagulation cascade as substrates, enzymes, or cofactors.

[0203] Table 4e provides a non-limiting list of coagulation factor sequences covered by the BPXTEN fusion protein of the present invention. Coagulation factors included in the BPXTEN of the present invention may be proteins that show at least about 80% sequence identity with the protein sequences selected from Table 4e, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0204] Table 4e: Coagulation factor polypeptide sequences

[0205] BPXTEN name SEQ ID NO: Amino acid sequence FIX precursor <![CDATA[ 1735 ]]> <![CDATA[ MQRVNMIMAESPGLITICLLGY LLSAECTVFLDHENANKILNRP KR YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT <!-- 56 -->]]> FIX Homo sapiens 1736 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 4 from patent US 20080214462 1737 MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 6 from Patent US 20080214462 1738 MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIFNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 8 from Patent US 20080214462 1739 MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNFNAAINTYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDATCLRSTKFTIFNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIVSWGEGCAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 2 from Patent US 7125841 1740 MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEPREVFENTEKITEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNPTEAETILDNITQGTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRAIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWARVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 1 from Patent US 20080167219 1741 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 2 from Patent US 20080167219 1742 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 3 from Patent US 20080167219 1743 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 4 from Patent US 20080167219 1744 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 5 from Patent US 20080167219 1745 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Sequence 6 from Patent US 20080167219 1746 YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDATCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDAPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLASTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLT Factor VII / VIIa 1747 ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP

[0206] "Factor IX" ("FIX") includes human factor IX protein and species and sequence variants of it having at least a portion of the biological receptor activity of mature factor IX. In some embodiments, a FIX peptide is a structural analog or peptide mimic of any FIX peptide described herein, including the sequence in Table 4e. In one specific example of the invention, FIX is human FIX. In another embodiment, FIX is a polypeptide sequence from Table 4e. Mature factor IX is a single-chain protein of 415 amino acid residues containing approximately 17% carbohydrates by weight (Schmidt 2003, Trends Cardiovasc Med, 13: 39).

[0207] In some cases, a coagulation factor is factor IX, a sequence variant of factor IX, or a portion of factor IX, such as the exemplary sequence in Table 4e, as well as any protein or polypeptide substantially homologous to it, whose biological properties result in the activity of factor IX.

[0208] "Factor VII" (FVII) means human proteins and species and sequence variants of those having at least a portion of the biological activity of activating factor VII. Factor VII and recombinant human FVIIa have been introduced for uncontrolled bleeding in hemophiliac patients (with factor VIII or IX deficiency) who have developed inhibitors against alternative clotting factors. Recombinant human factor VIIa has utility in treating uncontrolled bleeding in hemophiliac patients (with factor VIII or IX deficiency), including those who have developed inhibitors against alternative clotting factors. In some embodiments, the FVII peptide is the activated form (FVIIa), a structural analog or peptide mimic of any FVII peptide described herein, including the sequences in Table 4e. Factor VII and VIIa have been cloned as described in U.S. Patent No. 6,806,063 and U.S. Patent Application No. 20080261886.

[0209] Growth hormone protein

[0210] "Growth hormone" or "GH" refers to human growth hormone protein and its species and sequence variants, including but not limited to the 191-amino acid single-stranded human sequence of GH. This invention contemplates including in BPXTEN any GH homologous sequence, such as naturally occurring sequence fragments from primates, mammals (including domesticated animals), and non-natural sequence variants, that retain at least a portion of the biological activity or function of GH and / or can be used to prevent, treat, modulate, or improve GH-related diseases, defects, symptoms, or conditions. Non-mammalian GH sequences are well described in the literature. For example, sequence alignments for fish GH can be found in Genetics and Molecular Biology 2003, 26, pp. 295-300. Additionally, naturally occurring sequences homologous to human GH can be found using standard homology search techniques such as NCBI BLAST.

[0211] In one embodiment, the GH incorporated into a human or animal composition may be a recombinant polypeptide having a sequence corresponding to a protein found in nature. In another embodiment, the GH may be a sequence variant, fragment, homology, or mimic of a natural sequence that retains at least a portion of the biological activity of natural GH. Table 4f provides a non-limiting list of GH sequences from a wide variety of mammalian species covered by the BPXTEN fusion protein of the present invention. Any of these GH sequences or homologous derivatives constructed by rearranging individual mutations between species or families may be used in the fusion protein of the present invention. GH that can be incorporated into the BPXTEN fusion protein may include proteins that show at least about 80% sequence identity with the proteins selected from Table 4f, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0212] Table 4f: Amino acid sequences of growth hormones from animal species

[0213] Species GH Amino acid sequence SEQ ID NO. Human FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF 1750 Porcine FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1751 Alpaca FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERTYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGQILRQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1752 Camel FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERTYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGQILRQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1753 Horse FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDMELLRFSLLLIQSWLGPVQLLSRVFTNSLVFGTSDRVYEKLRDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1754 Elephant FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRPGQVLKQTYDKFDTNMRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1755 Red Fox FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLVLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1756 Dog FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1757 Cat FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRGGQILKQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1758 American mink FPAMPLSSLFANAVLRAQHLHQLAADTYKDFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDMELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGPILKQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1759 Fin whale FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1760 Dolphin FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNTQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1761 Hippopotamus FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNTQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1762 Rabbit FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDMELLRFSLLLIQSWLGPVQFLSRAFTNTLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRVGQLLKQTYDKFDTNLRGDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCVF 1763 Rat FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKEEAQQRTDMELLRFSLLLIQSWLGPVQFLSRIFTNSLMFGTSDRVYEKLKDLEEGIQALMQELEDGSPRIGQILKQTYDKFDANMRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFAESSCAF 1764 Mouse FPAMPLSSLFSNAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKEEAQQRTDMELLRFSLLLIQSWLGPVQFLSRIFTNSLMFGTSDRVYEKLKDLEEGIQALMQELEDGSPRVGQILKQTYDKFDANMRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1765 Hamster FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQTAFCFSETIPAPTGKEEAQQRSDMELLRFSLLLIQSWLGPVQFLSRIFTNSLMFGTSDRVYEKLKDLEEGIQALMQELEDGSPRVGQILKQTYDKFDTNMRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1766 Mole FPAMPLSNLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKEEAQQRSDMELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVFEKLKDLEEGIQALMRELEDGSLRAGQLLKQTYDKFDTNMRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1767 Guinea pig FPAMPLSSLFGNAVLRAQHLHQLAADTYKEFERTYIPEGQRYSIHNTQTAFCFSETIPAPTDKEEAQQRSDVELLHFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGTPRAGQILKQTYDKFDTNLRSNDALLKNYGLLSCFRKDLHRTETYLRV MKCRRFVESSCAF 1768 Cattle AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRV MKCRRFGEASCAF 1769 Sheep / Goat AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRV MKCRRFGEASCAF 1770 Red deer FPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRV MKCRRFGEASCAF 1771 Giraffe AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFSNSLVFGTSDRVYEKLKDLEEGILALMRELEDGTPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRV MKCRRFGEASCAF 1772 Mouse deer - 1 FPAMSLSGLFANAVLRVQHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDGPPRAGQILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRV MKCRRFGEASCAF 1773 Slow loris FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDMELLRFSLLLIQSWLGPVQLLSRVFTNSLVLGTSDRVYEKLKDLEEGIQALMRELEDGSPRVGQILKQTYDKFDTNLRSDDALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1774 Marmoset FPTIPLSRLLDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPASKKETQQKSNLELLRMSLLLIQSWFEPVQFLRSVFANSLLYGVSDSDVYEYLKDLEEGIQTLMGRLEDGSPRTGEIFMQTYRKFDVNSQNNDALLKNYGLLYCFRKDMDKVETFLRI VQCR-SVEGSCGF 1775 Brushtail possum FPAMPLSSLFANAVLRAQHLHQLVADTYKEFERTYIPEAQRHSIQSTQTAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLSPVQFLSRVFTNSLVFGTSDRVYEKLRDLEEGIQALMQELEDGSSRGGLVLKTTYDKFDTNLRSDEALLKNYGLLSCFKKDLHKAETYLRV MKCRRFVESSCAF 1776 Monkey (Rhesus monkey) FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGTSYSDVYDLLKDLEEGIQTLMGRLEDGSSRTGQIFKQTYSKFDTNSHNNDALLKNYGLLYCFRKDMDKIETFLRI VQCR-SVEGSCGF 1777

[0214] Cytokines

[0215] BP can be a cytokine or one or more cytokines. Cytokines are proteins released by cells (such as chemokines, interferons, lymphokines, interleukins, and tumor necrosis factor) that can influence cellular behavior. Cytokines can be produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by more than one type of cell. Cytokines can be involved in producing systemic or local immune regulatory effects.

[0216] Some cytokines can act as pro-inflammatory cytokines. Pro-inflammatory cytokines are those involved in inducing or amplifying inflammatory responses. They can work with various cells of the immune system, such as neutrophils and leukocytes, to generate an immune response. Some cytokines can also act as anti-inflammatory cytokines. Anti-inflammatory cytokines are those involved in reducing inflammatory responses. In some cases, anti-inflammatory cytokines can modulate pro-inflammatory cytokine responses. Some cytokines can function as both pro-inflammatory and anti-inflammatory cytokines.

[0217] The cytokines encompassed by the compositions of this invention can be effective in treating a wide range of diseases, including but not limited to cancer, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Alzheimer's disease, schizophrenia, viral infections (e.g., chronic hepatitis C, AIDS), allergic asthma, retinal neurodegenerative processes, metabolic disorders, insulin resistance, and diabetic cardiomyopathy. Cytokines can be particularly useful in treating inflammatory and autoimmune conditions.

[0218] Examples of cytokines that can be regulated by the systems and compositions disclosed herein include, but are not limited to, lymphokines, monokines, and conventional polypeptide hormones other than human growth hormone. Cytokines include parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro-relaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental prolactin; tumor necrosis factor-α; Müllerian duct inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); and neurotrophic factors. Growth factors, such as NGF-α; platelet-derived growth factor; transforming growth factor (TGF), such as TGF-α, TGF-β, TGF-β1, TGF-β2, and TGF-β3; insulin-like growth factor-I and-II; erythropoietin (EPO); Flt-3L; stem cell factor (SCF); bone-inducing factor; interferon (IFN), such as IFN-α, IFN-β, and IFN-γ; colony-stimulating factor (CSF), such as macrophage-CSF. (M-CSF); Granulocyte-Macrophage-CSF (GM-CSF); Granulocyte-CSF (G-CSF); Macrophage-Stimulating Factor (MSP); Interleukins (ILs), such as IL-1, IL-1a, IL-1b, IL-1RA, IL-18, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12b, IL-13, etc. IL-14, IL-15, IL-16, IL-17, and IL-20; tumor necrosis factors, such as CD154, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, and TRANCE; and other polypeptide factors, including LIF, oncokinase M (OSM), and kit ligand (KL). Cytokine receptors are receptor proteins that bind cytokines. Cytokine receptors can be membrane-bound or soluble.

[0219] Target polynucleotides can encode cytokines. Non-restricted examples of cytokines include 4-1BBL, activin βA, activin βB, activin βC, activin βE, artemin (ARTN), BAFF / BLyS / TNFSF138, BMP10, BMP15, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, bone morphogenetic protein 1 (BMP1), CCL1 / TCA3, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, and CCL17 / TARC. CCL18, CCL19, CCL2 / MCP-1, CCL20, CCL21, CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1, CCL5, CCL6, CCL7, CCL8, CCL9, CD153 / CD30L / TNFSF8, CD40L / CD154 / TNFSF5, CD40LG, CD70, CD70 / CD27L / TNFSF7, CLCF1, c-MPL / CD110 / TPOR, CNTF, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2 , CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, EDA-A1, FAM19A1, FAM19A2, FAM19A3, FAM19A4, FAM19A5, Fa s-ligand / FASLG / CD95L / CD178, GDF10, GDF11, GDF15, GDF2, GDF3, GDF4, GDF5, GDF6, GDF7, GDF8, GDF9, glial cell line-derived neurotrophic factor (GDNF), growth differentiation factor 1 (GDF1), IFNA1, IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA5 / IFNaG, IFNA7, IFNA8, IF NB1, IFNE, IFNG, IFNZ, IFNω / IFNW1, IL11, IL18, IL18BP, IL1A, IL1B, IL1F10, IL1F3 / IL1RA, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL1RL2, IL31, IL33, IL6, IL8 / CXCL8, Inhibin-A, Inhibin-B, Leptin, LIF, LTA / TNFB / TNFSF1, LTB / TNFC, Neuronal rank protein (NRTN), OSM, OX-40L / TNFSF4 / CD252, persephin (PSPN), RANKL / OPGL / TNFSF11 (CD254), TL1A / TNFSF15, TNFA, TNF-α / TNFA, TNFSF10 / TRAIL / APO-2L (CD253), TNFSF12, TNFSF13, TNFSF14 / LIGHT / CD258, XCL1, and XCL2. In some embodiments, the target gene encodes an immune checkpoint inhibitor. Non-limiting examples of such immune checkpoint inhibitors include PD-1, CTLA-4, LAG3, TIM-3, A2AR, B7-H3, B7-H4, BTLA, IDO, KIR, and VISTA. In some embodiments, the target gene encodes the α, β, γ, and / or δ chains of the T cell receptor (TCR).

[0220] In some cases, cytokines can be chemokines. Chemokines can be selected from, but are not limited to, the following groups: ARMCX2, BCA-1 / CXCL13, CCL11, CCL12 / MCP-5, CCL13 / MCP-4, CCL15 / MIP-5 / MIP-1δ, CCL16 / HCC-4 / NCC4, CCL17 / TARC, CCL18 / PARC / MIP-4, CCL19 / MIP-3b, CCL2 / MCP-1, CCL20 / MIP-3α / MIP3A, CCL21 / 6Ckine, CCL22 / MDC, CCL23 / MIP3, CCL24 / Eotaxin-2 / MPIF-2, CCL25 / TECK, CCL26 / Eotaxin-3, CCL27 / CTACK, CCL28, CCL3 / Mip1a, CCL4 / MIP1B, CCL4L1 / LAG-1, CCL5 / RANTES, CCL6 / C10, CCL8 / MCP-2, CCL9, CML5, CXCL1, CXCL10 / Crg-2, CXCL12 / SDF-1 β, CXCL14 / BRAK, CXCL15 / Lungkine, CXCL16 / SR-PSOX, CXCL17, CXCL2 / MIP-2, CXCL3 / GROγ, CXCL4 / PF4, CXCL5, CXCL6 / GCP-2, CXCL9 / MIG, FAM19A1, FAM19A2, FAM19A3, FAM19A4 / TAFA4, FAM19A5, Fractalkine / CX3CL1, I-309 / CCL1 / TCA-3, IL-8 / CXCL8, MCP-3 / CCL7, NAP-2 / PPBP / CXCL7, XCL2 and Armo IL10.

