Her-2 targeting bispecific compositions and methods for making and using the same

Abstract

To provide a bispecific T cell engager (TCE) cancer therapeutic agent having an increased therapeutic index.SOLUTION: (a) an extended recombinant polypeptide (XTEN), wherein the XTEN comprises a barcode fragment (BAR) that is releasable from the polypeptide upon digestion by a protease; And (c) a polypeptide having N - and C-terminal amino acids comprising a release segment (RS) located between the XTEN and the diabody construct, wherein the diabody construct specifically binds to CD3 and comprises CDR - L1, CDR - L2 and CDR - L3, and CDR - H1, CDR - H2 and CDR - H3, wherein the CDR - H3 comprises a specific amino acid sequence, wherein the first antigen-binding fragment (AF1) specifically binds to human epidermal growth factor-receptor 2 (). HER2 AF2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing Description: A computer-readable sequence listing has been submitted with this application by electronic filing and is incorporated herein by reference in its entirety. The sequence listing is contained in a file with the file name "789-601_20-1832-WO_ST25_FINAL.txt", created on June 14, 2021, and is 1502kb in size.

[0002] References This application is a continuation of U.S. Provisional Patent Application No. 63 / 044,301, filed June 25, 2020, entitled "BARCODED BISPECIFIC COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME," U.S. Provisional Patent Application No. 63 / 077,503, filed September 11, 2020, entitled "BARCODED BISPECIFIC COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME," U.S. Provisional Patent Application No. 63 / 108,783, filed November 2, 2020, entitled "HER2 TARGETED BISPECIFIC COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME," and U.S. Provisional Patent Application No. 63 / 108,783, filed November 2, 2020, entitled "HER2 TARGETED BISPECIFIC COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME." This application claims priority to U.S. Provisional Patent Application No. 63 / 166,857, filed March 26, 2021, entitled "HER2 TARGETED BISPECIFIC COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME," and U.S. Provisional Patent Application No. 63 / 196,408, filed June 3, 2021, entitled "HER2 TARGETED BISPECIFIC COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME," all of which are incorporated herein in their entireties. [Background technology]

[0003] background Bispecific T cell engagers (TCEs) represent a highly potent modality for cancer therapy, redirecting T cell cytotoxicity against tumors expressing selected tumor-associated antigens, bypassing the requirement for tumor antigen recognition by T cells. The activity of TCEs depends on their ability to activate T cells by effectively stimulating T cell receptors (TCRs). Their exceptional potency stems from the minimal requirement for initiating cytotoxicity: only three TCRs are stimulated and coalesce to form an immune synapse between the T cell and the target cell. In addition to their induction of cytotoxicity, their potency also involves cytokine-driven actions downstream of T cell activation, which enhance and amplify antitumor immune responses. Thus, TCEs hold promise for immunotherapy in patients whose tumors harbor insufficient mutations or otherwise escape immune surveillance. However, this modality is not without its own challenges, and the use of TCEs in solid tumors is limited by their exceptional potency in healthy tissues and on-target and off-tumor toxicity.

[0004] Although TCEs have been highly effective in inducing remission in patients with hematologic cancers, their use in solid tumors is limited by their extreme potency and on-target toxicity against normal tissues that express the target, even at low levels. Rare but impressive clinical responses have been observed in tumors that typically resist immunotherapy (e.g., microsatellite-stable colorectal and prostate cancers), but toxicities such as cytokine release syndrome at low doses have prevented dose escalation to demonstrate the clinical potential of this modality. The induction of grade 4 cytokine release syndrome in a patient treated with Ichnos ISB 1302 HER2 TCE at a dose of <1 μg / kg highlights the challenges faced by TCEs, even when directed against relatively tissue-restricted targets.

[0005] Clinical trials with blinatumomab (an approved CD3xCD19 bispecific antibody) revealed cytokine release syndrome (CRS) as one of the major safety-related adverse events. CRS and on-target toxicity at low drug doses significantly compromise the therapeutic index and potential of TCE modalities for solid tumors in the clinic. For example, a clinical trial with catumaxomab (CD3xEpCAM) was terminated at the 10 μg dose due to drug-induced liver failure. In another trial, a HER2-targeted TCE (Glenmark GBR1302), the drug dose was limited to less than 1 μg / kg due to the onset of G4 CRS. Pasotuxumab (a PSMA-targeted TCE) demonstrated favorable responses but was hampered by CRS at doses greater than 40 μg / day. The literature is replete with other examples of CRS and on-target toxicity challenges presented by TCEs.

[0006] Attempts to circumvent CRS have involved complex molecular design, but these have failed due to toxicity and / or enhanced immunogenicity. This presents a significant unmet need for new strategies that can overcome the therapeutic index challenge in solid tumors. If the potency of TCEs could be harnessed and the challenges of CRS and on-target toxicity could be controlled, it would be possible to generate powerful therapies that could potentially be used against a wide range of cancers.

[0007] Therapeutic agents, such as drug substances, contain polypeptides, and these polypeptides can be produced in ways that result in mixtures of polypeptides that can affect the activity of the drug substance. Mixtures of polypeptides often contain full-length polypeptides along with their size variants (e.g., truncated forms). The presence of variants that differ in size from the desired full-length product can affect the biological behavior of the drug substance and, therefore, the safety and / or efficacy of the polypeptide drug substance. For example, protein-based prodrugs for cancer therapy can be engineered with tumor-targeting activation mechanisms. More specifically, while a full-length therapeutic protein can be in an inactivated (non-cytotoxic) prodrug form, truncated variants of the full-length construct can lack protective sequences and become cytotoxic (active), thus "contaminating" the prodrug composition. In some cases, such shorter-length variants may pose a greater immunogenicity risk, have less selective toxicity to tumor cells, or exhibit a less desirable pharmacokinetic profile (e.g., resulting in a narrower therapeutic window) compared to the full-length protein. As a result, detecting and quantifying variations in protein structure can be important for evaluating the biological properties (e.g., clinical safety and pharmacological efficacy) of biopharmaceuticals and for developing new biopharmaceuticals (e.g., with increased efficacy and reduced side effects). Existing techniques and methods for identifying and quantifying the amount of "contaminating" truncation products can include one or more drawbacks, such as limited sensitivity, ease, efficiency, or effectiveness. Summary of the Invention [Means for solving the problem]

[0008] overview The present invention addresses a long-standing unmet need to provide TCE cancer therapeutics with an increased therapeutic index. In doing so, the present invention harnesses the therapeutic potential of TCEs by providing Xtenylated protease-activating bispecific T cell engagers (XPATs). XPATs represent a novel strategy for improving the toxicity profile of T cell engagers while maintaining their potency against solid tumors, thereby enabling a significant increase in the therapeutic index and an expansion of the target landscape of this powerful modality. In certain specific embodiments, the XPATs of the present invention target HER2-bearing tumors. More specifically, AMX-818 is a HER2-targeted, conditionally activated prodrug TCE designed to exploit dysregulated protease activity in tumors at the expense of healthy tissue where protease inhibition is effective, thereby expanding the safety margin and therapeutic index.

[0009] a polypeptide having an N-terminal amino acid and a C-terminal amino acid, the polypeptide comprising: (a) an extended recombinant polypeptide (XTEN), the XTEN comprising a barcode fragment (BAR) releasable from the polypeptide upon digestion with a protease; (b) a bispecific antibody construct (BsAb) that specifically binds to Cluster of Differentiation 3 T-cell receptor (CD3) and comprises light chain complementarity determining regions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3) and heavy chain complementarity determining regions 1 (CDR-H1), 2 (CDR-H2), and 3 (CDR-H3); and (c) a release segment (RS) located between the XTEN and the bispecific antibody construct, wherein the XTEN (i) comprises at least 100, or at least 150 amino acids, and (ii) at least 90% of its amino acid residues are selected from the group consisting of glycine (G), glycine (A), glycine (B), glycine (C), glycine (G), glycine (H), glycine (H), glycine (I), glycine (IJ ... (iii) the amino acids are lanine (A), serine (S), threonine (T), glutamate (E), or proline (P); (iii) the XTEN comprises at least four different amino acids that are G, A, S, T, E, or P; and (iv) the XTEN is formed from a plurality of non-overlapping sequence motifs, each of which is 9 to 14 amino acids in length; the plurality of non-overlapping sequence motifs comprises: (1) a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least twice in the XTEN; and (2) only non-overlapping sequence motifs that occur only once within the XTEN; the barcode fragment (BAR) comprises at least a portion of the non-overlapping sequence motif that occurs only once within the XTEN; and the barcode fragment (BAR) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease;Provided herein is a polypeptide, wherein the barcode fragment (BAR) does not include the N-terminal amino acid or the C-terminal amino acid of the polypeptide;

[0010] In certain embodiments, the set of non-overlapping sequence motifs each independently comprise an amino acid sequence identified herein by SEQ ID NOs: 179-200 and 1715-1722. In certain embodiments, the set of non-overlapping sequence motifs each independently comprise an amino acid sequence identified herein by SEQ ID NOs: 186-189. In certain embodiments, the set of non-overlapping sequence motifs comprises at least two, at least three, or all four of the sequence motifs SEQ ID NOs: 186-189. In certain embodiments, the XTEN comprises a length of 100-3,000, 150-3,000, 100-1,000, or 150-1,000 amino acid residues. In certain embodiments, the XTEN comprises 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 acid residues. In certain embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P).