[0221] Table 4g ​​provides a non-limiting list of such sequences of BP covered by the BPXTEN fusion protein of the present invention. The metabolic protein of the BPXTEN composition of the present invention can be a protein that shows at least about 80% sequence identity with the protein sequences selected from Table 4g, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0222] Table 4g: Cytokines used for conjugation

[0223] Protein Name (Synonyms) Amino Acid Sequence SEQ ID NO. Anti-CD3 See U.S. Patent Nos. 5,885,573 and 6,491,916 IL-1ra, human full-length MEICRGLRSHLITLLLFLFHSETICRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE 270 IL-1ra, canine METCRCPLSYLISFLLFLPHSETACRLGKRPCRMQAFRIWDVNQKTFYLRNNQLVAGYLQGSNTKLEEKLDVVPVEPHAVFLGIHGGKLCLACVKSGDETRLQLEAVNITDLSKNKDQDKRFTFILSDSGPTTSFESAACPGWFLCTALEADRPVSLTNRPEEAMMVTKFYFQKE 271 IL-1ra, rabbit MRPSRSTRRHLISLLLFLFHSETACRPSGKRPCRMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNAKLEERIDVVPLEPQLLFLGIQRGKLCLSCVKSGDKMKLHLEAVNITDLGKNKEQDKRFTFIRSNSGPTTTFESASCPGWFLCTALEADQPVSLTNTPDDSIVVTKFYFQED 272 IL-1ra, rat MEICRGPYSHLISLLLILLFRSESAGHIPAGKRPCKMQAFRIWDTNQKTFYLRNNQLIAGYLQGPNTKLEEKIDMVPIDFRNVFLGIHGGKLCLSCVKSGDDTKLQLEEVNITDLNKNKEEDKRFTFIRSETGPTTSFESLACPGWFLCTTLEADHPVSLTNTPKEPCTVTKFYFQED 273 IL-1ra, mouse MEICWGPYSHLISLLLILLFHSEAACRPSGKRPCKMQAFRIWDTNQKTFYLRNNQLIAGYLQGPNIKLEEKIDMVPIDLHSVFLGIHGGKLCLSCAKSGDDIKLQLEEVNITDLSKNKEEDKRFTFIRSEKGPTTSFESAACPGWFLCTTLEADRPVSLTNTPEEPLIVTKFYFQEDQ 274 Anakinra MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE 275 IL-10 MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 276

[0224] "IL-1ra" refers to the human IL-1 receptor antagonist protein and species and sequence variants of the same protein possessing at least a portion of the biological activity of mature IL-1ra, including the sequence variant anakinet®. Human IL-1ra is a mature glycoprotein of 152 amino acid residues. The IL-1ra-containing fusion protein of the present invention can be specifically used to treat any of the aforementioned diseases and conditions. IL-1ra has been cloned as described in U.S. Patent Nos. 5,075,222 and 6,858,409.

[0225] In some cases, BP can be IL-10. IL-10 can be a potent anti-inflammatory cytokine that inhibits the production of pro-inflammatory cytokines and chemokines. IL-10 can be used to treat autoimmune diseases and inflammatory conditions such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Alzheimer's disease, schizophrenia, allergic asthma, retinal neurodegenerative processes, and diabetes.

[0226] In some cases, IL-10 can be modified to improve stability and reduce prolytic degradation. Modification can involve the substitution of one or more amide bonds. In some cases, one or more amide bonds within the IL-10 backbone can be substituted to achieve the aforementioned effects. One or more amide bonds (‒CONH‒) in IL-10 can be replaced with isosteric bonds that are amide bonds, such as ‒CH2NH‒, ‒CH2S‒, ‒CH2CH2‒, ‒CH=CH‒ (cis and trans), ‒COCH2‒, ‒CH(OH)CH2‒, or ‒CH2SO‒. Furthermore, the amide bonds in IL-10 can also be replaced with reduced isosteric pseudopeptide bonds. See Couder et al. (1993) Int. J. Peptide Protein Res. 41:181-184, which is incorporated herein by reference in its entirety.

[0227] One or more acidic amino acids, including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl and heteroarylsulfonamides of 2,4-diaminopropionic acid, ornithine or lysine and tetrazolium-substituted alkyl amino acids; and side-chain amide residues, such as asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine; and hydroxyl-containing amino acids, including serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic substituted derivatives of serine or threonine, which may be substituted.

[0228] One or more hydrophobic amino acids in IL-10, such as alanine, leucine, isoleucine, valine, ortholeucine, (S)-2-aminobutyric acid, (S)-cyclohexylalanine, or other simple α-amino acids, may be substituted with amino acids, including but not limited to aliphatic side chains from C1-C10 carbons, including branched, cyclic and linear alkyl, alkenyl, or alkynyl substitutions.

[0229] In some cases, one or more hydrophobic amino acids in IL-10 may be replaced, for example, by aromatic-substituted hydrophobic amino acids, including phenylalanine, tryptophan, tyrosine, sulfotyrosine, biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 2-benzothiophene alanine, 3-benzothiophene alanine, histidine, and amino, alkylamino, dialkylamino, aza, halogenated (fluorine, chlorine, bromine, or iodine), or alkoxy (C1-C4) substituted forms of the aromatic amino acids listed above. Illustrative examples include: 2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2'-, 3'- or 4'-amino-, 2'-, 3'- or 4'-chloro-, 2-, 3- or 4-biphenylalanine, 2'-, 3'- or 4'-methyl-, 2-, 3- or 4-biphenylalanine and 2- or 3-pyridylalanine;

[0230] One or more hydrophobic amino acids in IL-10, such as phenylalanine, tryptophan, tyrosine, sulfotyrosine, biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 2-benzothiophene alanine, 3-benzothiophene alanine, and histidine, including amino, alkylamino, dialkylamino, aza, halogenated (fluorine, chlorine, bromine, or iodine) or alkoxy groups, may be substituted with aromatic amino acids, including 2-, 3-, or 4-aminophenyl. Alanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2'-, 3'- or 4'-amino-, 2'-, 3'- or 4'-chloro-, 2-, 3- or 4-biphenylalanine, 2'-, 3'- or 4'-methyl-, 2-, 3- or 4-biphenylalanine and 2- or 3-pyridylalanine.

[0231] Amino acids containing basic side chains, including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, and homoarginine, may be substituted. Alkyl, alkenyl, or aryl substituted derivatives of the aforementioned amino acids may also be substituted. Examples include N-ε-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine, N,N-γ,γ'-diethyl-homoarginine, α-methyl-arginine, α-methyl-2,3-diaminopropionic acid, α-methyl-histidine, and α-methyl-ornithine, wherein the alkyl group occupies the pre-R position of the α-carbon. Modified IL-10 may comprise an amide formed from any combination of the following: alkyl, aromatic, heteroaromatic, ornithine or 2,3-diaminopropionic acid, carboxylic acid, or any of many well-known activated derivatives, such as acyl chlorides, active esters, active azolides and related derivatives, lysine, and ornithine.

[0232] In some cases, IL-10 may contain one or more naturally occurring L-amino acids, synthetic L-amino acids, and / or D-enantiomers of amino acids. IL-10 polypeptides may contain one or more of the following amino acids: ω-aminodecanoic acid, ω-aminotetradecanoic acid, cyclohexylalanine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, δ-aminovaleric acid, tert-butylalanine, tert-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, leucine, naphthylalanine, ornithine, citrulline, 4-chlorophenylalanine, 2-fluorophenylalanine, pyridylalanine, 3-benzothioalanine, hydroxyproline, β-alanine, anthranilic acid, m-aminobenzoic acid, p-aminobenzoic acid, etc. The compounds include benzoic acid, m-aminomethylbenzoic acid, 2,3-diaminopropionic acid, α-aminoisobutyric acid, N-methylglycine (sarcosine), 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-2-thiophene alanine, methionine sulfoxide, arginine, N-acetyllysine, 2,4-diaminobutyric acid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine, ε-aminohexanoic acid, ω-aminohexanoic acid, ω-aminoheptanoic acid, ω-aminooctanoic acid, and 2,3-diaminobutyric acid.

[0233] IL-10 may contain cysteine ​​residues or cysteine ​​groups, which may act as a linker to another peptide via disulfide bonding or provide for cyclization of the IL-10 polypeptide. Methods of introducing cysteine ​​or cysteine ​​analogues are known in the art; see, for example, U.S. Patent No. 8,067,532. The IL-10 polypeptide may be cyclized. Other cyclization methods include introducing an oxime linker or a lanthanine linker; see, for example, U.S. Patent No. 8,044,175. Any combination of amino acids (or non-amino acid moieties) that can form cyclization bonds may be used and / or introduced. Cyclation bonds may be formed by any combination of amino acids having functional groups (or amino acids and ‒(CH2)). n CO‒ or (CH₂) n The functional group (C6H4‒CO‒) is generated, allowing for the introduction of bridging. Some examples are disulfides, and disulfide analogs such as ‒(CH2). n -Carba bridges, thioacetals, thioether bridges (cystathionine or lanathionine), and bridges containing esters and ethers.

[0234] IL-10 can be substituted with N-alkyl, aryl, or main chain crosslinks to construct lactams and other cyclic structures, C-terminal hydroxymethyl derivatives, O-modified derivatives, and N-terminal modified derivatives, including substituted amides such as alkylamides and hydrazides. In some cases, IL-10 peptides are inverse analogs.

[0235] IL-10 can be a natural protein, a peptide fragment of IL-10 having at least a portion of the biological activity of natural IL-10, or a modified peptide. IL-10 can be modified to improve intracellular uptake. One such modification can be the attachment of a protein transduction domain. The protein transduction domain can be attached to the C-terminus of IL-10. Alternatively, the protein transduction domain can be attached to the N-terminus of IL-10. The protein transduction domain can be attached to IL-10 via a covalent bond. The protein transduction domain can be selected from any sequence listed in Table 4h.

[0236] Table 4h. Exemplary protein transduction domains

[0237] SEQ ID NO Amino acid sequence 277 YGRKKRRQRRR; 278 RRQRRTSKLMKR 279 GWTLNSAGYLLGKINLKALAALAKKIL 280 KALAWEAKLAKALAKALAKHLAKALAKALKCEA 281 RQIKIWFQNRRMKWKK 282 YGRKKRRQRRR 283 RKKRRQRRR 284 YGRKKRRQRRR 285 RKKRRQRR 286 YARAAARQARA 287 THRLPRRRRRR 288 GGRRARRRRRR

[0238] The biosynthetic protein (BP) of human or animal compositions is not limited to natural full-length polypeptides, but also includes recombinant forms and biologically and / or pharmacologically active variants or fragments thereof. For example, those skilled in the art will understand that various amino acid substitutions can be prepared in the BP to produce variants without departing from the spirit of the invention regarding the biological activity or pharmacological properties of the BP. Examples of conserved substitutions of amino acids in the polypeptide sequence are shown in Table 5. However, in examples of BPXTENs in which the sequence identity of the BP is less than 100% compared to the specific sequence disclosed herein, the invention considers substitution for any of the other 19 natural L-amino acids for a given amino acid residue of a given BP, said amino acid residues may be located anywhere within the BP sequence, including adjacent amino acid residues. If any particular substitution results in an undesirable change in biological activity, the alternative amino acid can be employed, and the construct can be evaluated by the methods described herein, or by any techniques and guidelines regarding conserved and non-conserved mutations set forth, for example, in U.S. Patent No. 5,364,934, the contents of which are incorporated herein by reference in their entirety, or by methods generally known to those skilled in the art. Additionally, variants may include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the full-length natural amino acid sequence of the BP, which retain at least a portion of the biological activity of the natural peptide.

[0239] Table 5: Exemplary Conserved Amino Acid Substitutions

[0240] Original residue Exemplary substitution Ala (A) val; leu; ile Arg (R) lys; gin; asn Asn (N) gin; his; Iys; arg Asp (D) glu Cys (C) ser Gln (Q) asn Glu (E) asp Gly (G) pro His (H) asn: gin: Iys: arg xIle (I) leu; val; met; ala; phe: norleucine Leu (L) norleucine: ile: val; met; ala: phe Lys (K) arg: gin: asn Met (M) leu; phe; ile Phe (F) leu: val: ile; ala Pro (P) gly Ser (S) thr Thr (T) ser Trp (W) tyr Tyr (Y) trp: phe: thr: ser Val (V) ile; leu; met; phe; ala; norleucine

[0241] In some embodiments, the BP incorporated into the BPXTEN polypeptide may have a sequence that shows at least about 80% sequence identity with the sequences from Tables 4a-4h, or alternatively at least about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or 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 100% sequence identity with the sequences from Tables 4a-4h. In some embodiments, the BP incorporated into BPXTEN may be a bispecific sequence comprising a first binding domain and a second binding domain, wherein the first binding domain has specific binding affinity for tumor-specific markers or antigens of target cells, and shows at least about 80% sequence identity with the paired VL and VH sequences of anti-CD3 antibodies selected from Table 6f, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, or 100% sequence identity; and wherein the second binding domain, which has specific binding affinity for effector cells, shows at least about 80% sequence identity with the paired VL and VH sequences of the anti-target cell antibodies selected from Table 6a, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. The activity of the BPs of the foregoing embodiments can be evaluated using the assays or parameters as described herein, and those sequences that retain at least about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or more of activity compared to the corresponding native BP sequences are considered suitable for inclusion in human or animal BPXTENs. BPs found to retain suitable levels of activity can be linked to one or more XTEN peptides described above or anywhere else herein. In one embodiment, it was found that a BP retaining an appropriate level of activity could be linked to one or more XTEN peptides having at least about 80% sequence identity with sequences from Tables 3a-3b (e.g., at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity), resulting in a chimeric fusion protein.

[0242] T-cell binding agent

[0243] Other structural configurations of BPXTEN relate to XTEN-mediated protease-activated T-cell conjugates (“XPATs” or “XPATs”), wherein BP is a bispecific antibody (e.g., a bispecific T-cell conjugate). In some embodiments, the XPAT composition comprises a first portion including a first binding domain and a second binding domain, a second portion including a release segment, and a third portion including an XTEN-filling portion. In some embodiments, the XPAT composition has a configuration of formula Ia (described as N-terminus to C-terminus):

[0244] (Part 1) - (Part 2) - (Part 3) (Ia)

[0245] The first part comprises a bispecific scFv, wherein a first binding domain has a specific binding affinity for tumor-specific markers or antigens of target cells, and a second binding domain has a specific binding affinity for effector cells; the second part comprises a release segment (RS) capable of being cleaved by a mammalian protease (which may be tumor-specific or antigen-specific, and thus activated) as described more fully below; and the third part is a filler portion. In the foregoing embodiments, the binding domains of the first part may be in the following order: (VL-VH)1-(VL-VH)2, where “1” and “2” represent the first binding domain and the second binding domain, respectively, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, wherein the paired binding domains are linked by a polypeptide linker (as described more fully below). In one embodiment, substitutes for the first portions VL and VH are identified in Tables 6a-6f; substitutes for RS are identified in the sequences shown in Tables 8a-8b (as described more fully below); and substitutes for the filler portion are identified herein by: XTEN; albumin-binding domain; albumin; IgG-binding domain; a polypeptide consisting of proline, serine, and alanine; fatty acid; Fc domain; polyethylene glycol (PEG), PLGA; and hydroxyethyl starch. When desired, the filler portion is XTEN, having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences identified by the sequences shown in Tables 3a-3b. In the foregoing embodiments, the composition is a recombinant fusion protein. In another embodiment, portions are linked by chemical conjugation.

[0246] In another embodiment, the XPAT composition has the configuration of formula IIa (described as N-terminus to C-terminus):

[0247] (Part Three) - (Part Two) - (Part One) (IIa)

[0248] The first part is a bispecific binding domain comprising two scFvs, wherein the first binding domain has a specific binding affinity for tumor-specific markers or antigens of target cells, and the second binding domain has a specific binding affinity for effector cells; the second part comprises a release segment (RS) cleavable by mammalian proteases; and the third part is a filler portion. In the foregoing embodiments, the first binding domains may be in the order (VL-VH)1-(VL-VH)2, where “1” and “2” represent the first binding domain and the second binding domain, respectively, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, wherein the paired binding domains are linked by a peptide linker described herein. In one embodiment, substitutes for the first portions VL and VH are identified in Tables 6a-6f; substitutes for RS are identified in the sequences shown in Tables 8a-8b; and substitutes for the filler portion are identified herein by: XTEN; albumin-binding domain; albumin; IgG-binding domain; a polypeptide consisting of proline, serine, and alanine; a fatty acid; and an Fc domain. If desired, the filler portion is XTEN, having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the sequences shown in Tables 3a-3b. In the foregoing embodiments, the composition is a recombinant fusion protein. In another embodiment, portions are linked by chemical conjugation.

[0249] In another embodiment, the XPAT composition has a configuration of formula IIIa (described as N-terminus to C-terminus):

[0250] (Part 5) - (Part 4) - (Part 1) - (Part 2) - (Part 3) (IIIa)

[0251] The first part is a bispecific binding domain containing two scFvs, wherein the first binding domain has a specific binding affinity for tumor-specific markers or antigens of target cells, and the second binding domain has a specific binding affinity for effector cells; the second part contains a release segment (RS) cleavable by mammalian proteases; the third part is a filler portion; the fourth part contains a release segment (RS) cleavable by mammalian proteases, which may be the same as or different from the second part; and the fifth part is a filler portion, which may be the same as or different from the third part. In the foregoing embodiments, the binding domains of the first part may be in the order (VL-VH)1-(VL-VH)2, where "1" and "2" represent the first binding domain and the second binding domain, respectively, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, wherein the paired binding domains are linked by a peptide linker described herein. In the foregoing embodiments, the substitutes for RS were identified in the sequences set forth in Tables 8a-8b. In the foregoing embodiments, the substitutes for the filler portion were identified herein by: XTEN; albumin-binding domain; albumin; IgG-binding domain; a polypeptide consisting of proline, serine, and alanine; a fatty acid; and an Fc domain. If desired, the filler portion is XTEN, having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from those shown in Tables 3a-3b. In the foregoing embodiments, the composition is a recombinant fusion protein. In another embodiment, the portion is linked by chemical conjugation.

[0252] Based on their design and specific components, human or animal compositions advantageously provide bispecific therapeutic agents that, once cleaved by proteases associated with target tissue or disease-induced unhealthy tissue, exhibit higher selectivity, longer half-life, and result in less toxicity and fewer side effects. These human or animal compositions have an improved therapeutic index compared to bispecific antibody compositions known in the art. Such compositions can be used to treat certain diseases, including, but not limited to, cancers as described herein. Without being limited to any mechanistic theory, those skilled in the art will understand that the compositions of the invention achieve this reduction through a combination of mechanisms involving nonspecific interactions, including by positioning the binding domain to the steric hindrance of a large XTEN molecule, wherein the flexible, unstructured nature of the XTEN peptide, by tethering to the composition, enables it to oscillate and move around the binding domain, providing a blockage between the composition and tissue or cell, and by providing a reduction in the ability of the intact composition to penetrate cells or tissues due to its large molecular mass (contributed by both the actual molecular weight of the XTEN peptide and the large hydrodynamic radius of the unstructured XTEN peptide) compared to the size of the individual binding domains. However, in compositions designed in this way, when approaching a target tissue or cell carrying or secreting a protease capable of cleaving RS, or when the binding domain is internalized into the target cell or tissue after binding to a ligand, the bispecific binding domain is released from the bulk of the XTEN by the action of the protease, removing steric hindrance and allowing for greater freedom to exert its pharmacological effects. Human or animal compositions can be used to treat various conditions requiring selective delivery of the therapeutic bispecific antibody composition to cells, tissues, or organs. In one embodiment, the target tissue is cancer, which may be leukemia, lymphoma, or a tumor of an organ or system.

[0253] Combined structural domain

[0254] This disclosure considers the use of single-chain binding domains, such as, but not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single-domain antibodies, single-domain camelid antibodies, single-chain antibody molecules (scFv), and biantibodies capable of binding to ligands or receptors associated with antigens of effector cells and diseased tissues or cells, said diseased tissues or cells being cancerous, tumors, or other malignant tissues. In some embodiments, a bispecific antibody comprises a first binding domain having binding specificity to a target cell marker and a second binding domain having binding specificity to an effector cell antigen. In some embodiments, the first and second binding domains may be non-antibody scaffolds, such as anticalin, adnectin, fynomer, affilin, affinity molecules, centyrins, DARPin. In other embodiments, the binding domain for tumor cell targets is a variable domain of a T-cell receptor modified to bind to the MHC of a peptide fragment loaded with a protein overexpressed by tumor cells. In some embodiments, XPAT compositions are designed with the following considerations in mind: the localization of the target cathepsin and the presence of the same protease in healthy tissue where it is not intended to be targeted, and the presence of the target ligand in healthy tissue, but a greater presence in unhealthy target tissue, in order to provide a wide therapeutic window. A “therapeutic window” refers to the maximum difference between the minimum effective dose and the maximum tolerated dose of a given therapeutic composition. To help achieve a wide therapeutic window, the binding domain of the first portion of the composition is shielded by the proximity of the filling portion (e.g., an XTEN peptide), wherein the binding affinity of the intact composition for one or both ligands is reduced compared to the composition cleaved by a mammalian protease, thereby releasing the first portion from the shielding effect of the filling portion.

[0255] Regarding the single-chain binding domain, as well established in the art, an Fv is the smallest antibody fragment containing a complete antigen recognition and binding site, consisting of a dimer of a heavy chain variable domain (VH) and a light chain variable domain (VL) bound non-covalently. Within each VH and VL chain are three complementarity-determining regions (CDRs) that interact to define the antigen-binding site on the surface of the VH-VL dimer; the six CDRs of the binding domain confer antigen-binding specificity to the antibody or the single-chain binding domain. In some cases, scFvs are generated where each binding domain has 3, 4, or 5 CDRs. The scaffold sequence flanking the CDRs has a tertiary structure that is substantially conserved across species in native immunoglobulins, and scaffold residues (FRs) act to maintain the CDRs in their proper orientation. The constant domain is not required for binding function but helps stabilize the VH-VL interaction. In some embodiments, the domain of the polypeptide binding site can be a pair of VH-VL, VH-VH, or VL-VL domains from the same or different immunoglobulins; however, it is generally preferred to prepare single-chain binding domains using separate VH and VL chains from the parent antibody. The order of the VH and VL domains within the polypeptide chain is not limiting for the invention; the given order of domains can generally be reversed without loss of function, but it should be understood that the VH and VL domains are arranged in such a way that the antigen binding site can fold correctly. Therefore, the single-chain binding domains of bispecific scFv embodiments of human or animal compositions can be in the following order: (VL-VH) 1 -(VL-VH) 2 Where “1” and “2” represent the first and second binding structural domains, or (VL-VH), respectively. 1 -(VH-VL) 2 , or (VH-VL) 1 -(VL-VH) 2 , or (VH-VL) 1 -(VH-VL) 2 The paired binding domains are linked by peptide linkers as described below.

[0256] Therefore, the arrangement of the binding domains in the exemplary bispecific single-chain antibodies disclosed herein can be an arrangement in which the first binding domain is located at the C-terminus of the second binding domain. The arrangement of the V chains can be VH (target cell surface antigen)-VL (target cell surface antigen)-VL (effective cell antigen)-VH (effective cell antigen), VH (target cell surface antigen)-VL (target cell surface antigen)-VH (effective cell antigen)-VL (effective cell antigen), VL (target cell surface antigen)-VH (target cell surface antigen)-VL (effective cell antigen)-VH (effective cell antigen) or VL (target cell surface antigen)-VH (target cell surface antigen)-VL (effective cell antigen). For the arrangement where the second binding domain is located at the N-terminus of the first binding domain, the following orders are possible: VH (effective cell antigen) - VL (effective cell antigen) - VL (target cell surface antigen) - VH (target cell surface antigen), VH (effective cell antigen) - VL (effective cell antigen) - VH (target cell surface antigen) - VL (target cell surface antigen), VL (effective cell antigen) - VH (effective cell antigen) - VL (target cell surface antigen) - VH (target cell surface antigen), or VL (effective cell antigen) - VH (effective cell antigen) - VH (target cell surface antigen) - VL (target cell surface antigen). As used herein, "its N-terminus" or "its C-terminus" and their grammatical variations indicate relative positioning within the primary amino acid sequence, rather than placement at the absolute N-terminus or C-terminus of the bispecific single-chain antibody. Therefore, as a non-limiting example, "the first binding domain located at the C-terminus of the second binding domain" means that the first binding is located on the carboxyl side of the second binding domain within the bispecific single-chain antibody, and does not preclude the possibility that other sequences, such as His-tags or other compounds, such as radioisotopes, are located at the C-terminus of the bispecific single-chain antibody.

[0257] In one embodiment, the chimeric peptide assembly composition includes a first portion comprising a first binding domain and a second binding domain, wherein each binding domain is an scFv, and each scFv comprises a VL and a VH. In another embodiment, the chimeric peptide assembly composition includes a first portion comprising a first binding domain and a second binding domain, wherein the binding domain is a biantibody configuration, and each domain comprises a VL domain and a VH. In the foregoing embodiments, the first domain has binding specificity for tumor-specific markers or antigens of target cells, and the second binding domain has binding specificity for effector cell antigens. In one of the foregoing embodiments, the effector cell antigen is expressed on or within effector cells. In one embodiment, the effector cell antigen is expressed on T cells such as CD4+, CD8+, or natural killer (NK) cells. In another embodiment, the effector cell antigen is expressed on B cells, mast cells, dendritic cells, or myeloid cells. In one embodiment, the effector cell antigen is CD3, the cluster 3 antigen of cytotoxic T cells. In some of the foregoing embodiments, the first binding domain exhibits binding specificity for tumor-specific markers associated with tumor cells. In one embodiment, the binding domain has binding affinity for tumor-specific markers, wherein the tumor cells may include, but are not limited to, cells derived from: stromal cell tumors, fibroblast tumors, myofibroblast tumors, glial cell tumors, epithelial cell tumors, adipocyte tumors, immune cell tumors, vascular cell tumors, and smooth muscle cell tumors.In one embodiment, the tumor-specific marker or target cell antigen may be α4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, HER2, HER2neu, HER3, HER4, HER1 (EGFR), PD-L1, PSMA, CEA, TROP-2, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, βhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3; 9-O-acetyl-GD3, GM2, Globo H, fucose GM1, GD2, carbonic anhydrase IX, CD44v6, Nectin-4, or Sonic acid. Hedgehog (Shh), Wue-1, Plasma Cell Antigen 1, Melanoma Chondroitin Sulfate Proteoglycan (MCSP), CCR8, Prostate 6-Transmembrane Epithelial Antigen (STEAP), Mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6, Desmosome Core Protein 4, Fetal Acetylcholine Receptor (fnAChR), CD25, Cancer Antigen 19-9 (CA19-9), Cancer Antigen 125 (CA-125), Müllerian Inhibitory Substance Receptor Type II (MISIIR), Sialized Tn Antigen (s The antibodies include TN, fibroblast activation antigen (FAP), endothelial sialic acid protein (CD248), epidermal growth factor receptor variant III (EGFRvIII), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMP, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, and EphA2. In one embodiment, the first binding domain exhibiting binding affinity for CD70 is its native ligand CD27, rather than the antibody fragment. In another embodiment, the first binding domain exhibiting binding affinity for B7-H6 is its native ligand Nkp30, rather than the antibody fragment.

[0258] The scFv embodiment of the XPAT composition of the present invention comprises a first binding domain and a second binding domain, wherein the VL and VH domains are derived from monoclonal antibodies that are specifically capable of binding to tumor-specific markers or target cell antigens and effector cell antigens, respectively. In other cases, the first binding domain and the second binding domain each comprise six CDRs derived from a monoclonal antibody that is specifically capable of binding to target cell markers such as tumor-specific markers and effector cell antigens. In other embodiments, the first binding domain and the second binding domain of the first portion of the human or animal composition may have 3, 4, or 5 CHRs within each binding domain. In other embodiments, embodiments of the present invention comprise a first binding domain and a second binding domain, wherein each comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-H3 region, wherein each of these regions is derived from a monoclonal antibody capable of binding to tumor-specific markers or target cell antigens and effector cell antigens. In one embodiment, the present invention provides a chimeric peptide assembly composition wherein the second binding domain comprises VH and VL regions derived from a monoclonal antibody capable of binding human CD3. In another embodiment, the present invention provides a chimeric peptide assembly composition wherein the scFv second binding domain comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the paired VL and VH sequences of the anti-CD3 antibodies shown in Table 6a. In another aspect, embodiments of the second domain of the present invention comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-H3 regions, wherein each region is derived from a monoclonal antibody as shown in Table 6a. In the foregoing embodiments, the VH and / or VL domains may be configured as scFv, a biantibody, a single-domain antibody, or a single-domain camelid antibody.

[0259] In other embodiments, the second domain of the human or animal composition is derived from an anti-CD3 antibody as shown in Table 6a. In one of the foregoing embodiments, the second domain of the human or animal composition comprises paired VL and VH region sequences of an anti-CD3 antibody as shown in Table 6a. In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the second binding domain comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the paired VL and VH sequences of the huUCHT1 anti-CD3 antibody in Table 6a. In the foregoing embodiments, the VH and / or VL domains may be configured as scFv, part of a biantibody, a single-domain antibody, or a single-domain camelid antibody.

[0260] In other embodiments, the scFv of the first domain of the composition is derived from an antitumor cell antibody as shown in Table 6f. In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the first binding domain comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the paired VL and VH sequences of the antitumor cell antibodies shown in Table 6f. In one of the foregoing embodiments, the first domain of the composition comprises the paired VL and VH region sequences of the antitumor cell antibodies disclosed herein. In the foregoing embodiments, the VH and / or VL domains may be configured as scFv, a portion of a biantibody, a single-domain antibody, or a single-domain camelid antibody.