[0011] In certain embodiments, the XTEN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a sequence set forth in Table 3a. In certain embodiments, the barcode fragment (BAR) does not contain a glutamic acid, if present, immediately adjacent to another glutamic acid in the XTEN. In certain embodiments, the barcode fragment (BAR) has a glutamic acid at its C-terminus. In certain embodiments, the barcode fragment (BAR) has an N-terminal amino acid immediately preceded by a glutamic acid residue. In certain embodiments, the barcode fragment (BAR) is located a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10 to 150 amino acids in length, or 10 to 125 amino acids in length. In certain embodiments, the barcode fragment (BAR) is characterized in that (i) it does not contain a glutamic acid, if present, that is immediately adjacent to another glutamic acid in the XTEN; (ii) it has a glutamic acid at its C-terminus; (iii) it has an N-terminal amino acid that is immediately preceded by a glutamic acid residue; and (iv) it is located a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10 to 150 amino acids in length, or 10 to 125 amino acids in length. In certain embodiments, the glutamic acid residue that precedes the N-terminal amino acid of the barcode fragment (BAR) is not immediately adjacent to another glutamic acid residue.

[0012] In certain embodiments, the barcode fragment (BAR) does not include a second glutamic acid residue at a position other than the C-terminus of the barcode fragment, unless the second glutamic acid is immediately followed by a proline. In certain embodiments, the XTEN is located at the N-terminus of the bispecific antibody construct (BsAb), and the barcode fragment (BAR) is located within 200 amino acids, 150 amino acids, 100 amino acids, or 50 amino acids of the N-terminus of the polypeptide. In certain embodiments, the XTEN is located at the N-terminus of the bispecific antibody construct (BsAb), and the barcode fragment (BAR1) is located between 10 and 200 amino acids, between 30 and 200 amino acids, between 40 and 150 amino acids, or between 50 and 100 amino acids from the N-terminus of the protein. In certain embodiments, the XTEN is located at the C-terminus of the bispecific antibody construct (BsAb), and the barcode fragment (BAR) is located within 200 amino acids, 150 amino acids, 100 amino acids, or 50 amino acids of the C-terminus of the polypeptide. In certain embodiments, the XTEN is located at the C-terminus of the bispecific antibody construct (BsAb), and the barcode fragment (BAR) is located between 10 and 200 amino acids, between 30 and 200 amino acids, between 40 and 150 amino acids, or between 50 and 100 amino acids from the C-terminus of the protein. In certain embodiments, the barcode fragment (BAR) is at least 4 amino acids in length. In certain embodiments, the barcode fragment (BAR) is between 4 and 20 amino acids, between 5 and 15 amino acids, between 6 and 12 amino acids, or between 7 and 10 amino acids in length.

[0013] In certain embodiments, the barcode fragment (BAR) comprises an amino acid sequence set forth in Table 2. In certain embodiments, the XTEN has a length defined by a proximal end and a distal end, where (1) the proximal end is located closer to the bispecific antibody construct (BsAb) than the distal end, and (2) the barcode fragment (BAR) is located within a region of the XTEN spanning between 5% and 50%, between 7% and 40%, or between 10% and 30% of the length of the XTEN, measured from the distal end. In certain embodiments, the XTEN further comprises one or more additional barcode fragments, each of which differs in sequence and molecular weight from all other peptide fragments releasable from the polypeptide upon complete digestion of the polypeptide by the protease. In certain embodiments, the release segment (RS) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 7001-7626. In certain embodiments, the protease cleaves C-terminally to glutamic acid residues that are not followed by proline. In certain embodiments, the protease is a Glu-C protease.

[0014] In certain embodiments, the polypeptide is expressed as a fusion protein, and the fusion protein, in its uncleaved state, has the following structural configuration from N- to C-terminus: AF1-AF2-RS-XTEN, AF2-AF1-RS-XTEN, XTEN-RS-AF1-AF2, or XTEN-RS-AF2-AF1. In certain embodiments, the release segment (RS) is fused to the bispecific antibody construct (BsAb) via a spacer. In certain embodiments, the spacer comprises at least four amino acids, which may be glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). In certain embodiments, the spacer comprises an amino acid sequence having at least 80%, 90%, or 100% sequence identity to a sequence set forth in Table C. In certain embodiments, the CDR-H1 and the CDR-H2 of the first antigen-binding fragment (AF1) comprise the amino acid sequences of SEQ ID NOs: 8 and 9, respectively.

[0015] In certain embodiments, the CDR-L1 of AF1 comprises the amino acid sequence of SEQ ID NO: 1 or 2, the CDR-L2 of AF1 comprises the amino acid sequence of SEQ ID NO: 4 or 5, and the CDR-L3 of AF1 comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the CDR-L1 of AF1 comprises the amino acid sequence of SEQ ID NO: 1, the CDR-L2 of AF1 comprises the amino acid sequence of SEQ ID NO: 4 or 5, and the CDR-L3 of AF1 comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the CDR-L1 of AF1 comprises the amino acid sequence of SEQ ID NO: 2, the CDR-L2 of AF1 comprises the amino acid sequence of SEQ ID NO: 4 or 5, and the CDR-L3 of AF1 comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the CDR-L1 of the AF1 comprises the amino acid sequence of SEQ ID NO: 1, the CDR-L2 of the AF1 comprises the amino acid sequence of SEQ ID NO: 4, and the CDR-L3 of the AF1 comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the CDR-L1 of the AF1 comprises the amino acid sequence of SEQ ID NO: 2, the CDR-L2 of the AF1 comprises the amino acid sequence of SEQ ID NO: 5, and the CDR-L3 of the AF1 comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the first antigen-binding fragment (AF1) comprises four chain variable domain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4), each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 60, 64, 65, and 67, respectively. In certain embodiments, the first antigen-binding fragment (AF1) comprises four chain variable domain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4), each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 61, 64, 65, and 67, respectively.In certain embodiments, the first antigen-binding fragment further comprises four light chain variable domain framework regions (FR-L): FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 51, 52, 53, and 59, respectively. In certain embodiments, the first antigen-binding fragment further comprises four light chain variable domain framework regions (FR-L): FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 51, 52, 54, and 59, respectively.

[0016] In certain embodiments, the first antigen-binding fragment further comprises four light chain variable domain framework regions (FR-L): FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 51, 52, 55, and 59, respectively. In certain embodiments, the first antigen-binding fragment further comprises four light chain variable domain framework regions (FR-L): FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 51, 52, 56, and 59, respectively. In certain embodiments, the first antigen-binding fragment (AF1) further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4), wherein the FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, the FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, the FR-L3 comprises the amino acid sequence of SEQ ID NO: 53, 54, 55, or 56, the FR-L4 comprises the amino acid sequence of SEQ ID NO: 59, the FR-H1 comprises the amino acid sequence of SEQ ID NO: 60 or 61, the FR-H2 comprises the amino acid sequence of SEQ ID NO: 64, the FR-H3 comprises the amino acid sequence of SEQ ID NO: 65, and the FR-H4 comprises the amino acid sequence of SEQ ID NO: 67.

[0017] In certain embodiments, the first antigen-binding fragment (AF1) further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4), wherein the FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, the FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, the FR-L3 comprises the amino acid sequence of SEQ ID NO: 53, the FR-L4 comprises the amino acid sequence of SEQ ID NO: 59, the FR-H1 comprises the amino acid sequence of SEQ ID NO: 60, the FR-H2 comprises the amino acid sequence of SEQ ID NO: 64, the FR-H3 comprises the amino acid sequence of SEQ ID NO: 65, and the FR-H4 comprises the amino acid sequence of SEQ ID NO: 67.

[0018] In certain embodiments, the first antigen-binding fragment (AF1) further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4), wherein the FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, the FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, the FR-L3 comprises the amino acid sequence of SEQ ID NO: 54, the FR-L4 comprises the amino acid sequence of SEQ ID NO: 59, the FR-H1 comprises the amino acid sequence of SEQ ID NO: 61, the FR-H2 comprises the amino acid sequence of SEQ ID NO: 64, the FR-H3 comprises the amino acid sequence of SEQ ID NO: 65, and the FR-H4 comprises the amino acid sequence of SEQ ID NO: 67.

[0019] In certain embodiments, the first antigen-binding fragment (AF1) further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4), wherein the FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, the FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, the FR-L3 comprises the amino acid sequence of SEQ ID NO: 55, the FR-L4 comprises the amino acid sequence of SEQ ID NO: 59, the FR-H1 comprises the amino acid sequence of SEQ ID NO: 61, the FR-H2 comprises the amino acid sequence of SEQ ID NO: 64, the FR-H3 comprises the amino acid sequence of SEQ ID NO: 65, and the FR-H4 comprises the amino acid sequence of SEQ ID NO: 67.

[0020] In certain embodiments, the first antigen-binding fragment (AF1) further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4), wherein the FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, the FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, the FR-L3 comprises the amino acid sequence of SEQ ID NO: 56, the FR-L4 comprises the amino acid sequence of SEQ ID NO: 59, the FR-H1 comprises the amino acid sequence of SEQ ID NO: 61, the FR-H2 comprises the amino acid sequence of SEQ ID NO: 64, the FR-H3 comprises the amino acid sequence of SEQ ID NO: 65, and the FR-H4 comprises the amino acid sequence of SEQ ID NO: 67.

[0021] In certain embodiments, the first antigen-binding fragment (AF1) has a higher melting temperature (T) than the melting temperature of the anti-CD3 binding fragment of the first antigen-binding fragment in an in vitro assay. m or the T of a control bispecific antigen-binding construct of the test bispecific antigen-binding construct when the first antigen-binding fragment is incorporated into the test bispecific antigen-binding construct. m High T compared to mand the test bispecific antigen-binding construct comprises the first antigen-binding fragment and a reference antigen-binding fragment that binds to an antigen other than CD3, and the control bispecific antigen-binding construct consists of the anti-CD3 binding fragment consisting of the sequence of SEQ ID NO: 206 and the reference antigen-binding fragment. In certain embodiments, the T of the first antigen-binding fragment m is the T of the anti-CD3 binding fragment consisting of the sequence of SEQ ID NO: 206 m In certain embodiments, the first antigen-binding fragment (AF1) comprises a heavy chain variable region (VH I ) wherein said VH I comprises an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 102 or 105. In certain embodiments, the first antigen-binding fragment (AF1) comprises a light chain variable region (VL I ), wherein the VL I comprises or is identical to an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 101, 103, 104, 106 or 107. In certain embodiments, the VH I and the VL Iare linked by a linker comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table A. In certain embodiments, the first antigen-binding fragment (AF1) comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of any one of SEQ ID NOs: 201-205. In certain embodiments, the first antigen-binding fragment (AF1) specifically binds to human or cynomolgus monkey (cyno) CD3. In certain embodiments, the first antigen-binding fragment (AF1) specifically binds to human CD3. In certain embodiments, the first antigen-binding fragment (AF1) binds to a CD3 complex subunit that is the CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta unit of CD3. In certain embodiments, the first antigen-binding fragment (AF1) binds to the CD3 epsilon fragment of CD3. In some embodiments, the first antigen-binding fragment (AF1) exhibits an isoelectric point (pI) of less than or equal to 6.6. In some embodiments, the first antigen-binding fragment (AF1) exhibits an isoelectric point (pI) of between 6.0 and 6.6, inclusive.

[0022] In certain embodiments, the first antigen-binding fragment (AF1) exhibits an isoelectric point (pi) that is 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 having the sequence set forth in SEQ ID NO: 206. In certain embodiments, the first antigen-binding fragment (AF1) exhibits a dissociation constant (K) of between about 10 nM and about 400 nM, as determined in an in vitro antigen-binding assay involving human or cyno CD3 antigen. dIn certain embodiments, the first antigen-binding fragment (AF1) specifically binds to human or cyno CD3 with a dissociation constant (K) of less than about 10 nM, or less than about 50 nM, or less than about 100 nM, or less than about 150 nM, or less than about 200 nM, or less than about 250 nM, or less than about 300 nM, or less than about 350 nM, or less than about 400 nM, as determined in an in vitro antigen-binding assay. d In certain embodiments, the first antigen-binding fragment (AF1) specifically binds to human or cyno CD3 with a respective dissociation constant (K d ) and exhibits binding affinity to CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold weaker than the binding affinity of an antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 206.

[0023] In certain embodiments, the first antigen-binding fragment (AF1) is a chimeric or humanized antigen-binding fragment. In certain embodiments, the first antigen-binding fragment (AF1) is an Fv, Fab, Fab', Fab'-SH, a linear antibody, or a single-chain variable fragment (scFv). In certain embodiments, the second antigen-binding fragment (AF2) is an Fv, Fab, Fab', Fab'-SH, a linear antibody, a single-domain antibody, or a single-chain variable fragment (scFv). In certain embodiments, the first and second antigen-binding fragments are configured as (Fab')2 or a single-chain diabody. In certain embodiments, the second antigen-binding fragment (AF2) comprises a heavy chain variable region (VH) comprising an amino acid sequence identified herein by SEQ ID NOs: 778-783. II and a light chain variable region (VL) comprising the amino acid sequence identified herein by SEQ ID NOs: 878-883. II In certain embodiments, the VH II and the VL IIare linked by a linker comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table A. In certain embodiments, the first and second antigen-binding fragments are fused together by a peptide linker. In certain embodiments, the peptide linker comprises two or three amino acids that are glycine, serine, or proline. In certain embodiments, the peptide linker comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table B.

[0024] In certain embodiments, the XTEN is a first extended recombinant polypeptide (XTEN1) formed from a plurality of non-overlapping sequence motifs, the XTEN comprising a first plurality of non-overlapping sequence motifs; the BAR is a first barcode fragment (BAR1); the RS is a first release segment (RS1); the polypeptide further comprises: (d) a second extended recombinant polypeptide (XTEN2), the XTEN2 comprising a second barcode fragment (BAR2) that is releasable from the polypeptide upon digestion with the protease; and (e) a second release segment (RS2) positioned between the second XTEN (XTEN2) and the bispecific antibody construct (BsAb); EN2 is characterized in that it (i) contains at least 100 or at least 150 amino acids, (ii) at least 90% of its amino acid residues are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P); and (iii) contains at least four different amino acids that are G, A, S, T, E, or P, wherein the second barcode fragment (BAR2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease; and the second barcode fragment (BAR2) does not contain the N-terminal amino acid or the C-terminal amino acid of the polypeptide. In certain embodiments, the XTEN1 is located at the N-terminus of the bispecific antibody construct, and the XTEN2 is located at the C-terminus of the bispecific antibody construct. In certain embodiments, the XTEN1 is located at the C-terminus of the bispecific antibody construct and the XTEN2 is located at the N-terminus of the bispecific antibody construct.

[0025] In certain embodiments, the XTEN2 is formed from a second plurality of non-overlapping sequence motifs, each 9-14 amino acids in length, the second plurality of non-overlapping sequence motifs comprising: (1) a second set of non-overlapping sequence motifs that are repeated at least twice in the second XTEN; and (2) a non-overlapping sequence motif that appears only once in the second XTEN; and the second barcode fragment (BAR2) comprises at least a portion of the non-overlapping sequence motif that appears only once in the second XTEN. In certain embodiments, the second set of non-overlapping sequence motifs are each independently identified herein by SEQ ID NOs: 179-200 and 1715-1722. In certain embodiments, the second set of non-overlapping sequence motifs are each independently identified herein by SEQ ID NOs: 186-189. In certain embodiments, the second set of non-overlapping sequence motifs comprises at least two, at least three, or all four of the sequence motifs SEQ ID NOs: 186-189. In certain embodiments, the XTEN2 comprises a length of 100 to 3,000, 150 to 3,000, 100 to 1,000, or 150 to 1,000 amino acid residues, hi certain embodiments, the XTEN2 comprises 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 acid residues.

[0026] In certain embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN2 are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). In certain embodiments, the XTEN2 has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a sequence listed in Table 3a. In certain embodiments, the second barcode fragment (BAR2) does not contain a glutamic acid immediately adjacent to another glutamic acid, if present, in the XTEN2. In certain embodiments, the second barcode fragment (BAR2) has a glutamic acid at its C-terminus. In certain embodiments, the second barcode fragment (BAR2) has an N-terminal amino acid immediately preceded by a glutamic acid residue. In certain embodiments, the second barcode fragment (BAR2) is located a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10 to 150 amino acids in length, or 10 to 125 amino acids in length. In certain embodiments, the second barcode fragment (BAR2) (i) does not contain a glutamic acid, if present, immediately adjacent to another glutamic acid in the XTEN2; (ii) has a glutamic acid at its C-terminus; (iii) has an N-terminal amino acid immediately preceded by a glutamic acid residue; and (iv) is located a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10 to 150 amino acids in length, or 10 to 125 amino acids in length.

[0027] In certain embodiments, the glutamic acid residue preceding the N-terminal amino acid of the BAR2 is not immediately adjacent to another glutamic acid residue. In certain embodiments, the second barcode fragment (BAR2) does not include a second glutamic acid residue at a position other than the C-terminus of the second barcode fragment (BAR2), unless the second glutamic acid is immediately followed by a proline. In certain embodiments, the XTEN2 is located at the N-terminus of the bispecific antibody construct (BsAb), and the second barcode fragment (BAR2) is located within 200 amino acids, 150 amino acids, 100 amino acids, or 50 amino acids of the N-terminus of the polypeptide. In certain embodiments, the XTEN2 is located at the N-terminus of the bispecific antibody construct (BsAb), and the second barcode fragment (BAR2) is located between 10 and 200 amino acids, between 30 and 200 amino acids, between 40 and 150 amino acids, or between 50 and 100 amino acids from the N-terminus of the protein. In certain embodiments, the XTEN2 is located at the C-terminus of the bispecific antibody construct (BsAb), and the second barcode fragment (BAR2) is located within 200 amino acids, 150 amino acids, 100 amino acids, or 50 amino acids of the C-terminus of the polypeptide. In certain embodiments, the XTEN2 is located at the C-terminus of the bispecific antibody construct (BsAb), and the second barcode fragment (BAR2) is located between 10 and 200 amino acids, between 30 and 200 amino acids, between 40 and 150 amino acids, or between 50 and 100 amino acids from the C-terminus of the protein.

[0028] In certain embodiments, the second barcode fragment (BAR2) is at least 4 amino acids in length. In certain embodiments, the second barcode fragment (BAR2) is between 4 and 20 amino acids, between 5 and 15 amino acids, between 6 and 12 amino acids, or between 7 and 10 amino acids in length. In certain embodiments, the second barcode fragment (BAR2) comprises an amino acid sequence set forth in Table 2. In certain embodiments, the XTEN2 has a length defined by a proximal end and a distal end, wherein (1) the proximal end of the XTEN2 is located closer to the bispecific antibody construct (BsAb) than the distal end, and (2) the second barcode fragment (BAR2) is located within a region of the XTEN2 spanning between 5% and 50%, between 7% and 40%, or between 10% and 30% of the length of the XTEN2, measured from the distal end of the XTEN2. In certain embodiments, the XTEN2 further comprises one or more additional barcode fragments, each of which differs in sequence and molecular weight from all other peptide fragments releasable from the polypeptide upon complete digestion of the polypeptide by the protease. In certain embodiments, the first release segment (RS1) and the second release segment (RS2) are identical in sequence. In certain embodiments, the first release segment (RS1) and the second release segment (RS2) are not identical in sequence. In certain embodiments, the second release segment (RS2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 7001-7626. In certain embodiments, the first release segment (RS1) and the second release segment (RS2) are each substrates for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence.