[0261] In another embodiment, the chimeric polypeptide assembly composition includes a first portion comprising a first binding domain and a second binding domain, wherein the binding domain is a biantibody configuration, and each binding domain comprises a VL domain and a VH domain. In one embodiment, the biantibody embodiment of the present invention comprises a first binding domain and a second binding domain, wherein the VL and VH domains are derived from monoclonal antibodies having binding specificity to tumor-specific markers or target cell antigens and effector cell antigens, respectively. In another embodiment, the biantibody embodiment of the present invention comprises a first binding domain and a second binding domain, wherein each comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-H3 region, wherein each region is derived from a monoclonal antibody capable of binding to tumor-specific markers or target cell antigens and effector cell antigens. It is envisioned that the biantibody embodiment of the present invention comprises a first binding domain and a second binding domain, wherein the VL and VH domains are derived from monoclonal antibodies having binding specificity to tumor-specific markers or target cell antigens and effector cell antigens, respectively. In another aspect, the biantibody embodiments of the present invention comprise a first binding domain and a second binding domain, each comprising a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-H3 region, wherein each of these regions is derived from a monoclonal antibody capable of binding a tumor-specific marker or target cell antigen and effector cell antigen. In one embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the second binding domain of the biantibody comprises paired VH and VL regions derived from a monoclonal antibody capable of binding human CD3. In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the second binding domain of the biantibody comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the paired VL and VH sequences of anti-CD3 antibodies as shown in Table 6a. In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the second binding domain of the biantibody comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the VL and VH sequences of the huUCHT1 antibody as shown in Table 6a. In other embodiments, the second binding domain of the biantibody in the composition is derived from the anti-CD3 antibody described herein.In another embodiment, the present invention provides a chimeric polypeptide assembly composition wherein the first binding domain of the biantibody comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the VL and VH sequences of the antitumor cell antibodies shown in Table 6f. In other embodiments, the first binding domain of the biantibody in the composition is derived from the antitumor cell antibodies described herein.

[0262] Therapeutic monoclonal antibodies from which VL and VH and CDR domains may be derived for use in human or animal compositions are known in the art. Sequences of the aforementioned antibodies can be obtained from publicly available databases, patents, or references. Furthermore, non-limiting examples of monoclonal antibodies and VH and VL sequences derived from anti-CD3 antibodies are described in Table 6a, and non-limiting examples of monoclonal antibodies targeting cancer, tumors, or target cell markers and VH and VL sequences are described in Table 6f.

[0263] Anti-CD3 binding domain

[0264] In some embodiments, the present invention provides a chimeric polypeptide assembly comprising a binding domain of a first portion having binding affinity for T cells. In one embodiment, the binding domain of a second portion comprises VL and VH sequences derived from a monoclonal antibody against a CD3 antigen. In another embodiment, the binding domain comprises VL and VH sequences derived from monoclonal antibodies against CD3ε and CD3δ. Monoclonal antibodies against CD3neu are known in the art. Exemplary, non-limiting examples of VL and VH sequences of monoclonal antibodies against CD3 are illustrated in Table 6a. In one embodiment, the present invention provides a chimeric polypeptide assembly comprising a binding domain having binding affinity for CD3, said binding domain comprising the anti-CD3 VL and VH sequences shown in Table 6a. In another embodiment, the present invention provides a chimeric polypeptide assembly comprising a binding domain of a first portion having binding affinity for CD3ε, said binding domain comprising the anti-CD3ε VL and VH sequences shown in Table 6a. In another embodiment, the present invention provides a chimeric peptide assembly composition wherein the second binding domain of the first portion of the scFv comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the paired VL and VH sequences of the huUCHT1 anti-CD3 antibody in Table 6a. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a binding domain having binding affinity for CD3, said binding domain comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, wherein each is derived from the respective anti-CD3 VL and VH sequences shown in Table 6a. In another embodiment, the present invention provides a chimeric peptide assembly composition comprising a binding domain having binding affinity for CD3, the binding domain comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, wherein the CDR sequence is RASQDIRNYLN (SEQ ID NO: 8034), YTSRLES (SEQ ID NO: 8035), QQGNTLPWT (SEQ ID NO: 8036), GYSFTGYTMN (SEQ ID NO: 8037), LINPYKGVST (SEQ ID NO: 8038), and SGYYGDSDWYFDV (SEQ ID NO: 8039).

[0265] The CD3 complex is a group of cell surface molecules that bind to the T-cell antigen receptor (TCR) and function in TCR cell surface expression and signal transduction cascades, which are initiated when a peptide:MHC ligand binds to the TCR. Typically, when an antigen binds to the T-cell receptor, CD3 signals through the cell membrane into the cytoplasm within the T cell. This leads to T-cell activation, which rapidly divides to produce sensitized new T cells to attack the specific antigens exposed to the TCR. The CD3 complex contains the CD3ε molecule, along with four other membrane-bound peptides (CD3-γ, -δ, -ζ, and -β). In humans, CD3-ε is encoded by the CD3E gene on chromosome 11. The intracellular domain of each CD3 chain contains an immune receptor tyrosine-based activation motif (ITAM), which acts as a nucleation site for intracellular signal transduction mechanisms following T-cell receptor binding.

[0266] Many therapeutic strategies modulate T-cell immunity by targeting TCR signaling, particularly anti-human CD3 monoclonal antibodies (mAbs) widely used in immunosuppressive regimens. The CD3-specific mouse mAb OKT3 was the first mAb approved for human use (Sgro, C. Side-effects of a monoclonal antibody, muromonab CD3 / orthocloneOKT3: bibliographic review. Toxicology 105:23-29, 1995), and is widely used clinically as an immunosuppressant in transplantation (Chatenoud, Clin. Transplant 7:422-430, (1993); Chatenoudd, Nat. Rev. Immunol. 3:123-132 (2003); Kumar, Transplant. Proc. 30:1351-1352 (1998)), type 1 diabetes, and psoriasis. Importantly, anti-CD3 mAbs can induce partial T cell signaling and clonal anergy (Smith, JA, Nonmitogenic Anti-CD3 Monoclonal Antibodies Deliver a Partial T Cell Receptor Signal and Induce Clonal Anergy J. Exp. Med. 185:1413-1422 (1997)). OKT3 has been described in the literature as a T cell mitogen and a potent T cell killer (Wong, JT. The mechanism of anti-CD3 monoclonal antibodies. Mediation of cytolysis by inter-T cell bridging. Transplantation 50:683-689 (1990)). In particular, Wong's research confirmed that target killing can be achieved by bridging CD3 T cells and target cells, and that for bivalent anti-CD3 MABs, neither FcR-mediated ADCC nor complement fixation is required to lyse target cells.

[0267] OKT3 exhibits time-dependent mitogenic and T-cell cytotoxic activity; following early activation of T cells leading to cytokine release, OKT3 subsequently blocks all known T-cell functions upon further administration. It is precisely because of this subsequent blockade of T-cell function that OKT3 has been found to have such widespread use as an immunosuppressant in therapeutic regimens aimed at reducing or even eliminating allogeneic tissue rejection. Other CD3 molecule-specific antibodies are disclosed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50, WO2005 / 118635 and WO2007 / 033230, which describe anti-human monoclonal CD3ε antibodies. U.S. Patent 5,821,337 describes the VL and VH sequences of mouse anti-CD3 monoclonal Ab UCHT1 (muxCD3, Shalaby et al., J. Exp. Med. 175, 217-225 (1992) and a humanized variant of this antibody (hu UCHT1). U.S. Patent Application 20120034228 discloses a binding domain capable of binding to epitopes of human and non-chimpanzee primate CD3ε chains.

[0268] Table 6a: Anti-CD3 monoclonal antibodies and their sequences

[0269] Clone Name Antibody Name Target VH Sequence SEQ IDNO: (Full Length) SEQ IDNO: (CDR) VL Sequence SEQ IDNO: (Full Length) SEQ IDNO: (CDR) huOKT3 CD3 <![CDATA[QVQLVQSGGGVVQPGRSLRLSCKAS GYT FTRYTMH WVRQAPGKGLEWIG YINPSRG YTNYNQK VKD RFTISRDNSKNTAFLQMDSLRPEDTGVYFCAR YYDDHYC LDY WGQGTPVTVSS]]> 8040 8900,8901,8902 <![CDATA[DIQMTQSPSSLSASVGDRVTITC SASSS VSYMN WYQQTPGKAPKRWIY D TSKLAS GVPSRFSGSGSGTDYTFTISSLQPEDIATYYC QQWS SNPFT FGQGTKLQITR]]> 8049 8908,8909,8910 huUCHT1 CD3 <![CDATA[EVQLVESGGGLVQPGGSLRLSCAAS GYS FTGYTMN WVRQAPGKGLEWVA LINPYKG VST YNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCAR SGYYGDS DWYFDV WGQGTLVTVSS]]> 8041 8037,8038,8039 <![CDATA[DIQMTQSPSSLSASVGDRVTITC RASQD IRNYLN WYQQKPGKAPKLLIY YTSRLES GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQG NTLPWT FGQGTKVEIK]]> 8050 8034,8035,8036 hu12F6 CD3 <![CDATA[QVQLVQSGGGVVQPGRSLRLSCKAS GYT FTSYTMH WVRQAPGKGLEWIG YINPSSG YTKYNQK FKD RFTISADKSKSTAFLQMDSLRPEDTGVYFCAR WQDYDVY FDY WGQGTPVTVSS]]> 8042 8903,8904,8905 <![CDATA[DIQMTQSPSSLSASVGDRVTMTC RASSS VSYMH WYQQTPGKAPKPWIY A TSNLAS GVPSRFSGSGSGTDYTLTISSLQPEDIATYYC QQWS SNPPT FGQGTKLQITR]]> 8051 8911,8912,8913 mOKT3 CD3 <![CDATA[QVQLQQSGAELARPGASVKMSCKAS GYT FTRYTMH WVKQRPGQGLEWIG YINPSRG YTNYNQK FKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYC LDY WGQGTTLTVSS]]> 8043 8900,8906,8902 <![CDATA[QIVLTQSPAIMSASPGEKVTMTC SASSS VSYMN WYQQKSGTSPKRWIY D TSKLAS GVPAHFRGSGSGTSYSLTISGMEAEDAATYYC QQWS SNPFT FGSGTKLEINR]]> 8052 8908,8909,8910 MT103 blinatumomab CD3 <![CDATA[DIKLQQSGAELARPGASVKMSCKTS GYT FTRYTMH WVKQRPGQGLEWIG YINPSRG YTNYNQK FKD KATLTTDKSSSTAYMQLSSLTSEDSAVYYCAR YYDDHYC LDY WGQGTTLTVSS]]> 8044 8900,8906,8902 <![CDATA[DIQLTQSPAIMSASPGEKVTMTC RASSS VSYMN WYQQKSGTSPKRWIY D TSKVAS GVPYRFSGSGSGTSYSLTISSMEAEDAATYYC QQWS SNPLT FGAGTKLELK]]> 8053 8914,8915,8916 MT110 solitomab CD3 <![CDATA[DVQLVQSGAEVKKPGASVKVSCKAS GYT FTRYTMH WVRQAPGQGLEWIG YINPSRG YTNYADS VKG RFTITTDKSTSTAYMELSSLRSEDTATYYCAR YYDDHYC LDY WGQGTTVTVSS]]> 8045 8900,8907,8902 <![CDATA[DIVLTQSPATLSLSPGERATLSC RASQS VSYMN WYQQKPGKAPKRWIY D TSKVAS GVPARFSGSGSGTDYSLTINSLEAEDAATYYC QQWS SNPLT FGGGTKVEIK]]> 8054 8917,8915,8916 CD3.7 CD3 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS 8046 QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 8055 CD3.8 CD3 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 8047 QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL 8056 CD3.9 CD3 EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 773 ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL 772 CD3.10 CD3 EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 8048 QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL 8057

[0270] *The underlined sequence (if it exists) is the CDR within VL and VH.

[0271] CD3 cell antigen binding fragment

[0272] In another aspect, this disclosure relates to an antigen-binding fragment (AF2) having a specific binding affinity for effector cell antigens, which can be incorporated into any human or animal composition examples described herein. In some cases, the effector cell antigen is expressed on the surface of effector cells selected from plasma cells, T cells, B cells, cytokine-induced killer cells (CIK cells), mast cells, dendritic cells, regulatory T cells (RegT cells), helper T cells, myeloid cells, and NK cells.

[0273] Various AF2 binding fragments of effector cell antigens have specific utility for pairing with antigen-binding fragments in compositional form, which have binding affinity for EGFR antigens associated with diseased cells or tissues, in order to achieve cell killing of diseased cells or tissues. Binding specificity can be determined by complementarity-determining regions or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, binding specificity is determined by both light chain CDRs and heavy chain CDRs. A given combination of heavy chain CDRs and light chain CDRs provides a given binding pocket that imparts greater affinity and / or specificity to effector cell antigens compared to other reference antigens. A bispecific composition having a first antigen-binding fragment (AF1) against EGFR linked by a short, flexible peptide linker to a second antigen-binding fragment (AF2) that has binding specificity to effector cell antigens is bispecific, wherein each antigen-binding fragment has specific binding affinity to its respective ligand. Those skilled in the art will understand that in such compositions, AF1 against EGFR of diseased tissue is used in combination with AF2 against effector cell markers to bring effector cells close to cells of diseased tissue in order to achieve cell lysis of diseased tissue cells. Furthermore, AF1 and AF2 are incorporated into a specially designed polypeptide containing a cleavable release segment and XTEN to impart prodrug properties to the composition. When the composition is near diseased tissue having a protease capable of cleaving the release segment at one or more sites in the release segment sequence, the composition becomes activated by releasing the fused AF1 and AF2 after the release segment is cleaved.

[0274] In one embodiment, the AF2 of the human or animal composition has binding affinity for effector cell antigens expressed on the surface of T cells. In another embodiment, the AF2 of the human or animal composition has binding affinity for CD3. In yet another embodiment, the AF2 of the human or animal composition has binding affinity for members of the CD3 complex, said members comprising all known CD3 subunits of the CD3 complex, either individually or in independent combinations; for example, CD3ε, CD3δ, CD3γ, CD3ζ, CD3α, and CD3β. In yet another embodiment, AF2 has binding affinity for CD3ε, CD3δ, CD3γ, CD3ζ, CD3α, or CD3β.

[0275] The origin of the antigen-binding fragments considered in this disclosure may be derived from naturally occurring antibodies or fragments thereof, non-naturally occurring antibodies or fragments thereof, humanized antibodies or fragments thereof, synthetic antibodies or fragments thereof, hybrid antibodies or fragments thereof, or modified antibodies or fragments thereof. Methods for generating antibodies against a given target marker are well known in the art. For example, monoclonal antibodies may be prepared using a hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by a recombinant DNA method (US Patent No. 4,816,567). The structures of antibodies and their fragments, variable regions (VH and VL) of the antibody heavy and light chains, single-chain variable regions (scFv), complementarity-determining regions (CDRs), and domain antibodies (dAbs) are well understood. Methods for generating polypeptides having desired antigen-binding fragments with binding affinity for a given antigen are known in the art.

[0276] Those skilled in the art will understand that the use of the term "antigen-binding fragment" in the context of the compositions disclosed herein is intended to include a portion or fragment of an antibody that retains the ability to bind an antigen, which is a ligand of the corresponding intact antibody. In such embodiments, the antigen-binding fragment may be, but is not limited to, a CDR and intercalation framework region, a variable or hypervariable region of the antibody light chain and / or heavy chain (VL, VH), a variable fragment (Fv), a Fab' fragment, an F(ab')2 fragment, a Fab fragment, a single-chain antibody (scAb), a VHH camelid antibody, a single-chain variable fragment (scFv), a linear antibody, a single-domain antibody, a complementarity-determining region (CDR), a domain antibody (dAb), a single-domain heavy chain immunoglobulin of type BHH or BNAR, a single-domain light chain immunoglobulin, or other polypeptides known in the art containing an antibody fragment capable of binding an antigen. Antigen-binding fragments having CDR-H and CDR-L may be configured from the N-terminus to the C-terminus in a (CDR-H)-(CDR-L) or (CDR-H)-(CDR-L) orientation. The VL and VH of the two antigen-binding fragments can also be configured as single-chain biantibody configurations; that is, the VL and VH of AF1 and AF2 are configured with linkers of appropriate length to allow for biantibody arrangement.