[0029] In certain embodiments, the polypeptide is expressed as a fusion protein that, in its uncleaved state, has a structural arrangement identified herein from N-terminus to C-terminus as XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN1-RS1-diabodyRS2-XTEN2, or XTEN2-RS2-diabodyRS1-XTEN1, wherein the diabody comprises a light chain variable region (VL) of the AF1. I ), the heavy chain variable region of AF1 (VH I ), the light chain variable region (VL II ), and the heavy chain variable region (VH II In certain embodiments, the spacer of the first release segment (RS1) is a first spacer, and the second release segment (RS2) is fused to the bispecific antibody construct (BsAb) via a second spacer. In certain embodiments, the second spacer comprises at least four amino acids that are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). In certain embodiments, the second spacer comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table C.

[0030] In certain embodiments, the XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3a, and the BsAb comprises light chain complementarity determining regions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3) and heavy chain complementarity determining regions 1 (CDR-H1), 2 (CDR-H2), and 3 (CDR-H3), wherein the CDR-H1, the CDR-H2, and the CDR-H3 comprise the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively, and a light chain variable region (VL) identified herein by SEQ ID NOs: 778-783. II ) and heavy chain variable regions (VH) identified herein by SEQ ID NOs: 878-883 II wherein the RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 7001-7626; the XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3a; and the RS2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 7001-7626. The polypeptides comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein, wherein the polypeptide has a structural arrangement identified herein by, from N-terminus to C-terminus, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, or XTEN2-RS2-AF1-AF2-RS1-XTEN1.

[0031] In certain embodiments, the polypeptide has a terminal half-life at least two-fold longer than that of a bispecific antibody construct not linked to any XTEN. In certain embodiments, the polypeptide is less immunogenic than a bispecific antibody construct not linked to any XTEN, as determined by measuring the production of IgG antibodies that selectively bind to the bispecific antibody construct after administration of an equivalent dose to a subject. In certain embodiments, the polypeptide exhibits an apparent molecular weight index of greater than about 3, greater than about 4, greater than about 5, or greater than about 6 under physiological conditions. In certain embodiments, the polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein, such as the sequences in Table D.

[0032] In certain embodiments, pharmaceutical compositions comprise the above polypeptide and one or more pharmaceutically suitable excipients.In certain embodiments, said pharmaceutical compositions are formulated for administration to humans or animals by any clinically appropriate route and formulation.In certain embodiments, said pharmaceutical compositions are in liquid form or frozen.In certain embodiments, said pharmaceutical compositions are in a pre-filled syringe for single injection.In certain embodiments, said pharmaceutical compositions are formulated as lyophilized powder that is reconstituted before administration.

[0033] In certain embodiments, the composition is present in pharmaceutical combination with at least one additional therapeutic agent selected from the group consisting of an antibody, an antibody fragment, an antibody conjugate, a cytotoxic agent, a toxin, a radionuclide, an immunomodulatory agent, a photoactive therapeutic agent, a radiosensitizer, a hormonal agent, an anti-angiogenic agent, and combinations thereof.

[0034] The additional therapeutic agent is a PD-1 / PD-L1(2) inhibitor, which is an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody.

[0035] In certain embodiments, the PD-1 / PD-L1(2) inhibitor is an anti-PD-1 antibody selected from the group including nivolumab (Opdivo, BMS-936558, MDX1106), pembrolizumab (Keytruda, MK-3475, lambrolizumab), pidilizumab (CT-011), PDR-001, JS001, STI-A1110, AMP-224, and AMP-514 (MEDI0680).

[0036] In one embodiment, the PD-1 / PD-L1(2) inhibitor can be an anti-PD-L1 antibody selected from the group including atezolizumab (Tecentriq, MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX1105), and LY3300054. In another embodiment, the PD-1 / PD-L1(2) inhibitor is an anti-PD-L2 antibody.

[0037] In certain embodiments, the combination is a combination pack containing components separate from each other, hi certain embodiments, the components are administered simultaneously or sequentially for use in the treatment of the same disease in separate dosage forms.

[0038] In certain embodiments, the polypeptides described herein are part of a pharmaceutical combination for use as a medicament for treating a hyperproliferative disorder selected from the group consisting of breast cancer, respiratory tract cancer, brain cancer, reproductive cancer, gastrointestinal cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, and distant metastases thereof.

[0039] In certain embodiments, the polypeptides described herein are used in the preparation of a medicament for treating a disease in a subject, hi certain embodiments, the disease is cancer.

[0040] In certain embodiments, there is a method of treating a disease in a subject, comprising administering to said subject in need thereof one or more therapeutically effective doses of a pharmaceutical composition or pharmaceutical combination.

[0041] In one embodiment, the disease is cancer. In certain embodiments, the cancer is selected from the group consisting of glioblastoma, melanoma, cholangiocarcinoma, small cell lung cancer, colorectal cancer, prostate cancer, vaginal cancer, angiosarcoma, non-small cell lung cancer, appendix cancer, squamous cell carcinoma, salivary gland duct carcinoma, adenoid cystic carcinoma, small intestine cancer, and gallbladder cancer.

[0042] Provided is a method for treating a disease of a subject, comprising administering one or more therapeutically effective doses of the pharmaceutical composition described above to the subject in need thereof.In certain embodiments, the pharmaceutical composition is administered to the subject as one or more therapeutically effective doses in the course of treatment that is effective for treatment.In certain embodiments, the dose is administered to humans or animals by any clinically appropriate route and formulation.In certain embodiments, the subject is a mouse, a rat, a monkey or a human.

[0043] Also provided herein is a nucleic acid comprising a polynucleotide sequence encoding a polypeptide described herein, or the reverse complement of said polynucleotide sequence. Further provided herein is an expression vector comprising said polynucleotide sequence described herein and a recombinant regulatory sequence operably linked to said polynucleotide sequence. Provided herein is a host cell comprising said expression vector. In certain embodiments, the host cell is a prokaryote. In certain embodiments, the host cell is E. coli. In certain embodiments, the host cell is a mammalian cell.

[0044]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein merely illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0045] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0046] Various features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings, in which:

[0047] [Figure 1]Figure 1 shows a mixture of Xtenylated protease-activated T cell engager ("XPAT") polypeptides with XTENs of various lengths. Full-length XPAT (top) contains a 288-amino acid XTEN at the N-terminus and an 864-amino acid XTEN at the C-terminus. In XPAT, various truncations can occur at either or both the N- and C-terminal XTENs, for example, during fermentation, purification, or other steps in product preparation. While products with limited truncations (i.e., near the distal end of the XTEN) may function in a manner similar to full-length constructs, severe truncations (i.e., near the proximal end of the XTEN) may have significantly different pharmacological properties than their full-length counterparts. The presence of truncations poses challenges in quantifying pharmacologically active and inactive variants in XPAT products. As illustrated in Figure 1 using full-length XPAT, each XTEN has a proximal end and a distal end, with the proximal end being located closer to the biologically active polypeptide (e.g., a T cell engager, cytokine, monoclonal antibody (mAb), antibody fragment, or other protein to be XTENized) than the distal end.

[0048] [Figure 2]Figure 2 shows a mixture of XPAT polypeptides with barcoded XTENs of various lengths. In full-length XPAT (top), the 288-amino acid-long N-terminal XTEN contains three cleavably fused barcode sequences, "NA," "NB," and "NC," from distal to proximal ends, while the 864-amino acid-long C-terminal XTEN contains three cleavably fused barcode sequences, "CC," "CB," and "CA," from proximal to distal ends. Each barcode is positioned to indicate a pharmacologically relevant length of the corresponding XTEN. For example, a minor N-terminal truncation product of XPAT lacks the barcode "NA" (e.g., due to truncation) but possesses more proximal barcodes, "NB" and "NC," and may exhibit substantially the same pharmacological properties as the full-length construct. In contrast, a major N-terminal truncation product of XPAT lacks all three N-terminal barcodes (e.g., due to truncation) and may have distinctly different pharmacological activity from the full-length construct. A unique proteolytic cleavage sequence has been identified from the biologically active polypeptide of XPAT (wherein the tandem scFvs make up the active portion of the T cell engager). Because it is present in full-length variants of XPAT (including full-length XPAT, minor truncations, and major truncations), this unique proteolytic cleavage sequence can be used as a reference to quantify the amount of various cleavage products relative to the total amount of biologically active protein.

[0049] [Figure 3]Figure 3 illustrates a possible design for a barcoded XTEN by inserting a barcode-generating sequence into a generic (or regular) XTEN. The exemplary generic (or regular) XTEN (top) contains non-overlapping 12-mer motifs of the sequence "BCDABDCDABDCBDCDABDCB," with sequence motifs "A," "B," "C," and "D" occurring 3, 6, 5, and 7 times, respectively. A Glu-C protease digest of the exemplary generic XTEN (top panel) yields no unique peptides except at both ends ("NT" and "CT"). Insertion of a barcode-generating sequence "X" (e.g., a unique 12-mer) into the XTEN results in a unique proteolytic cleavage sequence (or barcode sequence) that is not present anywhere in the XTEN. The barcode-generating sequence "X" can be positioned so that the resulting barcode marks a pharmacologically relevant length of the XTEN. For example, an XTEN lacking a barcode due to cleavage may be functionally different from a corresponding XTEN with a barcode. Those skilled in the art will understand that the barcode generating sequence ("X") can be the barcode sequence itself. Alternatively, the barcode generating sequence ("X") can be different from the resulting barcode sequence. For example, the barcode sequence can overlap with, and thus contain, part of a preceding or following 12-mer motif.