[0277] The various CD3-binding AF2s of this disclosure have been specifically modified to enhance their stability in the peptide examples described herein. Antibody protein aggregation continues to be a significant issue for its developability and remains a major focus area in antibody production. Antibody aggregation can be triggered by the partial unfolding of its domains, leading to monomer-monomer binding, followed by nucleation and aggregate growth. Although the aggregation tendency of antibodies and antibody-based proteins can be influenced by external experimental conditions, they are strongly dependent on inherent antibody properties such as those determined by their sequence and structure. While it is well known that proteins are only slightly stable in their folded state, it is often less understood that most proteins are inherently prone to aggregation in their unfolded or partially unfolded state, and the resulting aggregates can be very stable and long-lived. Reduction in aggregation tendency has also been shown to be accompanied by increased expression titers, demonstrating that reducing protein aggregation is beneficial throughout the development process and can lead to more efficient clinical research pathways. For therapeutic proteins, aggregates are a significant risk factor for detrimental immune responses in patients and can form via a variety of mechanisms. Controlling aggregation can improve protein stability, manufacturability, loss rate, safety, formulation, titer, immunogenicity, and solubility. Intrinsic properties of proteins, such as size, hydrophobicity, electrostatics, and charge distribution, play a significant role in protein solubility. The low solubility of therapeutic proteins with hydrophobic surfaces has been shown to make formulation development more difficult and can lead to weak biodistribution in vivo, undesirable pharmacokinetic behavior, and immunogenicity. Reducing the overall surface hydrophobicity of candidate monoclonal antibodies can also provide benefits and cost savings related to purification and dosing regimens. Individual amino acids can be identified through structural analysis as having aggregation potential in antibodies and can be located in CDRs and framework regions. In particular, residues can be predicted to be at high risk of causing hydrophobicity problems in a given antibody. In one embodiment, this disclosure provides AF2 with the ability to specifically bind CD3, wherein the AF2 has at least one amino acid substitution of a hydrophobic amino acid in a framework region relative to a parent antibody or antibody fragment, wherein the hydrophobic amino acid is selected from isoleucine, leucine, or methionine. In another embodiment, CD3 AF2 has at least two amino acid substitutions of a hydrophobic amino acid in one or more framework regions, wherein the hydrophobic amino acid is selected from isoleucine, leucine, or methionine.

[0278] In designing the sequence of AF2 in the embodiments described herein, variations with respect to the net charge of the polypeptide were considered, particularly variations with respect to the antibody or antibody fragment constituting the specific embodiments of the invention set forth herein, wherein individual amino acid substitutions were prepared relative to the parent antibody used as a starting point. Related to these design considerations is the isoelectric point (pI) of the polypeptide, which is the pH at which the antibody or antibody fragment has no net charge. Antibodies or antibody fragments typically have a net positive charge, which tends to be associated with increased blood clearance and tissue retention, and generally a shorter half-life, while a net negative charge results in reduced tissue uptake and a longer half-life. This charge can be manipulated by mutations in the framework residues. The isoelectric point of the polypeptide can be determined arithmetically (e.g., computationally) or experimentally by in vitro determination. In some embodiments, the isoelectric points of AF1 and AF2 are designed within specific ranges of each other, thereby promoting stability.

[0279] In one embodiment, this disclosure provides AF2 for use in any of the peptide embodiments described herein, comprising CDR-L and CDR-H, wherein the AF2 (a) specifically binds to differentiation cluster 3 (CD3) of the T cell receptor; and (b) comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences having SEQ ID NO: 742, 743, and 744, respectively. In another embodiment, this disclosure provides AF2 for use in any of the peptide embodiments described herein, comprising CDR-L and CDR-H, wherein the AF2 (a) specifically binds to differentiation cluster 3 (CD3) of the T cell receptor; (b) comprises CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences of SEQ ID NO: 742, 743, and 744, respectively; and (c) comprises CDR-L, wherein the CDR-L comprises CDR-L1 having an amino acid sequence of SEQ ID NO: 735 or 736, CDR-L2 having an amino acid sequence of SEQ ID NO: 738 or 739, and CDR-L3 having an amino acid sequence of SEQ ID NO: 740.In another embodiment, the aforementioned AF2 embodiment of this paragraph further includes a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 includes FR-L1 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or identical to the amino acid sequence of SEQ ID NO: 746, FR-L2 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or identical to the amino acid sequence of SEQ ID NO: 747, and ... The amino acid sequence of any one of SEQ ID NO: 748-751 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to FR-L3; the amino acid sequence of SEQ ID NO: 754 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to FR-L4; the amino acid sequence of SEQ ID NO: 755 or SEQ ID NO: 756 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to FR-H1; and the amino acid sequence of SEQ ID NO: The amino acid sequence of SEQ ID NO: 759 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is the same as FR-H2; the amino acid sequence of SEQ ID NO: 760 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is the same as FR-H3; and the amino acid sequence of SEQ ID NO: 764 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is the same as FR-H4.In another embodiment, AF2 used in any of the peptide embodiments described herein comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 comprises FR-L1 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or equivalent to the amino acid sequence of SEQ ID NO: 746, and FR-L2 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or equivalent to the amino acid sequence of SEQ ID NO: 747 ... The amino acid sequence of NO:748 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or equivalent FR-L3 with SEQ ID NO:754; the amino acid sequence of SEQ ID NO:755 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or equivalent FR-L4 with SEQ ID NO:755; and the amino acid sequence of SEQ ID NO:755 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or equivalent FR-H1 with SEQ ID NO:754; and the amino acid sequence of SEQ ID NO:754 ... The amino acid sequence of 759 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or equivalent FR-H2; the amino acid sequence of SEQ ID NO: 760 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or equivalent FR-H3; and the amino acid sequence of SEQ ID NO: 764 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or equivalent FR-H4.In another embodiment, AF2 used in any of the peptide embodiments described herein comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 comprises FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or equivalent to the amino acid sequence of SEQ ID NO: 746, and FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or equivalent to the amino acid sequence of SEQ ID NO: 747 ... The amino acid sequence of NO:749 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is identical to FR-L3; the amino acid sequence of SEQ ID NO:754 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is identical to FR-L4; the amino acid sequence of SEQ ID NO:755 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is identical to FR-H1; and ...4 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is identical to FR-H1. The amino acid sequence of SEQ ID NO: 759 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is the same as FR-H2; the amino acid sequence of SEQ ID NO: 760 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is the same as FR-H3; and the amino acid sequence of SEQ ID NO: 764 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is the same as FR-H4.In another embodiment, the AF2 of the human or animal polypeptide embodiment described herein comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 comprises FR-L1 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or identical to the amino acid sequence of SEQ ID NO: 746, FR-L2 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or identical to the amino acid sequence of SEQ ID NO: 747, FR-L3 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or identical to the amino acid sequence of SEQ ID NO: 750, and ...47, and FR-L3 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, The amino acid sequence of ID NO: 754 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or equivalent FR-L4 with SEQ ID NO: 755; the amino acid sequence of SEQ ID NO: 755 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or equivalent FR-H1 with SEQ ID NO: 759; and the amino acid sequence of SEQ ID NO: 759 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or equivalent FR-H2 with SEQ ID NO: 755. The amino acid sequence of NO:760 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is the same as FR-H3; and the amino acid sequence of SEQ ID NO:764 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is the same as FR-H4.In another embodiment, the AF2 of the human or animal polypeptide embodiment described herein comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 comprises FR-L1 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or identical to the amino acid sequence of SEQ ID NO: 746, FR-L2 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or identical to the amino acid sequence of SEQ ID NO: 747, FR-L3 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or identical to the amino acid sequence of SEQ ID NO: 751, and FR-L3 showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or identical to the amino acid sequence of SEQ ID NO: 747 ... The amino acid sequence of NO: 754 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is identical to FR-L4; the amino acid sequence of SEQ ID NO: 756 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is identical to FR-H1; the amino acid sequence of SEQ ID NO: 759 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is identical to FR-H2; and the amino acid sequence of SEQ ID NO: 754 ... The amino acid sequence of NO:760 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is the same as FR-H3; and the amino acid sequence of SEQ ID NO:764 shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is the same as FR-H4.

[0280] In another embodiment, this disclosure provides AF2 for use in any peptide embodiment described herein, wherein AF2 comprises a variable weight (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence of SEQ ID NO: 766 or SEQ ID NO: 769. In another embodiment, this disclosure provides AF2 for use in any peptide embodiment described herein, wherein AF2 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence of any one of SEQ ID NO: 765, 767, 768, 770, or 771. In another embodiment, this disclosure provides AF2 for use in any of the polypeptide embodiments described herein, wherein the AF2 comprises a variable weight (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence of SEQ ID NO: 766 or SEQ ID NO: 769, and a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence of any one of SEQ ID NO: 765, 767, 768, 770, or 771.

[0281] In another embodiment, this disclosure provides AF2 for use in any of the polypeptide embodiments described herein, wherein the AF2 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any of the amino acid sequences in SEQ ID NO:776-780.

[0282] In another aspect, this disclosure provides AF2 antigen-binding fragments that bind to CD3 protein complexes, exhibiting enhanced stability compared to CD3-binding antibodies or antigen-binding fragments known in the art. Furthermore, the CD3 antigen-binding fragments of this disclosure are designed to confer a higher degree of stability to chimeric bispecific antigen-binding fragment compositions in which they are integrated, resulting in improved expression and recovery of the fusion protein, increased shelf life, and enhanced stability when administered to humans or animals. In one method, the CD3 AF2 of this disclosure is designed to have a higher degree of thermal stability compared to certain CD3-binding antibodies and antigen-binding fragments known in the art. As a result, CD3 AF2 used as a component of chimeric bispecific antigen-binding fragment compositions in which they are integrated exhibits advantageous pharmaceutical properties, including high thermal stability and low aggregation tendency, leading to improved expression and recovery during manufacturing and storage, and promoting a long serum half-life. Biophysical properties, such as thermal stability, are often limited by antibody variable domains, which vary considerably in their inherent properties. High thermal stability is often associated with high expression levels and other desirable properties, including less susceptibility to aggregation (Buchanan A et al., Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression. MAbs2013;5:255). Thermal stability is measured by the "melting temperature" (T0). m The melting temperature is determined by [the method described above], defined as the temperature at which the next half of the molecules denature. The melting temperature of each heterodimer is an indicator of its thermal stability. [The text then abruptly shifts to a different topic:] Determining T m The in vitro determination of the heterodimer is known in the art, including the methods described in the examples below. The melting point of the heterodimer can be measured using techniques such as differential scanning calorimetry (Chen et al. (2003) Pharm Res 20:1952-60; Ghirlando et al. (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of the heterodimer can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9) or as described in the examples below.

[0283] The thermal denaturation curves of the CD3-binding fragment and the anti-CD3 bispecific antibody comprising the said anti-CD3-binding fragment, combined with the reference of this disclosure, show that the constructs of this disclosure are more resistant to thermal denaturation than the antigen-binding fragment comprising the sequence shown in SEQ ID NO:781 or the control bispecific antibody (wherein the control bispecific antigen-binding fragment comprises SEQ ID NO:781) and the reference antigen-binding fragment in conjunction with the EGFR examples described herein. In one embodiment, the peptide of any human or animal composition example described herein comprises the anti-CD3 AF2 of the examples described herein, wherein the Tf of the antigen-binding fragment comprising the sequence of SEQ ID NO:781 is determined, as determined by the increase in melting temperature in an in vitro assay, to be more resistant to thermal denaturation. m In comparison, the T of AF2 m The temperature is at least 2°C higher, or at least 3°C ​​higher, or at least 4°C higher, or at least 5°C higher, or at least 6°C higher, or at least 7°C higher, or at least 8°C higher, or at least 9°C higher, or at least 10°C higher.

[0284] In another embodiment, the peptide of any human or animal composition embodiment described herein comprises AF2 that specifically binds to human or cynomolgus monkey CD3, with a dissociation constant (K... d The concentration is approximately 10 nM to approximately 400 nM, or approximately 50 nM to approximately 350 nM, or approximately 100 nM to approximately 300 nM, as determined in an in vitro antigen binding assay containing human or cynomolgus monkey CD3 antigen. In another embodiment, the polypeptide of any human or animal composition embodiment described herein comprises AF2 that specifically binds to human or cynomolgus monkey CD3, with a dissociation constant (K0). d (Worse than about 10 nM, or about 50 nM, or about 100 nM, or about 150 nM, or about 200 nM, or about 250 nM, or about 300 nM, or about 350 nM, or weaker than about 400 nM, as determined in an in vitro antigen binding assay. For clarity, K) d The binding of the 400 antigen-binding fragment to its ligand is weaker than that of K. d The antigen-binding fragment is 10 nM. In another embodiment, the polypeptide of any human or animal composition embodiment described herein comprises AF2 that specifically binds to human or cynomolgus monkey CD3, with a binding affinity of up to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or up to 1 / 10 of the antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 781, as determined by the respective dissociation constant (K0) in an in vitro antigen binding assay. dThe binding affinity for CD3 is determined by the specific parameters. In another embodiment, this disclosure provides a bispecific polypeptide comprising AF2, which, relative to the AF1 EGFR embodiments described herein incorporated into human or animal polypeptides, exhibits a binding affinity for CD3 of at most 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or at most 1 / 1000, as determined by the respective dissociation constants (K0) in an in vitro antigen binding assay. d The binding affinity of a human or animal composition for a target ligand can be determined using the following methods: binding or competitive binding assays, such as the Biacore assay with a chip-based binding receptor or binding protein as described in U.S. Patent 5,534,617, or an ELISA assay, the assay described in the examples herein, a radioreceptor assay, or other assays known in the art. The binding affinity constant can then be determined using standard methods, such as the Scatchard analysis described by van Zoelen et al., Trends Pharmacol Sciences (1998) 19) 12): 487, or other methods known in the art.