[0050] [Figure 4-1]Figures 4A-4B illustrate quantification of cleavage levels for N-terminal XTEN. Figure 4A demonstrates that barcoded XTEN (bottom panel) can be constructed by replacing a sequence motif (e.g., the third sequence motif from the N-terminus, "D") in a generic XTEN (top panel) with a barcode-generating motif "X." In this example, the barcode-generating motif ("X") is itself a unique proteolytically cleavable barcode sequence. As shown in the bottom panel of Figure 4A, the barcode is positioned such that all severely cleaved forms of XTEN lack the barcode, and all limitedly cleaved forms of XTEN contain the barcode. Figure 4B illustrates the relative abundance of various cleavage products in two different mixtures of XPAT. In one of the mixtures, the barcode is present in 99% of the constructs containing biologically active protein. In the other mixture, 13% of the constructs lack the barcode (e.g., due to cleavage). 4A-4B illustrate the use of barcoded XTEN to distinguish between two polypeptide mixtures that have substantially similar average molecular weights but distinctly different pharmacological activities. [Figure 4-2] Same as above.

[0051] [Figure 5-1] Figures 5A-5B and 6A-6C illustrate the dose-dependent cytotoxicity of masked (Xtenylated) and unmasked bispecific T cell engagers against target cells with varying levels of target antigen expression. Proteolytically unmasked (deXtenylated) bispecifics demonstrate potent cytotoxicity against various tumor lines with EC50s in the single-digit pM range when tested, for example, with SK-OV-3 and BT-474 cells. Xtenylation further demonstrates robust masking capabilities, for example, protecting the bispecific T cell engagers from immune synapse formation, thereby resulting in reduced toxicity as indicated by a rightward shift in the concentration-response curve.

[0052] [Figure 5-2]5A-5B illustrate the effective masking by XTEN of Xtenylated protease-activated T cell engagers ("XPAT") in general, and of HER2-XPAT in particular. For example, we observed dose-dependent cytotoxicity of Xtenylated (masked) HER2-XPAT (e.g., as described in Table D) and the corresponding de-Xtenylated (unmasked, activated) HER2-PAT against two different HER2-expressing (e.g., cancer) cell lines. Unmasked de-Xtenylated PAT (shown as filled circles) yielded EC50 values ​​of 3.4 picomolar (pM) (SKOV3 cells) (Figure 5A) and 4.8 pM (BT474 cells) (Figure 5B), respectively, while the corresponding masked Xtenylated PAT (shown as filled squares) yielded EC50 values ​​of 44,474 pM (SKOV3 cells) and 49,370 pM (BT474 cells), indicating at least a 104-fold masking effect.

[0053] [Figure 6-1] Figure 6A shows the effective masking of bispecific T cell engagers by XTEN when they come into contact with non-cancerous tissue (cardiomyocytes). More specifically, Figure 6A shows the cytotoxicity of Xtenylated (masked) HER2-XPAT (e.g., as described in Table D) and the corresponding de-Xtenylated (unmasked, activated) HER2-PAT against cardiomyocytes. While cardiomyocyte death by T cell-induced cytolysis was observed in response to unmasked, de-Xtenylated PAT (with an EC50 concentration of approximately 64 pM), in contrast to tumor cells, cardiomyocytes remained insensitive to killing by masked Xtenylated PAT at concentrations as high as 1 micromolar (μM).

[0054] [Figure 6-2]Figures 6B-6C illustrate the robust masking of bispecific T cell engagers by XTEN in that target cells express the target antigen at relatively moderate or low levels. For example, Figure 6B illustrates the cytotoxic effect of XTEN-ylated and de-XTEN-ylated protease-activated T cell engagers (PATs) on MCF-7, a cancer cell line with low levels of HER2 expression, where masking with XTEN polypeptides reduced the T cell-mediated cytotoxicity of the tested HER2-XPAT by approximately 10-fold. As another example, we measured the cytotoxicity of XTEN-ylated (masked) HER2-XPAT and the corresponding de-XTEN-ylated (unmasked, activated) HER2-PAT on MDA-MB-453, another cancer cell line with moderate levels of HER2 expression (Figure 6C). [Figure 6-3] Same as above.

[0055] [Figure 7-1]Figures 7A-7D illustrate the proteolytic activation of Xtenylated bispecifics and the robust tumor regression induced thereby in subjects. Figure 7A illustrates comparable efficacy induced by equimolar administration of a cleavable Xtenylated HER2 T cell engager ("HER2-XPAT," shown as a hexagon) and the corresponding unmasked HER2 T cell engager ("HER2-PAT," shown as a triangle) in tumor-bearing mice (e.g., BT-474). (** indicates p<0.01). Notably, between the two tested Xtenylated bispecifics, tumor regression was observed with the cleavable Xtenylated construct (shown as a hexagon) but not with the non-cleavable Xtenylated counterpart (shown as a diamond), indicating that proteolytic unmasking (deXtenylation) is a prerequisite for efficacy. Figure 7B illustrates the efficacy of a cleaving Xtenylated bispecific (e.g., HER2-XPAT) against large tumors with a single dose (e.g., 2.1 milligrams per kilogram (mpk)). The non-cleaving construct used in this experiment is identical to the corresponding cleaving construct, except that the release site is replaced with a non-cleaving sequence of similar length made up of GASTEP amino acids (glycine, alanine, serine, threonine, glutamate, and / or proline). Figure 7C shows the efficacy of two different concentrations (15 nmol / Kg and 36 nmol / Kg) of masked HER2-XPAT (filled triangles) compared to unmasked HER2-PAT (filled squares) and non-cleaving XPAT (filled diamonds). Data for tumors treated with vehicle and vehicle + PBMC are also shown. FIG. 7D shows the cleavage percentage of HER2-PAT in vivo in BT-474 tumor-bearing mice. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above.

[0056] [Figure 8]Figure 8 illustrates lymphocyte margination induced in subjects by administration of Xtenylated HER2-XPAT. For example, upon intravenous infusion of a single dose (e.g., 25 mg / kg at a dose volume of 10 ml / kg) of Xtenylated HER2-XPAT (e.g., as described in Table D), a decrease in lymphocyte counts (hematology) was observed in both female and male monkey subjects (represented by triangles and squares, respectively) starting 6 hours after administration and continuing for at least 24-72 hours after administration.

[0057] [Figure 9-1] Figures 9A-9B. Figure 9A illustrates the stability of Xtenylated protease-activated T cell engager (PAT) in the plasma circulation of subjects (e.g., cynomolgus monkeys) (e.g., at a 25 mg / kg dose). The clearance of Xtenylated PAT was not significantly increased relative to its non-cleavable form, indicating minimal peripheral cleavage. Equivalent pharmacokinetics were observed between the tested cleavable HER2-XPAT (filled triangles and filled squares) and its non-cleavable counterpart (open triangles and open squares). The non-cleavable constructs used in this experiment were identical to the corresponding cleavable constructs, except that the release site was replaced with a non-cleavable sequence of similar length made up of GASTEP amino acids (glycine, alanine, serine, threonine, glutamate, and / or proline). Figure 9B shows the low frequency of proteolytic metabolites of HER2-XPAT in circulation, even 96 hours after administration. [Figure 9-2] Same as above.

[0058] [Figure 10-1] Figures 10A-10C. Figure 10A shows single-dose, single-subject HER2-XPAT dose escalation, demonstrating that all doses up to 42 mg / kg were tolerated. Figure 10B shows a single-subject HER2-PAT tapering scheme in which the maximum tolerated dose of unmasked HER2-PAT was 0.2 mg / kg. Figure 10C shows plasma concentrations of masked HER2-PAT (450-fold higher tolerated Cmax) compared to unmasked HER2-PAT. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0059] [Figure 11-1] Figures 11A-11E. Figures 11A and 11B show peripheral T cell activation data demonstrating the lack of peripheral T cell activation in response to HER2-XPAT compared to HER2-PAT. Figures 11C-11E show levels of cytokines IL-6 (Figure 11C), TNF-α (Figure 11D), and IFN-γ (Figure 11E) in subjects treated with various concentrations of HER2-XPAT and HER2-PAT, demonstrating that HER2-XPAT does not induce cytokine release, even at 50 mg / kg. Note: normal ranges for cytokine levels: IL-6 ≦6 pg / ml, TNF-α 1-10 pg / ml, IFN-γ ≦10 pg / ml. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 11-5] Same as above.

[0060] [Figure 12] Figure 12. AMX-818 incubated ex vivo in plasma samples from NHPs and humans showed minimal cleavage to unmasked TCE, even under inflammatory conditions. AMX-818 with a fluorescent label (DyL650) attached to a tandem scFv was incubated in the indicated plasma samples at 37°C for 7 days. The samples were then run on a gel, and metabolites similar in size to the unmasked active form of AMX-818 were quantified using the LI-COR detector. Human inflammatory disease samples were derived from patients with rheumatoid arthritis, lupus, inflammatory bowel disease, and multiple sclerosis. Human cancer samples were derived from patients with lung, breast, and colon tumors. Sample size: healthy NHP N=4, healthy humans N=4, "inflammatory" NHP N=6, "inflammatory" humans N=27, cancer humans N=11.

[0061] [Figure 13-1] Figures 13A-13B provide additional data supporting the preferred safety profile of the HER2-XPAT of the present invention. The data in Figure 13A show comparable PK between the HER2-XPAT and non-cleavable HER2-PAT formats, demonstrating that the protease release site remains highly stable in the circulation of cynomolgus monkeys, even at high doses. Figure 13B shows that even at high doses of HER2 XPAT, systemic accumulation of metabolites lacking one or both XTEN masks is very limited. [Figure 13-2] Same as above.