[0285] In a related aspect, this disclosure provides AF2, which binds to CD3 and is incorporated into a chimeric bispecific polypeptide composition designed to have an isoelectric point (pI) that imparts enhanced stability to the composition of this disclosure compared to corresponding compositions known in the art that contain CD3-binding antibodies or antigen-binding fragments. In one embodiment, the polypeptide of any human or animal composition embodiment described herein comprises CD3-binding AF2, wherein the AF2 exhibits a pI between 6.0 and 6.6, including the endpoints. In another embodiment, the polypeptide of any human or animal composition embodiment described herein comprises CD3-binding AF2, wherein the AF2 exhibits a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the pI of a reference antigen-binding fragment comprising the sequence shown in SEQ ID NO: 781. In another embodiment, the peptide of any human or animal composition embodiment described herein comprises an AF2 fused to an AF1 that binds to an EGFR antigen and binds to CD3, wherein the pI exhibited by the AF2 is within at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units of the pI of the AF1 that binds to the EGFR antigen or its epitope. In another embodiment, the peptide of any human or animal composition embodiment described herein comprises an AF2 fused to an AF1 that binds to an EGFR antigen and binds to CD3, wherein the pI exhibited by the AF2 is within at least about 0.1 to about 1.5, or at least about 0.3 to about 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 to about 0.9 pH units of the pI of the AF1. Specifically, this design, with the pIs of two of the antigen-binding fragments within such a range, is expected to confer a higher degree of stability to the chimeric bispecific antigen-binding fragment composition in which they are integrated, resulting in improved expression and enhanced recovery of the fusion protein in a soluble, non-aggregated form, increased shelf life of the formulated chimeric bispecific peptide composition, and enhanced stability when the composition is administered to humans or animals. In other words, placing AF2 and AF1 within a relatively narrow pI range allows for the selection of buffers or other solutions in which both AF2 and AF1 are stable, thereby promoting the overall stability of the composition.

[0286] In some embodiments, the VL and VH of the antigen-binding fragment are fused via a relatively long linker comprising 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids, which, when linked together, possess flexible properties. In one embodiment, the VL and VH of any scFv embodiment described herein are linked via a relatively long linker of hydrophilic amino acids, wherein the linker is GSGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEGEG (SEQ ID NO: 8058).

[0287] TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 8059),

[0288] GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 8060, or

[0289] GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 8061). In another embodiment, AF1 and AF2 are linked together by a short linker having 3, 4, 5, 6, or 7 amino acids of hydrophilic amino acids. In one embodiment, the short linker sequence is SGGGGS (SEQ ID NO: 8062), GGGGS (SEQ ID NO: 8063), GGSGGS (SEQ ID NO: 8064), GGS, or GSP. In another embodiment, this disclosure provides a composition comprising a single-chain biantibody, wherein, upon folding, a first domain (VL or VH) pairs with a last domain (VH or VL) to form a scFv, and two intermediate domains pair to form another scFv, wherein the first and second domains, as well as the third and last domains, are fused together by one of the aforementioned short linkers, and the second and third variable domains are fused together by one of the aforementioned relatively long linkers. As those skilled in the art will understand, the selection of short and relatively long adapters is to prevent incorrect pairing of adjacent variable domains, thereby facilitating the formation of single-chain biantibody conformations of VL and VH containing a first antigen-binding fragment and a second antigen-binding fragment.

[0290] Table 6b. Exemplary CD3 CDR Sequences

[0291] Construct CDR region Amino acid sequence SEQ ID NO: 3.23, 3.30, 3.31, 3.32 CDR-L1 RSSNGAVTSSNYAN 735 3.24 CDR-L1 RSSNGEVTTSNYAN 736 3.33, 3.9 CDR-L1 RSSTGAVTTSNYAN 737 3.23, 3.30, 3.31, 3.32, 3.9, 3.33 CDR-L2 GTNKRAP 738 3.24 CDR-L2 GTIKRAP 739 3.23, 3.24, 3.30, 3.31, 3.32 CDR-L3 ALWYPNLWVF 740 3.33, 3.9 CDR-L3 ALWYSNLWVF 741 3.23, 3.24, 3.30, 3.31, 3.32, 3.9 CDR-H1 GFTFNTYAMN 742 3.23, 3.24, 3.30, 3.31, 3.32, 3.9 CDR-H2 RIRSKYNNYATYYADSVKD 743 3.23. 3.24, 3.30, 3.31, 3.32 CDR-H3 HENFGNSYVSWFAH 744 3.9 CDR-H3 HGNFGNSYVSWFAY 745

[0292] Table 6c. Exemplary CD3 FR sequences

[0293] Construct FR region Amino acid sequence SEQ ID NO: 3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33 FR-L1 ELVVTQEPSLTVSPGGTVTLTC 746 3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33 FR-L2 WVQQKPGQAPRGLIG 747 3.23, 3.24 FR-L3 GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC 748 3.30 FR-L3 GTPARFSGSSLGGKAALTLSGVQPEDEAVYYC 749 3.31 FR-L3 GTPARFSGSLLGGSAALTLSGVQPEDEAVYYC 750 3.32 FR-L3 GTPARFSGSSLGGSAALTLSGVQPEDEAVYYC 751 3.9 FR-L3 GTPARFSGSLLGGKAALTLSGVQPEDEAEYYC 752 3.33 FR-L3 GTPARFSGSSLGGSAALTLSGVQPEDEAEYYC 753 3.23, 3.24, 3.30, 3.31, 3.32, 3.9 FR-L4 GGGTKLTVL 754 3.23, 3.24 FR-H1 EVQLLESGGGIVQPGGSLKLSCAAS 755 3.30, 3.31, 3.32 FR-H1 EVQLQESGGGIVQPGGSLKLSCAAS 756 3.33 FR-H1 EVQLQESGGGLVQPGGSLKLSCAAS 757 3.9 FR-H1 EVQLLESGGGLVQPGGSLKLSCAAS 758 3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33 FR-H2 WVRQAPGKGLEWVA 759 3.23, 3.24, 3.30, 3.31, 3.32 FR-H3 RFTISRDDSKNTVYLQMNNLKTEDTAVYYCVR 760 3.9 FR-H3 RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR 762 3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33 FR-H4 WGQGTLVTVSS 764

[0294] Table 6d: Exemplary VL and VH sequences

[0295] Construct Region Amino acid sequence SEQ ID NO: 3.23 VL ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL 765 3.23, 3.24 VH EVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 766 3.24 VL ELVVTQEPSLTVSPGGTVTLTCRSSNGEVTTSNYANWVQQKPGQAPRGLIGGTIKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL 767 3.30 VL ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL 768 3.30, 3.31, 3.32 VH EVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 769 3.31 VL ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGSAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL 770 3.32 VL ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL 771 3.9 VL ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL 772 3.9 VH EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 773 3.33 VL ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL 774 3.33 VH EVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 775

[0296] Table 6e: Exemplary scFv sequences

[0297] Construct Amino acid sequence SEQ ID NO: 3.23 ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 776 3.24 ELVVTQEPSLTVSPGGTVTLTCRSSNGEVTTSNYANWVQQKPGQAPRGLIGGTIKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 777 3.30 ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 778 3.31 ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGSAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 779 3.32 ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS 780 3.9 ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 781 3.33 ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 782 4.11 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 783 4.12 QAGLTQPPSASGTPGQRVTLSCSGSYSNIGTYYVYWYQQLPGTAPKLLIYSNDQRLSGVPDRFSGSKSGTSASLAISGLQSEDEAAYYCAAWDDSLNGWAFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 784 4.13 QPGLTQPPSASGTPGQRVTLSCSGRSSNIGSYYVYWYQHLPGMAPKLLIYRNSRRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDDSLKSWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 785 4.14 QSVLTQPPSASGTPGQRVTISCSGSSSNIGTNYVYWYQQFPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSGSLAISGLQSEDEADYSCAAWDDSLNGWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 786 4.15 QPGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 787 4.16 QAVLTQPPSASGTPGQRVTISCSGSSSNIGSYYVYWYQQVPGAAPKLLMRLNNQRPSGVPDRFSGAKSGTSASLVISGLRSEDEADYYCAAWDDSLSGQWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 788 4.17 QAGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDASLSGWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLVQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS 789

[0298] Anti-EpCAM binding domain

[0299] In some embodiments, the present invention provides a chimeric peptide assembly composition comprising a binding domain having binding affinity for the tumor-specific marker EpCAM. In one embodiment, the binding domain comprises VL and VH regions derived from a monoclonal antibody against EpCAM. Monoclonal antibodies against EpCAM are known in the art. Exemplary non-limiting examples of EpCAM monoclonal antibodies and their VL and VH sequences are illustrated in Table 6f. In one embodiment, the present invention provides a chimeric peptide assembly composition comprising a binding domain having binding affinity for the tumor-specific marker EpCAM, said binding domain comprising the anti-EpCAM VL and VH sequences shown in Table 6f. In another embodiment, the present invention provides a chimeric peptide assembly composition wherein a first portion of the first binding domain comprises VH and VL regions, wherein each VH and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the paired VL and VH sequences of the 4D5MUCB anti-EpCAM antibody shown in Table 6f. In another embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a binding domain having binding affinity for a tumor-specific marker, the binding domain comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, wherein each is derived from the respective VL and VH sequences shown in Table 6f.