[0062] [Figure 14-1] Figures 14A-14D. AMX818 and 818-PAT induce surface expression of PD-1 on T cells in response to SKOV3 tumor cells. Surface PD-1 expression was assessed by flow cytometry on CD4+ and CD8+ T cells after 48 hours of co-incubation of PBMCs and SKOV3 cells at an effector:target ratio of 5:1 with the indicated concentrations of test article. Figures 14A and 14C show surface PD1 expression on CD4+ T cells in the presence of AMX818 and 818-PAT. Figures 14B and 14D show surface PD1 expression on CD8+ T cells in the presence of AMX818 and 818-PAT. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 14-4] Same as above.

[0063] [Figure 15-1] Figures 13A-13C. AMX818 and 818-PAT induce surface expression of PD-L1 on T cells in response to SKOV3 tumor cells. Figures 15A and 15B show PD-L1 expression on SKOV3 cells at a 5:1 effector:target ratio for the indicated concentrations of test article. Figure 15C shows surface HER2 expression on SKOV3 tumor cells. [Figure 15-2] Same as above. [Figure 15-3]Same as above.

[0064] [Figure 16-1] Figures 16A-16C. XPAT is preferentially cleaved to unmasked TCE in human tumors implanted in live mice, with minimal cleavage observed in healthy tissues. A single dose of 1.8 mg / kg (13 nM) red fluorescently labeled (Alexa) XPAT was injected into mice implanted with human tumors (Figure 16A). Two days later, tumors and healthy organs were harvested and in vivo protease cleavage was measured (results shown in Figure 16B). Green fluorescently labeled (Dyl800) XPAT was added after tissue harvest in the presence of protease inhibitors to account for artifactual cleavage that may result from the release of unrelated intracellular proteases during processing. By comparing the cleavage products generated by red and green fluorescently labeled XPAT, we were able to determine the cleavage that occurred in vivo in various tissues and artifactual cleavage that occurred during tissue processing. Overall, by day 2 post-injection in tumor-bearing mice, 20% of XPAT within the tumor is activated (n=31 across 9 tumor types, FIG. 16C). [Figure 16-2] Same as above. [Figure 16-3] Same as above.

[0065] The patent or application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the fee. DETAILED DESCRIPTION OF THE INVENTION

[0066] Detailed Description of the Preferred Embodiments There is a significant unmet need in cancer treatment. While TCEs have been shown to be effective in inducing remission of certain cancers, their extremely potent potency in healthy tissues and on-target, off-tumor toxicity have prevented them from becoming widely used therapeutics. One explanation is that TCEs bridge T cells and tumor cells, leading to T cell-mediated tumor cell activation and the initiation of a cytokine amplification cascade that may promote further tumor cell death and provide long-term immunity. TCE-activated T cells release cytolytic perforin / granzymes in an antigen-MHC recognition-independent manner. This generates a two-fold response: direct tumor cell death and amplified tumor killing by initiating a potent cytokine response from tumor cells. Direct tumor cell death results in the release of tumor antigens. Cytokine responses include, among others, increased interferon-γ, which stimulates CD8 T cell activity and antigen presentation by APCs; increased IL2, which leads to increased proliferation of activated T cells; and increased CXCL9 and 10 responses, which increase T cell recruitment. Taken together, the release of tumor antigens and the initiation of cytokine responses can lead to the activation of endogenous T cell responses, resulting in epitope spreading and potentially inducing long-lasting immunity.

[0067] The toxicity challenges associated with TCEs arise from the fact that most tumor targets are also expressed to some degree in healthy tissues, and that normal cells can also mount cytokine responses that result in cytokine release syndrome (CRS). These two potent responses of healthy tissues to T cell activation by TCEs result in a general lack of therapeutic index for these agents.

[0068] The present invention overcomes the shortcomings of existing TCEs by providing a conditionally activated TCE, XPAT, or XTEN-ylated protease-activated bispecific T cell engager (referred to herein as HER2-XPAT and exemplified as AMX818) that targets HER2. More specifically, the XPAT of the present invention exploits the dysregulated protease activity present in tumors versus healthy tissue, thereby enabling an expanded therapeutic index. The XPAT core consists of two single-chain antibody fragments (scFvs) targeting CD3 and a tumor target (in an exemplary embodiment, the tumor target is HER2). Attached to the core are two unstructured polypeptide masks (XTENs), which structurally reduce target engagement with either the tumor target and / or CD3, thereby extending protein half-life. The properties of the XTEN polymer also minimize the potential for immunogenicity. Its lack of stable tertiary structure is unfavorable for antibody binding, and the absence of hydrophobic, aromatic, and positively charged residues that serve as anchor residues for peptide MHC II binding reduces the likelihood of binding to T cell epitopes. In humans, minimal immunogenicity of XTEN polymers has been observed in association with extended half-life forms of human growth hormone and factor VIII in >200 patients treated with XTEN-containing drugs. A protease cleavage site at the base of the XTEN mask allows proteolytic activity of XPAT in the tumor microenvironment, leaching off a small, highly potent TCE that can redirect cytotoxic T cells to kill target-expressing tumor cells. In healthy tissues, where protease activity is tightly regulated, XPAT should remain largely inactive as an intact prodrug, thus extending its therapeutic index compared to unmasked TCEs.

[0069] In addition to local activation, the short half-life of the unmasked PAT form should further broaden the therapeutic index and further enhance the potency of T cell immunity for improved eradication of solid tumors. The release site used in XPAT can be cleaved by proteases across a wide range of tumors, collectively responsible for the hallmarks of all cancers: growth, survival and death, angiogenesis, invasion and metastasis, inflammation, and immune evasion. Thus, the TCE activity of XPAT is localized to tumors by exploiting its enhanced protease activity, which is upregulated at all stages of cancer and tumor development but tightly regulated in healthy tissues.

[0070] An exemplary HER2-XPAT component, AMX-818, was optimized to achieve the desired balance of providing sufficient protection in healthy tissues while retaining the necessary potency in tumors across a broad range of cancers. To reduce the potential for T cell activation by the prodrug, we selected a longer XTEN polymer mask (576 amino acids compared to 256 amino acids at the HER2 site) as well as a lower binding affinity to the a-CD3 domain. To ensure sufficient activation of AMX-818 in tumors, we engineered a protease release site at the base of the XTEN to be cleaved by at least eight different proteases from three different classes reported to be overexpressed or dysregulated in cancer. These include several matrix metalloproteinases (MMPs), matriptase, uPA, and the cysteine ​​protease legumain. As a safety checkpoint, co-engagement of both CD3 and HER2 by AMX-818 is required for T cell activation. T cell activation should not occur if AMX-818 is not masked in inflamed tissue where HER2 expression is absent, or if AMX-818 encounters HER2 expressed in healthy tissue where proteases are tightly regulated. This AND gate feature is predicted to result in preferential activation in tumors where both elevated protease activity and high HER2 expression are present.

[0071] Thus, the presence of XTEN on XPAT results in a drug with a long half-life, weak target engagement, and negligible T cell activation. When XTEN is removed by the action of proteases in the tumor microenvironment, this preferential activation of XPAT generates an activated drug (PAT without XTEN) with a short half-life, optimal target engagement, and highly efficient T cell activation, resulting in a potent activated drug with an improved therapeutic index. The HER2-XPAT of the present invention can improve the toxicity profile while maintaining the potency of T cell engagers against solid tumors, thus enabling a significant increase in the therapeutic index and an expansion of the target landscape of this powerful modality.

[0072] Summary of data generated from AMX-818, an exemplary HER2-XPAT The target binding and in vitro bioactivity of AMX-818 have been characterized in multiple studies. Equilibrium binding analysis using surface plasmon resonance demonstrated highly comparable affinities for AMX-818 and its metabolites between human HER2 and CD3 and cynomolgus monkey HER2 and CD3, supporting the use of cynomolgus monkeys as a species for toxicity and PK studies. Proteolytically activated AMX-818 (PAT) bound to human and cynomolgus monkey HER2 with affinities of 2.4 nM and 2.0 nM, respectively, and to human and cynomolgus monkey CD3 with affinities of 26.3 nM and 21.5 nM, respectively. Masking of AMX-818 reduced its affinity for HER2 by 10-fold and its affinity for CD3 by approximately 6-fold for both species. AMX-818 bound to human and cynomolgus HER2 with affinities of 24.9 nM and 20.1 nM, while the CD3 affinities for human and cynomolgus were 160 nM and 140.3 nM, respectively.

[0073] The activity of TCEs depends on their ability to activate T cells by effectively stimulating T cell receptors (TCRs). Their exceptional potency stems from the minimal requirement for initiating cytotoxicity: only three TCRs, which are primed and coalesce to form an immune synapse between the T cell and target cell. While T cell engagers are primarily known to induce cytotoxicity, their potency also involves cytokine-driven actions downstream of T cell activation, which enhance and amplify antitumor immune responses. T cell activation by AMX-818, its prototypical form AMX-818-P1, and its proteolytic metabolites was characterized in vitro using Jurkat NFAT-reporter cells and primary human PBMCs in the presence of BT-474 (breast) and SKOV-3 (ovarian) tumor cells, which highly express HER2. Human T cells were also assessed for upregulation of the surface activation marker CD69 and the inhibitory receptor PD-1 by flow cytometry. As an indirect measure of T cell activation, upregulation of PD-L1, a ligand for PD-1, was assessed on the surface of SKOV3 tumor targets, as it is induced in response to IFN-γ secreted by activated T cells.