[0300] Table 6f. Anti-target cell monoclonal antibodies and sequences

[0301] Product Name Antibody Name Target VH Sequence SEQ ID NO: (Full Length) SEQ ID NO: (CDR) VL Sequence SEQ ID NO: (Full Length) SEQ ID NO: (CDR) Tysabri™ natalizumab Alpha 4 Integrin <![CDATA[QVQLVQSGAEVKKPGASVKVSCKASGFNIK DTYIH WVRQAPGQRLEWMG RIDPANGYTKYDPKFQG RVTITADTSASTAYMELSSLRSEDTAVYYCAR EGYYGNYGVYAMDY WGQGTLVTVSS]]> 8065 8918,8919,8920 <![CDATA[DIQMTQSPSSLSASVGDRVTITC KTSQDINKYMA WYQQTPGKAPRLLIH YTSALQ PGIPSRFSGSGSGRDYTFTISSLQPEDIATYYC LQYDNLWT FGQGTKVEIK]]> 8164 9178,9179,9180 REGN910 nesvacumab Ang2 <![CDATA[EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYDIH WVRQATGKGLEWVSAI GPAGDTYYPGSV KGRFTISRENAKNSLYLQMNSLRAGDTAVYYCAR GLITFGGLIAPFD YWGQGTLVTVSS]]> 8066 8921,8922,8923 <![CDATA[EIVLTQSPGTLSLSPGERATLSCRA SQSVSSTYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QHYDNSQ TFGQGTKVEIK]]> 8165 9181,9182,9183 hMFE23 CEA <![CDATA[QVKLEQSGAEVVKPGASVKLSCKAS GFNIKDS YMWLRQGPGQRLEWIGWI DPENGD TEYAPKFQGKATFTTDTSANTAYLGLSSLRPEDTAVYCNEG TPTGPYYFD YWGQGTLVTVSS]]> 8067 8924,8925,8926 <![CDATA[ENVLTQSPSSMSASVGDRVNIACSA SSSVS YMHWFQQKPGKSPKLWIYSTSN LAS GVPSRFSGSGSGTDYSLTISSMQPEDAATYYCQQ RSSYPL TFGGGTKLEIK]]> 8166 9184,9185,9186 M5A (humanized T84.66) CEA <![CDATA[EVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYMH WVRQAPGKGLEWVA RIDPANGNSKYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCAP FGYYVSDYAMAY WGQGTLVTVSS]]> 8068 8927,8928,8929 <![CDATA[DIQLTQSPSSLSASVGDRVTITC RAGESVDIFGVGFLH WYQQKPGKAPKLLIY RASNLES GVPSRFSGSGSRTDFTLTISSLQPEDFATYYC QQTNEDPYT FGQGTKVEIK]]> 8167 9187,9188,9189 M5B (humanized T84.66) CEA <![CDATA[EVQLVESGGGLVQPGGSLRLSCAASGFNIK DTYM HWVRQAPGKGLEWVA RIDPANGNSKYVPKFQG RATISADTSKNTAYLQMNSLRAEDTAVYYCAP FGYYVSDYAMAY WGQGTLVTVSS]]> 8069 8930,8931,8929 <![CDATA[DIQLTQSPSSLSASVGDRVTITC RAGESVDIFGVGFLH WYQQKPGKAPKLLIY RASNLES GVPSRFSGSGSRTDFTLTISSLQPEDFATYYC QQTNEDPYT FGQGTKVEIK]]> 8167 9187,9188,9189 CEA-Cide Labetuzumab (MN-14) CEACAM5 <![CDATA[EVQLVESGGGVVQPGRSLRLSCSASGFDFT TYWMS WVRQAPGKGLEWIG EIHPDSSTINYAPSLKD RFTISRDNAKNTLFLQMDSLRPEDTGVYFCAS LYFGFPWFAY WGQGTPVTVSS]]> 8070 8932,8933,8934 <![CDATA[DIQLTQSPSSLSASVGDRVTITC KASQDVGTSVA WYQQKPGKAPKLLIY WTSTRHT GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYSLYRS FGQGTKVEIK]]> 8168 9190,9191,9192 CEA-Scan arcitumomab CEACAM5 <![CDATA[EVKLVESGGGLVQPGGSLRLSCATS GFTFTDYYMN WVRQPPGKALEWLG FIGNKANGYTTEYSAS VKGRFTISRDKSQSILYLQMNTLRAESDSA TYYCTRDR GLRFYFDYWGQGTTLTVSS]]> 8071 8935,8936,8937 <![CDATA[QTVLSQSPAILSASPGEKVTMTC RASSSVTYIHW YQQKPGSSPKSWIYA TSNLASG VPARFSGSGSGTSYSLTISRVEAEDAATYYC QHWSSKPPT FGGGTKLEIKR]]> 8169 9193,9194,9195 MT110 CEACAM5 <![CDATA[EVQLVESGGGLVQPGRSLRLSCAASGFTVS SYWMH WVRQAPGKGLEWVG FIRNKANGGTTEYAASVKG RFTISRDDSKNTLYLQMNSLRAEDTAVYYCAR DRGLRFYFDY WGQGTTVTVSS]]> 8072 8938,8939,8940 <![CDATA[QAVLTQPASLSASPGASASLTC TLRRGINVGAYSIY WYQQKPGSPPQYLLR YKSDSDKQQGS GVSSRFSASKDASANAGILLISGLQSEDEADYYC MIWHSGASAV FGGGTKLTVL]]> 8170 9196,9197,9198 MT103 blinatumomab CD19 <![CDATA[QVQLQQSGAELVRPGSSVKISCKASGYAFS SYWMN WVKQRPGQGLEWIG QIWPGDGDTNYNGKFKG KATLTADESSSTAYMQLSSLASEDSAVYFCAR RETTTVGRYYYAMDY WGQGTTVTVSS]]> 8073 8941,8942,8943 <![CDATA[DIQLTQSPASLAVSLGQRATISC KASQSVDYDGDSY LNWYQQIPGQPPKLLI YDASNLVS GIPPRFSGSGSGTDFTLNIHPVEKVDAATYHC QQSTEDPWT FGGGTKLEIM]]> 8171 9199,9200,9201 Arzerra ofatumumab CD20 <![CDATA[EVQLVESGGGLVQPGRSLRLSCAASGFTFN DYAMH WVRQAPGKGLEWVS TISWNSGSIGYADSVKG RFTISRDNAKKSLYLQMNSLRAEDTALLYYCAK DIQYGNYYYGMDV WGQGTTVTVSS]]> 8074 8944,8945,8946 <![CDATA[EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPIT FGQGTRLEIK]]> 8172 9202,9203,9204 Bexxar™ tositumomab CD20 <![CDATA[QAYLQQSGAELVRPGASVKMSCKASGYTFT SYNMH WVKQTPRQGLEWIG AIYPGNGDTSYNQKFKG KATLTVDKSSSTAYMQLSSLTSEDSAVYFCAR VVYYSNSYWYFDV WGTGTTVTVSG]]> 8075 8947,8948,8949 <![CDATA[QIVLSQSPAILSASPGEKVTMTC RASSSVSYMHW YQQKPGSSPKPWIY APSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYC QQWSFNPPT FGAGTKLELK]]> 8173 9205,9206,9207 GAZYVA Obinutuzumab CD20 <![CDATA[QVQLVQSGAEVKKPGSSVKVSCKASGYAFS YSWIN WVRQAPGQGLEWMG RIFPGDGDTDYNGKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR NVFDGYWLVY WGQGTLVTVSS]]> 8076 8950,8951,8952 <![CDATA[DIVMTQTPLSLPVTPGEPASISC RSSKSLLHSNGITYLY WYLQKPGQSPQLLIY QMSNLVS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC AQNLELPYT FGGGTKVEIK]]> 8174 9208,9209,9210 Ocrelizumab / 2H7 v16 CD20 <![CDATA[EVQLVESGGGLVQPGGSLRLSCAAS GYTFTSYN MHWVRQAPGKGLEWVGA IYPGNGDT SYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR VVYYSNSYWYFDV WGQGTLVTVSS]]> 8077 8953,8954,8949 <![CDATA[DIQMTQSPSSLSASVGDRVTITC RASSSVSYMH WYQQKPGKAPKPLIY APSNLAS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSFNPPT FGQGTKVEIK]]> 8175 8911,9206,9207 Rituxan™ rituximab CD20 <![CDATA[QVQLQQPGAELVKPGASVKMSCKAS GYTFTSYN MHWVKQTPGRGLEWIGA IYPGNGDT SYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYC ARSTYYGGDWYFNV WGAGTTVTVSA]]> 8078 8953,8954,8955 <![CDATA[QIVLSQSPAILSASPGEKVTMTCRAS SSVSY IHWFQQKPGSSPKPWIY ATS NLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYC QQWTSNPPT FGGGTKLEIK]]> 8176 9211,9212,9213 Zevalin™ ibritumomabtieuxetan CD20 <![CDATA[QAYLQQSGAELVRPGASVKMSCKAS GYTFTSYN MHWVKQTPRQGLEWIG AIYPGNGDTSYNQKF KGKATLTVDKSSSTAYMQLSSLTSEDSAVYFCAR VVYYSNSYWYFDV WGTGTTVTVSA]]> 8079 8953,8956,8949 <![CDATA[QIVLSQSPAILSASPGEKVTMTC RASSSVSYMH WYQQKPGSSPKPWIY APSNLAS GVPARFSGSGSGTSYSLTISRVEAEDAATYYC QQWSFNPPT FGAGTKLELK]]> 8173 8911,9206,9207 Mylotarg Gemtuzumab(hP67.6) CD33 <![CDATA[QLVQSGAEVKKPGSSVKVSCKAS GYTITDSNIH WVRQAPGQSLEWIG YIYPYNGGTDYNQKFKN RATLTVDNPTNTAYMELSSLRSEDTDFYYCVN GNPWLAY WGQGTLVTVSS]]> 8080 8957,8958,8959 <![CDATA[DIQLTQSPSTLSASVGDRVTITC RASESLDNYGIRFLT WFQQKPGKAPKLLMY AASNQGS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQTKEVPWS FGQGTKVEVK]]> 8177 9214,9215,9216 Daratumumab CD38 <![CDATA[EVQLLESGGGLVQPGGSLRLSCAVS GFTFNSFA MSWVRQAPGKGLEWVSA ISGSGGGT YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFC AKDKILWFGEPVFDY WGQGTLVTVSS]]> 8081 8960,8961,8962 <![CDATA[EIVLTQSPATLSLSPGERATLSCRAS QSVSSY LAWYQQKPGQAPRLLIY DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPT FGQGTKVEIK]]> 8178 9217,9218,9219 1F6 CD70 <![CDATA[QIQLVQSGPEVKKPGETVKISCKAS GYTFTNYGMN WVKQAPGKGLKWMG WINTYTGEPTYADAFKG RFAFSLETSASTAYLQINNLKNEDTATYFCAR DYGDYGMDY WGQGTSVTVSS]]> 8082 8963,8964,8965 <![CDATA[DIVLTQSPASLAVSLGQRATISC RASKSVSTSGYSFMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATY YC QHSREVPWT FGGGTKLEIK]]> 8179 9220,9221,9222 2F2 CD70 <![CDATA[QVQLQQSGTELMTPGASVTMSCKTS GYTFSTYWIE WVKQRPGHGLEWIG EILGPSGYTDYNEKFKA KATFTADTSSNTAYMQLSSLASEDSAVYYCAR WDRLYAMDY WGGGTSVTVSS]]> 8083 8966,8967,8968 <![CDATA[DIVLTQSPASLTVSLGQKTTISC RASKSVSTSGYSFMH WYQLKPGQSPKLLIY LASDLPS GVPARFSGSGSGTDFTLKIHPVEEEDAATY YC QHSREIPYT FGGGTKLEIT]]> 8180 9220,9223,9224 2H5 CD70 <![CDATA[QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYIMH WVRQAPGKGLEWVA VISYDGRNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAED TAVYYCAR DTDGYDFDY WGQGTLVTVSS]]> 8084 8969,8970,8971 <![CDATA[EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLI YDASNRA TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQ RTNWPLT FGGGTKVEIK]]> 8181 9202,9225,9226 10B4 CD70 <![CDATA[QIQLVESGGGVVQPGRSLRLSCAASGFTFG YYAMH WVRQAPGKGLEWVA VISYDGSIKYYADSVKG RFTISRDNSKNTLYLQMNSLRAED TAVYYCAR EGPYSNYLDY WGQGTLVTVSS]]> 8085 8972,8973,8974 <![CDATA[AIQLTQSPSSLSASVGDRVTITC RASQGISSALA WYQQKPGKAPKFLIY DASSLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQ FNSYPFT FGPGTKVDIK]]> 8182 9227,9228,9229 8B5 CD70 <![CDATA[QVQLVESGGGVVQPGRSLRLSCATSGFTFS DYGMH WVRQAPGKGLEWVA VIWYDGSNKYYADSVKG RFTISRDNSKKTLSLQMNSLRAED TAVYYCAR DSIMVRGDY WGQGTLVTVSS]]> 8086 8975,8976,8977 <![CDATA[DIQMTQSPSSLSASVGDRVTITC RASQGISSWLA WYQQKPEKAPKSLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQ YNSYPLT FGGGTKVEIK]]> 8183 9230,9231,9232 18E7 CD70 <![CDATA[QVQLVESGGGVVQPGRSLRLSCAASGFTFS DHGMH WVRQAPGKGLEWVA VIWYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAED TAVYYCAR DSIMVRGDY WGQGTLVTVSS]]> 8087 8978,8976,8977 <![CDATA[DIQMTQSPSSLSASVGDRVTITC RASQGISSWLA WYQQKPEKAPKSLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQ YNSYPLT FGGGTKVEIK]]> 8183 9230,9231,9232 69A7 CD70 <![CDATA[QVQLQESGPGLVKPSETLSLTCTVSGGSVS SDYYYWS WIRQPPGKGLEWLG YIYYSGSTNYNPSLKS RVTISVDTSKNQFSLKLRSVTTA DTAVYYCARGDGDYGGNCFDYWGQGTLVTVSS]]> 8088 8979,8980,8981 <![CDATA[EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIF DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQ RSNWPLT FGGGTKVEIK]]> 8184 9202,9203,9233 CE-355621 cMET <![CDATA[QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYGFS WVRQAPGQGLEWMG WISASNGNTYYAQKLQG RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR VYADYADY WGQGTLVTVSS]]> 8089 8982,8983,8984 <![CDATA[DIQMTQSPSSVSASVGDRVTITC RASQGINTWLA WYQQKPGKAPKLLIY AASSLKS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQANSFPLT FGGGTKVEIK]]> 8185 9234,9235,9236 LY2875358 emibetuzumab cMET <![CDATA[QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDYY MHWVRQAPGQGLEWMGR VNPNRRGTT YNQKFEGRVTMTTDTSTSTAYMELRSLRSDDTAVYYC ARANWLDY WGQGTTVTVSS]]> 8090 8985,8986,8987 <![CDATA[DIQMTQSPSSLSASVGDRVTITCSVS SSVSSIY LHWYQQKPGKAPKLLIY STS NLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QVYSGYPLT FGGGTKVEIK]]> 8186 9237,9238,9239 MetMAb onartuzumab cMET <![CDATA[EVQLVESGGGLVQPGGSLRLSCAASGYTFT SYWLH WVRQAPGKGLEWVG MIDPSNSDTRFNPNFKD RFTISADTSKNTAYLQMNSLRAEDTAVYYC ATYRSYVTPLDY WGQGTLVTVSS]]> 8091 8988,8989,8990 <![CDATA[DIQMTQSPSSLSASVGDRVTITC KSSQSLLYTSSQKNYLA WYQQKPGKAPKLLIY WASTRES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYYAYPWT FGQGTKVEIK]]> 8187 9240,9241,9242 tremelimumab(CP-675206, or11.2.1) CTLA4 <![CDATA[QVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYGMH WVRQAPGKGLEWVA VIWYDGSNKYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYCAR DPRGATLYYYYYGMDV WGQGTTVTVSS]]> 8092 8991,8992,8993 <![CDATA[DIQMTQSPSSLSASVGDRVTITC RASQSINSYLD WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYYSTPFT FGPGTKVEIK]]> 8188 9243,9231,9244 Yervoy Ipilimumab10D1 CTLA4 <![CDATA[QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYTMH WVRQAPGKGLEWVT FISYDGNNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAIYYCAR TGWLGPFDY WGQGTLVTVSS]]> 8093 8994,8995,8996 <![CDATA[EIVLTQSPGTLSLSPGERATLSC RASQSVGSSYLA WYQQKPGQAPRLLIY GAFSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPWT FGQGTKVEIK]]> 8189 9245,9246,9247 AGS16F H16-7.8 ENPP3 <![CDATA[QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGGYY WSWIRQHPGKGLEWIG IIYYSGSTYYNPSLKS RVTISVDTSKNQFSLKLNSVTAADTAVFYCAR VAIVTTIPGGMDV WGQGTTVTVSS]]> 8094 8997,8998,8999 <![CDATA[EIVLTQSPDFQSVTPKEKVTITC RASQSIGISLH WYQQKPDQSPKLLIK YASQSFS GVPSRFSGSGSGTDFTLTINSLEAEDAATYYC HQSRSFPWT FGQGTKVEIK]]> 8190 9248,9249,9250 MT110 solitomab EpCAM <![CDATA[EVQLLEQSGAELVRPGTSVKISCKASGYAFT NYWLG WVKQRPGHGLEWIG DIFPGSGNIHYNEKFKG KATLTADKSSSTAYMQLSSLTFEDSAVYFCAR LRNWDEPMDY WGQGTTVTVSS]]> 8095 9000,9001,9002 <![CDATA[ELVMTQSPSSLTVTAGEKVTMSC KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLIY WASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNDYSYPLT FGAGTKLEIK]]> 8191 9251,9241,9252 MT201 Adecatumumab EpCAM <![CDATA[EVQLLESGGGVVQPGRSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA VISYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK DMGWGSGWRPYYYYGMDV WGQGTTVTVSS]]> 8096 9003,9004,9005 <![CDATA[ELQMTQSPSSLSASVGDRVTITC RTSQSISSYLN WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFTLTISSLQPEDSATYYC QQSYDIPYT FGQGTKLEIK]]> 8192 9253,9241,9254 Panorex EdrecolomabMab CO17-1A EpCAM <![CDATA[QVQLQQSGAELVRPGTSVKVSCKAS GYAFTNYL IEWVKQRPGQGLEWIGV INPGSGGT NYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFC ARDGPWFAY WGQGTLVTVSA]]> 8097 9006,9007,9008 <![CDATA[NIVMTQSPKSMSMSVGERVTLTCKAS ENVVTY VSWYQQKPEQSPKLLIY GAS NRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHC GQGYSYPYT FGGGTKLEIK]]> 8193 9255,9256,9257 tucotuzumab EpCAM <![CDATA[QIQLVQSGPELKKPGETVKISCKAS GYTFTNYGMN WVRQAPGKGLKWMG WINTYTGEPTYAD DFKGRFVFSLETSASTAFLQLNNLRSEDTATYFCVRFI SKGDY WGQGTSVTVSS]]> 8098 8963,9009,9010 <![CDATA[QILLTQSPAIMSASPGEKVTMTC SASSSVS YMLWYQQKPGSSPKPWIF DTSNLAS GFPARFSGSGSGTSYSLIISSMEAEDAATYYC HQRSGYPYT FGGGTCLAIK]]> 8194 9258,9259,9260 UBS-54 EpCAM <![CDATA[VQLQQSDAELVKPGASVKISCKAS GYTFTDHAIH WVKQNPEQGLEWIG YFSPGNDDFKYNE RFKGKATLTADKSSSTAYVQLNSLTSEDSAVYFCTR SLNMAY WGQGTSVTVSS]]> 8099 9011,9012,9013 <![CDATA[DIVMTQSPDSLAVSLGERATINC KSSQSVLYSSNNKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSYPLT FGGGTKVKES]]> 8195 9261,9241,9262 3622W94 323 / A3 EpCAM <![CDATA[EVQLVQSGPEVKKPGASVKVSCKAS GYTFTNYGMN WVRQAPGQGLEWMG WINTYTGEPTYGE DFKGRFAFSLDTSASTAYMELSSLRSEDTAVYFCARFG NYVDY WGQGSLVTVSS]]> 8100 8963,9014,9015 <![CDATA[DIVMTQSPLSLPVTPGEPASISC RSSINKKGSNGITY LYWYLQKPGQSPQLLIYQMSNLASGVPDRF SGSGS GTDFTLKISRVEAEDVGVYYC AQNLEIPRT FGQGTKVEIK]]> 8196 9263,9264,9265 4D5MOCBv2 EpCAM <![CDATA[EVQLVQSGPGLVQPGGSVRISCAASGYTFT NYGMN WVKQAPGKGLEWMG WINTYTGESTYADSFKG RFTFSLDTSASAAYLQINSLRAEDTAVYYCAR FAIKGD YWGQGTLLTVSS]]> 8101 9016,9017,9018 <![CDATA[DIQMTQSPSSLSASVGDRVTITC RSTKSLLHSNGITYLY WYQQKPGKAPKLLIY QMSNLAS GVPSRFSSSGSGTDFTLTISSLQPEDFATYYC AQNLEIPRT FGQGTKVEIK]]> 8197 9266,9267,9265 4D5MOCB EpCAM <![CDATA[EVQLVQSGPGLVQPGGSVRISCAASGYTFT NYGMN WVKQAPGKGLEWMG WINTYTGESTYADSFKG RFTFSLDTSASAAYLQINSLRAEDTAVYYCAR FAIKGDY WGQGTLLTVSS]]> 8101 9016,9017,9019 <![CDATA[DIQMTQSPSSLSASVGDRVTITC RSTKSLLHSNGITYLY WYQQKPGKAPKLLIY QMSNLAS GVPSRFSSSGSGTDFTLTISSLQPEDFATYYC AQNLEIPRT FGQGTKVELK]]> 8198 9266,9267,9265 MEDI-547 1C1 EphA2 <![CDATA[EVQLLESGGGLVQPGGSLRLSCAASGFTFS HYMMA WVRQAPGKGLEWVS RIGPSGGPTHYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGYDSG YDYVAVAGPAEYFQH WGQGTLVTVSS]]> 8102 9020,9021,9022 <![CDATA[DIQMTQSPSSLSASVGDRVTITC RASQSISTWLA WYQQKPGKAPKLLIY KASNLHT GVPSRFSGSGSGTEFSLTISGLQPDDFATYYC QQYNSYSRT FGQGTKVEIK]]> 8199 9268,9269,9270 MORAb-003 farletuzumab FOLR1 <![CDATA[EVQLVESGGGVVQPGRSLRLSCSAS GFTFSGYG LSWVRQAPGKGLEWVAM ISSGGSYT YYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFC ARHGDDPAWF AYWGQGTPVTVSS]]> 8103 9023,9024,9025 <![CDATA[DIQLTQSPSSLSASVGDRVTITCSVS SSISSNN LHWYQQKPGKAPKPWIY GTS NLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYC QQWSSYPYMYT FGQGTKVEIK]]> 8200 9271,9272,9273 M9346A huMOV19(vLCv1.00) FOLR1 <![CDATA[QVQLVQSGAEVVKPGASVKISCKASGYTFT GYFMN WVKQSPGQSLEWIG RIHPYDGDTFYNQKFQG KATLTVDKSSNTAHMELLSLTSEDFAVYYCTR YDGSRAMDY WGQGTTVTVSS]]> 8104 9026,9027,9028 <![CDATA[DIVLTQSPLSLAVSLGQPAIISC KASQSVSFAGTSLMH WYHQKPGQQPRLLIY RASNLEA GVPDRFSGSGSKTDFTLNISPVEAEDAATYYC QQSREYPYT FGGGTKLEIK]]> 8201 9274,9275,9276 M9346A huMOV19(vLCv1.60) FOLR1 <![CDATA[QVQLVQSGAEVVKPGASVKISCKASGYTFT GYFMN WVKQSPGQSLEWIG RIHPYDGDTFYNQKFQG KATLTVDKSSNTAHMELLSLTSEDFAVYYCTR YDGSRAMD YWGQGTTVTVSS]]> 8105 9026,9027,9029 <![CDATA[DIVLTQSPLSLAVSLGQPAIISC KASQSVSFAGTSLMH WYHQKPGQQPRLLIY RASNLEA GVPDRFSGSGSKTDFTLTISPVEAEDAATYYC QQSREYPYT FGGGTKLEIM]]> 8202 9274,9275,9276 26B3.F2 FOLR1 <![CDATA[GPELVKPGASVKISCKASDYSFT GYFMN WVMQSHGKSLEWIG R IFPYNGDTFYNQKFKG RATLTVDKSSSTAHMELRSLASEDSAVYFCAR GTHYFDY WGQGTTLTVSS]]> 8106 9026,9030,9031 <![CDATA[PASLSASVGETVTITC RTSENIFSYLA WYQQKQGISPQLLVY NAKTLAE GVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC QHHYAFPWT FGGGSKLEIK]]> 8203 9277,9278,9279 RG7686 GC33 GPC3 <![CDATA[QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYEMH WVRQAPGQGLEWMG ALDPKTGDTAYSQKFKG RVTLTADKSTSTAYMELSSLTSED TAVYYCTR FYSYTY WGQGTLVTVSS]]> 8107 9032,9033,9034 <![CDATA[DVVMTQSPLSLPVTPGEPASISC RSSQSLVHSNGNTYLH WYLQKPGQSPQLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGV YYC SQNTHVPPT FGQGTKLEIK]]> 8204 9280,9281,9282 4A6 GPC3 <![CDATA[EVQLVQSGAEVKKPGESLKISCKGSGYSFT SYWIA WVRQMPGKGLEWMG IIFPGDSDTRYSPSFQG QVTISADRSIRTAYLQWSSLKASD TALYYCAR TREGYFDY WGQGTLVTVSS]]> 8108 9035,9036,9037 <![CDATA[EIVLTQSPGTLSLSPGERATLSC RAVQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC Q QYGSSPT FGGGTKVEIK]]> 8205 9283,9182,9284 11E7 GPC3 <![CDATA[EVQLVQSGAEVKKPGESLKISCKGSGYSFT NYWIA WVRQMPGKGLEWMG IIYPGDSDTRYSPSFQG QVTISADKSIRTAYLQWSSLKASD TAMYYCAR TREGYFDY WGQGTLVTVSS]]> 8109 9038,9039,9037 <![CDATA[EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC Q QYGSSPT FGGGTKVEIK]]> 8206 9285,9182,9284 16D10 GPC3 <![CDATA[EVQLVQSGADVTKPGESLKISCKVSGYRFT NYWIG WMRQMSGKGLEWMG IIYPGDSDTRYSPSFQG HVTISADKSINTAYLRWSSLKASD TAIYYCAR TREGFFDY WGQGTPVTVSS]]> 8110 9040,9039,9041 <![CDATA[EILLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPT FGQGTKVEIK]]> 8207 9285,9182,9284 AMG-595 HER1(EGFR) <![CDATA[QVQLVESGGGVVQSGRSLRLSCAAS GFTFRNYGMH WVRQAPGKGLEW VAVIWYDGSDKYYADSVRG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGY DILTGNPRDFDY WGQGTLVTVSS]]> 8111 9042,9043,9044 <![CDATA[DTVMTQTPLSSHVTLGQPASISC RSSQSLVHSDGNTYLS WLQQRPGQPPRLLIY RISRRFS GVPDRFSGSGAGTDFTLEISRVEAEDVGVYYC MQSTHVPRT FGQGTKVEIK]]> 8208 9286,9287,9288 Erubitux™ cetutximab HER1(EGFR) <![CDATA[QVQLKQSGPGLVQPSQSLSITCTVS GFSLTNYG VHWVRQSPGKGLEWLGV IWSGGNT DYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYC ARALTYYDYEFAY WGQGTLVTVSA]]> 8112 9045,9046,9047 <![CDATA[DILLTQSPVILSVSPGERVSFSCRAS QSIGTN IHWYQQRTNGSPRLLIK YAS ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK]]> 8209 9289,9290,9291 GA201 Imgatuzumab HER1(EGFR) <![CDATA[QVQLVQSGAEVKKPGSSVKVSCKASGFTFT DYKIH WVRQAPGQGLEWMG YFNPNSGYSTYAQKFQG RVTITADKSTSTAYMELSSLRSEDTAVYYCAR LSPGGYYVMDA WGQGTTVTVSS]]> 8113 9048,9049,9050 <![CDATA[DIQMTQSPSSLSASVGDRVTITC RASQGINNYLN WYQQKPGKAPKRLIY NTNNLQT GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LQHNSFPT FGQGTKLEIK]]> 8210 9292,9293,9294 Humax zalutumumab HER1(EGFR) <![CDATA[QVQLVESGGGVVQPGRSLRLSCAASGFTFS TYGMH WVRQAPGKGLEWVA VIWDDGSYKYYGDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYCAR DGITMVRGVMKDYFDY WGQGTLVTVSS]]> 8114 9051,9052,9053 <![CDATA[AIQLTQSPSSLSASVGDRVTITC RASQDISSALV WYQQKPGKAPKLLIY DASSLES GVPSRFSGSESGTDFTLTISSLQPEDFATYYC QQFNSYPLT FGGGTKVEIK]]> 8211 9295,9228,9296 IMC-11F8 necitumumab HER1(EGFR) <![CDATA[QVQLQESGPGLVKPSQTLSLTCTVSGGSIS SGDYYWS WIRQPPGKGLEWIG YIYYSGSTDYNPSLKS RVTMSVDTSKNQFSLKVNSVTAADTAVYYCAR VSIFGVGTFDY WGQGTLVTVSS]]> 8115 9054,9055,9056 <![CDATA[EIVMTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC HQYGSTPLT FGGGTKAEIK]]> 8212 9202,9203,9297 MM-151 P1X HER1(EGFR) <![CDATA[QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG SIIPIFGTVNYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYCAR DPSVNLYWYFDL WGRGTLVTVSS]]> 8116 9057,9058,9059 <![CDATA[DIQMTQSPSTLSASVGDRVTITC RASQSISSWWA WYQQKPGKAPKLLIY DASSLES GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYHAHPTT FGGGTKVEIK]]> 8213 9298,9228,9299 MM-151 P2X HER1(EGFR) <![CDATA[QVQLVQSGAEVKKPGSSVKVSCKASGGTFG SYAIS WVRQAPGQGLEWMG SIIPIFGAANPAQKSQG RVTITADESTSTAYMELSSLRSEDTAVYYCAK MGRGKVAFDI WGQGTMVTVSS]]> 8117 9057,9060,9061 <![CDATA[DIVMTQSPDSLAVSLGERATINC KSSQSVLYSPNNKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYGSPIT FGGGTKVEIK]]> 8214 9300,9241,9301 MM-151 P3X HER1(EGFR) <![CDATA[QVQLVQSGAEVKKPGASVKVSCKASGYAFT SYGIN WVRQAPGQGLEWMG WISAYNGNTYYAQKLRG RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR DLGGYGSGSVPFDP WGQGTLVTVSS]]> 8118 9062,9063,9064 <![CDATA[EIVMTQSPATLSVSPGERATLSC RASQSVSSNLA WYQQKPGQAPRLLIY GASTRAT GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QDYRTWPRRV FGGGTKVEIK]]> 8215 9302,9303,9304 TheraCIM nimotuzumab HER1(EGFR) <![CDATA[QVQLQQSGAEVKKPGSSVKVSCKASGYTFT NYYIY WVRQAPGQGLEWIG GINPTSGGSNFNEKFKT RVTITADESSTTAYMELSSLRSEDTAFYFCTR QGLWFDSDSDGRGFDF WGQGTTVTVSS]]> 8119 9065,9066,9067 <![CDATA[DIQMTQSPSSLSASVGDRVTITC RSSQNIVHSNGNTYLD WYQQTPGKAPKLLIY KVSNRFS GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC FQYSHVPWT FGQGTKLQIT]]> 8216 9305,9281,9306 Vectibix™ panitumimab HER1(EGFR) <![CDATA[QVQLQESGPGLVKPSETLSLTCTVS GGSVSSGDYY WTWIRQSPGKGLEWIGH IYYSGNT NYNPSLKSRLTISIDTSKTQFSLKLSSVTAADTAIYYC VRDRVTGAFDI WGQGTMVTVSS]]> 8120 9068,9069,9070 <![CDATA[DIQMTQSPSSLSASVGDRVTITCQAS QDISNY LNWYQQKPGKAPKLLIY DAS NLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFC QHFDHLPLA FGGGTKVEIK]]> 8217 9307,9218,9308 07D06 HER1(EGFR) <![CDATA[QIQLVQSGPELKKPGETVKISCKAS GYTFTEYP IHWVKQAPGKGFKWMGM IYTDIGKP TYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFC VRDRYDSLFDY WGQGTTLTVSS]]> 8121 9071,9072,9073 <![CDATA[DVVMTQTPLSLPVSLGDQASISCRSS QSLVHSNGNTY LHWYLQKPGQSPKLLIY KVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVPWT FGGGTKLEIK]]> 8218 9309,9310,9311 12D03 HER1(EGFR) <![CDATA[EMQLVESGGGFVKPGGSLKLSCAASGFAFS HYDMS WVRQTPKQRLEWVA YIASGGDITYYADTVKG RFTISRDNAQNTLYLQMSSLKSEDTAMFYCSR SSYGNNGDALDF WGQGTSVTVSS]]> 8122 9074,9075,9076 <![CDATA[DVVMTQTPLSLPVSLGDQASISC RSSQSLVHSNGNTYLH WYLQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQSTHVLT FGSGTKLEIK]]> 8219 9280,9281,9312 C1 HER2 <![CDATA[QVQLVESGGGLVQPGGSLRLSCAASGFTFS SYAMG WVRQAPGKGLEWVS SISGSSRYIYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK MDASGSYFNF WGQGTLVTVSS]]> 8123 9077,9078,9079 <![CDATA[QSPSFLSAFVGDRITITC RASPGIRNYLA WYQQKPGKAPKLLIY AASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNSYPLS FGGGTKVEIK]]> 8220 9313,9314,9315 Erbicin HER2 <![CDATA[QVQLLQSAAEVKKPGESLKISCKGSGYSFT SYWIG WVRQMPGKGLEWMG IIYPGDSDTRYSPSFQG QVTISADKSISTAYLQWSSLKASDTAVYYCAR WRDSPL WGQGTLVTVSS]]> 8124 9080,9039,9081 <![CDATA[QAVVTQEPSFSVSPGGTVTLTC GLSSGSVSTSYYPS WYQQTPGQAPRTLIY STNTRSS GVPDRFSGSILGNKAALTITGAQADDESDYYC VLYMGSGQYV FGGGTKLTVL]]> 8221 9316,9317,9318 Herceptin trastuzumab HER2 <![CDATA[EVQLVESGGGLVQPGGSLRLSCAAS GFNIKDTY IHWVRQAPG...