[0074] AMX-818 (PAT) inhibited Jurkat NFAT-luciferase reporter T cells in the presence of BT-474 cells with an EC in the 70 pM range. 50Although activated at high levels, responses to masked AMX-818 and AMX-818-P1 were significantly attenuated by four orders of magnitude, with maximal responses reduced by 80–90% compared to those of activated AMX-818(PAT). T cell activation by AMX-818(PAT) was not observed in the absence of HER2+ BT-474 tumor cells, demonstrating that monovalent engagement of CD3 was not sufficient for activation and that co-engagement of both CD3 and tumor targets was required for effective T cell receptor (TCR) stimulation. The masked AMX-818 metabolites alone, AMX-818(1x-C) and AMX-818(1x-N), exhibited intermediate activity. AMX-818-NoClvSite did not induce detectable T cell activation, suggesting that the minimal responses observed with AMX-818 were likely driven by proteolytic cleavage.

[0075] AMX-818(PAT) and the prodrug AMX-818 induced CD69 and PD-1 expression on the surface of both CD4+ and CD8+ T cell subsets and PD-L1 expression on SKOV3 tumor cells to a similar extent. However, the dose-response curve for AMX-818 was shifted by an average of 400-650-fold compared to that of AMX-818(PAT), further demonstrating the effective functional masking of the XTEN mask on AMX-818.

[0076] AMX-818 and its metabolites were characterized for their cytotoxic activity and induction of proinflammatory cytokines. Peripheral blood mononuclear cells (PBMCs) were used as effector cells, and the HER2-highly expressing BT-747 breast tumor line was selected as the tumor target cell. Because cardiac tissue is known to express HER2 and, although rare, cardiotoxicity has been observed in patients treated with some HER2-targeted therapies, primary cardiomyocytes (HER2 low to moderate) were selected to represent a more physiological cytolytic target for AMX-818 and its proteolytic metabolites. Luminescence-based cytotoxicity assays were performed at a 1:1 effector:target cell ratio, and cell-free supernatants were collected for measurement of TCE-induced cytokines.

[0077] AMX-818 (PAT) exhibited highly potent cytotoxicity against BT-474 tumor cells, with half-maximal inhibitory concentrations (IC) ranging from 5 to 11 pM. 50 ) values ​​demonstrated near-complete target cell killing. When human cardiomyocytes, which have lower HER2 expression—cells not expected to exhibit dysregulated protease activity—were used as targets, the cytotoxic response was approximately 13-fold lower, and maximal killing was incomplete, reaching only 50-60%. The cytotoxic response by AMX-818 against both BT-474 and cardiomyocytes was strongly attenuated, with mean IC 50The values ​​shifted 2500- to 3000-fold, demonstrating effective functional masking of the prodrug by its XTEN polymer mask. Masking provides synergistic protection from cytotoxicity that far exceeds their combined effect on reducing target binding by preventing the formation of a functional immune synapse necessary to initiate target cell killing. AMX-818 and its prototype, AMX-818-P1, exhibited comparable cytotoxicity consistent with their nearly identical composition. The cytotoxicity of the singly masked proteolytic metabolites, AMX-818(1x-N) and AMX-818(1x-C), was intermediate between AMX-818(PAT) and AMX-818, demonstrating partial protection by the singly masked metabolites. The cytotoxicity observed with AMX-818 was likely due to proteolytic cleavage, based on the further reduction in activity provided by AMX-818-NoClvSite, a form lacking both protease cleavage sites.

[0078] In general, the relative potencies of AMX-818 and its metabolites in inducing cytokine secretion in supernatants from cytotoxicity assays closely resembled those observed for cytotoxicity against both BT-474 and cardiomyocyte target cells (AMX-818(PAT) was the most potent, with the masked form being the least potent). 50 The values ​​were on average higher than those for the cytotoxic response, demonstrating that cytotoxicity is the more sensitive assay between the two. The response of AMX-818 was reduced by several orders of magnitude compared to that of AMX-818(PAT) in the presence of both BT-474 and cardiomyocytes, while the masked metabolites AMX-818(1x-N) and AMX-818(1x-C) alone showed intermediate responses.

[0079] In the presence of cardiomyocytes, the maximum cytokine levels induced by AMX-818 at the highest concentration tested (300 nM) were significantly reduced compared with those induced by AMX-818(PAT), except for IL-6. In the presence of both BT-474 and cardiomyocytes, elevated IL-6 levels were detected at 300 nM for both AMX-818 and AMX-818-NoClvSite, and these levels were in most cases 2- to 6-fold higher than the maximum levels produced by AMX-818(PAT). Notably, this contrasts with what was observed in cynomolgus monkeys, where peak systemic levels of IL-6 were >9-fold higher at the MTD of 0.2 mg / kg for AMX-818 than at the MTD of 42 mg / kg for AMX-818.

[0080] When cytokine induction was assessed in the absence of HER2-expressing target cells, the cytokines IL-2, IL-4, TNF-α, and IFN-γ were not induced in human PBMC cultures treated with suspension and plate-coated AMX-818 or AMX-818(PAT). At the highest concentration tested (500 nM), soluble AMX-818 induced low IL-10 levels from all PBMC donors. IL-6 was a notable exception; at 500 nM, in soluble form, AMX-818 induced IL-6 levels from all donors by an average of 4.6-fold increase over levels induced by the anti-CD3 antibody positive control. High IL-6 levels were also induced from two of five donors in the wet-plate-coated format, and lower IL-6 levels were seen even with the highest concentration of AMX-818(PAT). Importantly, however, such elevated IL-6 levels were not accompanied by increases in TNF-α, IFN-γ, and IL-2, cytokines normally associated with IL-6 secretion under CRS conditions, and were not observed in cynomolgus monkeys administered AMX-818 at doses up to 50 mg / kg. In contrast, activated AMX-818 (PAT) induced high IL-6 levels in cynomolgus monkeys at doses ≤0.3 mg / kg, which were accompanied by increases in additional proinflammatory cytokines.

[0081] In vivo pharmacology, PK, and toxicity studies were conducted to characterize the efficacy and safety of AMX-818 and its metabolites. In addition to standard toxicity endpoints, the proteolytic stability of AMX-818 in the circulation was assessed in cynomolgus monkeys administered high doses of AMX-818 or under proinflammatory conditions, and in vitro after prolonged incubation in plasma from patients with cancer or systemic autoimmune disease. While preferential cleavage of AMX-818 in tumors clearly results in protease-dependent efficacy, its peripheral stability in NHPs provides a larger margin of safety compared to AMX-818(PAT), predicting an increased therapeutic index. Collectively, these data support the conditional potency of AMX818, allowing tumor-localized activity through simultaneous stability and effective masking in circulation and peripheral tissues.

[0082] Several in vivo efficacy studies were performed to evaluate the impact of AMX-818 in redirecting T cells to kill HER2-expressing tumors. Because AMX-818 is not cross-reactive with mouse HER2 or CD3, immunodeficient mice were inoculated with human HER2-expressing xenograft tumors and engrafted with human PBMCs (hPBMCs) as a source of effector T cells. The antitumor activity of AMX-818 was evaluated in the HER2-high-expressing BT-474 breast model (approximately 975,000 HER2 receptors) and the HER2-low-expressing HT-55 colorectal model (25,000 receptors).

[0083] In the BT474 model, administration of an equimolar dose of 2.1 mg / kg of AMX-818 and its prototype, AMX-818-P1, or 0.9 mg / kg of unmasked AMX-818(PAT) robustly and completely induced tumor regression. The antitumor efficacy of AMX-818 depended on protease cleavage of its mask, as demonstrated by the lack of significant tumor growth inhibition in mice treated with a form of AMX-818 lacking its protease release site (AMX-818-NoClvSite). AMX-818-P1 and AMX-818(PAT) induced comparable activation of T cells in the tumor microenvironment, as assessed by upregulation of the activation markers CD25 and CD69 on CD4 and CD8+ T cells by flow cytometry. Importantly, consistent with the requirement for dual engagement of both HER2 and CD3 for T cell activation and their redirection to killing, T cells were not activated in the periphery, even by unmasked AMX-818 (PAT), in which human HER2 is not expressed.

[0084] A single 2.1 mg / kg dose of AMX-818 inhibited large established BT-474 tumors (478 mm 3 The efficacy of this antibody was sufficient to induce tumor regression within 4 days of administration in mice bearing HER2-binding scFv-1 (mean tumor volume of 1000 mcg). Efficacy depended on both HER2 expression and T cells, as demonstrated by the lack of activity of the non-tumor-binding variant NB-XPAT (a version of AMX-818 in which its HER2-binding domain was replaced with a non-HER2-binding scFv) or the lack of activity of AMX-818 upon administration in tumor-bearing mice lacking hPBMCs. These findings further support the necessity of dual engagement for AMX-818's activity and provide a safety measure that AMX-818 should be cleaved in normal tissues in the absence of HER2.

[0085] In mice bearing HER2-low-expressing HT-55 tumors, AMX-818 induced complete tumor regression in all mice at 5.1 mg / kg (103% TGI, p<0.01) and 70% tumor growth inhibition at 2.1 mg / kg, demonstrating dose- and protease-dependent efficacy. The efficacy of AMX-818 in a model expressing 25,000 HER2 receptors offers the potential to treat patients suffering from multiple cancer types, including tumors with low HER2 expression levels. Finally, a mean of 25.2% of AMX-818 (PAT) and fluorescently labeled AMX-818 were preferentially unmasked in tumors compared with <2% in combined heart, brain, and liver tissues after 2 days of incubation in BT-474 tumor-bearing mice. This supports the notion that local protease dysregulation is a major occupancy factor in tumors, while protease inhibition predominates in normal tissues.