Claims

1. A polypeptide comprising an extended recombinant polypeptide (XTEN), comprising: (a) Extended recombinant polypeptide (XTEN), which contains (i) a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif in the set is repeated at least twice in the XTEN peptide; and (ii) An additional non-overlapping sequence motif that appears only once within the XTEN polypeptide; and (b) A first barcode fragment that can be released from the polypeptide after partial or complete digestion by the protease, wherein the first barcode fragment is part of the XTEN polypeptide, which is contained within the XTEN polypeptide and is different in sequence and molecular weight from the sequence motif of all other peptide fragments that can be released from the polypeptide after complete digestion by the protease. The barcode fragments do not include the N-terminal or C-terminal amino acids of the polypeptide.

2. The polypeptide of claim 1, wherein the barcode fragment does not include glutamic acid immediately adjacent to another glutamic acid in the XTEN polypeptide.

3. The polypeptide according to any one of claims 1–2, wherein the barcode fragment has a glutamic acid at its C-terminus.

4. The polypeptide according to any one of claims 1–3, wherein the barcode fragment has an N-terminal amino acid preceding a glutamic acid residue.

5. The polypeptide of claim 4, wherein the glutamic acid residue preceding the N-terminal amino acid is not immediately adjacent to another glutamic acid residue.

6. The polypeptide according to any one of claims 1–4, wherein the barcode fragment does not include glutamic acid residues at positions other than the C-terminus of the barcode fragment, unless the glutamic acid is immediately followed by proline.

7. The polypeptide according to claims 1–6, wherein the barcode fragment is located 10 to 150 amino acids from the N-terminus or C-terminus of the polypeptide.

8. The polypeptide according to any one of claims 1–7, wherein the sequence motifs of the set of non-overlapping sequence motifs are identified by SEQ ID NO: 182-203 and 1715-1722.

9. The polypeptide according to claim 8, wherein the sequence motif of the set of non-overlapping sequence motifs is identified by SEQ ID NO: 186-189.

10. The polypeptide of claim 9, wherein the set of non-overlapping sequence motifs comprises at least two, at least three, or all four of the sequence motifs identified by SEQ ID NO:186-189.

Images