[0086] Terminology As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0087] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0088] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0089] As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including, but not limited to, glycine and both the D or L optical isomers and amino acid analogs and peptidomimetics. Standard one- or three-letter codes are used to represent amino acids.

[0090] A "host cell" includes an individual cell or cell culture that can be or has been a recipient of a subject vector. A host cell includes the progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomics of total DNA complement) to the original parent cell due to naturally occurring or genetically engineered mutations.

[0091] A "chimeric" protein contains at least one fusion polypeptide containing regions in a sequence that is different from their naturally occurring positions. The regions may normally be present in separate proteins and brought together in the fusion polypeptide, or they may normally be present in the same protein but arranged in a new configuration within the fusion polypeptide. Chimeric proteins may be produced, for example, by chemical synthesis, or by translation of a polynucleotide in which the peptide regions are encoded in the desired relationship.

[0092] "Conjugated," "linked," "fused," and "fusion" are used interchangeably herein. These terms refer to the joining of two or more chemical elements or components by whatever means, including chemical conjugation or recombinant means.

[0093] The terms "polynucleotide," "nucleic acid," "nucleotide," and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may have any three-dimensional structure and may perform any function, known or unknown. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0094] As used herein, a polynucleotide having "homology" or being "homologous" is one that hybridizes under stringent 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 to the sequence.

[0095] The terms "percent identity" and "% identity," when applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using standard algorithms. Such algorithms can insert gaps in a standardized, reproducible manner into the sequences to be compared to optimize the alignment between two sequences, thereby achieving a more meaningful comparison of the two sequences. Percent identity can be measured over the length of the entire defined polynucleotide sequence, e.g., as defined by a particular SEQ ID NO:, or over a shorter length, e.g., over the length of a fragment taken from a larger, defined polynucleotide sequence, e.g., a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210, or at least 450 contiguous residues. It is understood that such lengths are merely exemplary, and that any fragment length supported by the sequences set forth in the tables, figures, or sequence listing herein can be used to describe the length over which percent identity can be measured.

[0096] "Amino acid sequence identity percentage (%)" for the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a query sequence that are identical to the amino acid residues of a second reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage, and any conservative substitutions are not considered part of the sequence identity.Alignment for determining amino acid sequence identity percentage can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Percent identity may be measured over the length of the entire defined polypeptide sequence, e.g., as defined by a particular SEQ ID NO:, or over a shorter length, e.g., over the length of a fragment taken from the larger defined polypeptide sequence, e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. It will be understood that such lengths are exemplary only, and that any fragment length supported by the sequences shown in the tables, figures, or sequence listing herein can be used to describe the length over which percent identity may be measured.

[0097] As used herein, the "repetitiveness" of an XTEN sequence refers to its 3-mer repetitiveness, which can be measured by a computer program or algorithm or by other means known in the art. The 3-mer repetitiveness of an XTEN is assessed by determining the number of occurrences of overlapping 3-mer sequences within a polypeptide. For example, a 200-amino acid residue polypeptide has 198 overlapping three-amino acid sequences (3-mers), but the number of unique 3-mer sequences will depend on the amount of repetition within the sequence. A score can be generated that reflects the degree of 3-mer repetitiveness within the entire polypeptide sequence (hereinafter referred to as the "subsequence score"). In the context of the present invention, the "subsequence score" refers to the sum of the occurrences of each unique 3-mer frame throughout the entire 200-amino acid sequence of the polypeptide, divided by the absolute number of unique 3-mer subsequences within that 200-amino acid sequence. Examples of such subsequence scores derived from the first 200 amino acids of repetitive and non-repetitive polypeptides are provided in Example 73 of International Patent Application Publication No. WO2010 / 091122A1, which is incorporated by reference herein in its entirety. In some embodiments, the present invention provides BPXTENs that each comprise XTENs that may have a subsequence score of less than 16, or less than 14, or less than 12, or more preferably less than 10.

[0098] The term "substantially non-repetitive XTEN," as used herein, refers to (1) XTEN that have few or no instances of four consecutive amino acids of the same amino acid type in the XTEN sequence, and (2) XTEN that have a subsequence score (as defined in the previous paragraph herein) of 12, or 10 or less, or XTEN in which there is no pattern in the N-terminal to C-terminal ordering of sequence motifs that make up the polypeptide sequence.

[0099] "Vector" refers to a nucleic acid molecule that transfers an inserted nucleic acid molecule into and / or between host cells, preferably a nucleic acid molecule that self-replicates in a suitable host. The term includes vectors that function primarily for the insertion of DNA or RNA into a cell, replicating vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Vectors that provide more than one of the above functions are also included. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" usually implies a suitable host cell comprised of an expression vector that can function to produce a desired expression product.

[0100] The term "t" 1 / 2 ", as used herein, means ln(2) / K el The terminal half-life is calculated as K el is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration versus time curve. Half-life typically refers to the time required for half of the amount of an administered substance accumulated in a living organism to be metabolized or eliminated by normal biological processes. 1 / 2 ", "terminal half-life", "elimination half-life" and "circulating half-life" are used interchangeably herein.

[0101] The terms "antigen," "target antigen," or "immunogen" are used interchangeably herein to refer to the structure or binding determinant to which an antibody fragment or antibody fragment-based therapeutic binds or has specificity.

[0102] The term "payload" as used herein refers to a protein or peptide with biological or therapeutic activity, which corresponds to the pharmacophore of a small molecule. Examples of payloads include, but are not limited to, cytokines, enzymes, hormones, and growth factors. The payload may further include genetically fused or chemically conjugated moieties, such as chemotherapeutic agents, antiviral compounds, toxins, or imaging agents. These conjugated moieties can be joined to the remainder of the polypeptide by a linker that can be cleavable or non-cleavable.

[0103] As used herein, "treatment" or "treating" or "alleviating" or "relieving" are used interchangeably herein. These terms refer to an approach to obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or preventative benefit. "Therapeutic benefit" refers to the eradication or amelioration of the underlying disorder being treated. Therapeutic benefit is also achieved by the eradication or amelioration of one or more of the physiological symptoms associated with the underlying condition, such that the subject experiences improvement, even though the subject may still be suffering from the underlying disorder. For preventative benefit, the composition may be administered to a subject at risk of developing a particular condition, or to a subject who reports one or more physiological symptoms of a disease, even if the disease has not been diagnosed.

[0104] "Therapeutic effect," as used herein, refers to a physiological effect produced by a fusion polypeptide of the present disclosure other than the ability to induce the production of antibodies against an antigenic epitope possessed by the biologically active protein, including, but not limited to, curing, alleviating, ameliorating, or preventing a condition in a human or other animal, or otherwise enhancing the physical or mental well-being of a human or animal. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0105] The terms "therapeutically effective amount" and "therapeutically effective dose," as used herein, refer to an amount of a biologically active protein, either alone or as part of a fusion protein composition, that, when administered to a subject in one or multiple doses, is capable of exerting any detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of a condition or pathology. Such an effect need not necessarily be beneficial. A condition may also refer to a disorder or disease.

[0106] The term "therapeutically effective dose regimen," as used herein, refers to a schedule for sequentially administered doses of a biologically active protein, either alone or as part of a fusion protein, wherein the doses are given in therapeutically effective amounts to produce a sustained beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease state or condition.

[0107] Fusion Polypeptides Disclosed herein is a polypeptide (or fusion polypeptide) comprising one or more extended recombinant polypeptides (XTEN(s) (as described more fully herein below), a bispecific antibody construct (BsAb) linked to the XTEN(s), and one or more release segments (RS), wherein the release segment is positioned between the XTEN and the bispecific antibody construct (BsAb), the polypeptide (or fusion polypeptide) having an N-terminal amino acid and a C-terminal amino acid.

[0108] In some embodiments, the polypeptide comprises a first XTEN (e.g., as described in the "Extended Recombinant Polypeptides (XTEN)" section below, or elsewhere herein). In some embodiments, the polypeptide further comprises a second XTEN (e.g., as described in the "Extended Recombinant Polypeptides (XTEN)" section below, or elsewhere herein). In some embodiments, the polypeptide comprises an XTEN at or near its N-terminus (an "N-terminal XTEN"). In some embodiments, the polypeptide comprises an XTEN at or near its C-terminus (a "C-terminal XTEN"). In some embodiments, the polypeptide comprises both an N-terminal XTEN and a C-terminal XTEN. In some embodiments, the first XTEN is an N-terminal XTEN and the second XTEN is a C-terminal XTEN. In some embodiments, the first XTEN is a C-terminal XTEN and the second XTEN is an N-terminal XTEN.

[0109] Because a bispecific antibody (BsAb), a biologically active polypeptide ("BP"), is linked to one or more XTENs within the polypeptide, the polypeptide may be referred to as an XTEN-containing fusion polypeptide: "BPXTEN."

[0110] The XTEN may include one or more barcode fragments (as described more fully below) that are releasable (or configured to be released) from the XTEN upon digestion of the fusion polypeptide (or BPXTEN) by a protease. In some embodiments, each barcode fragment differs in sequence and molecular weight from all other peptide fragments (including all other barcode fragments, if present) that are releasable from the polypeptide upon complete digestion of the polypeptide by a protease.

[0111] The (fusion) polypeptide may include one or more reference fragments (as described more fully below) that are releasable (configured to be released) from the polypeptide, e.g., upon protease digestion that releases barcode fragments from the polypeptide. In some embodiments, each reference fragment may be a single reference fragment that differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon digestion of the polypeptide with a protease.

[0112] In some embodiments, the polypeptide has an N-terminal amino acid and a C-terminal amino acid. The polypeptide is a bispecific antibody construct (BsAb) comprising: (a) an extended recombinant polypeptide (XTEN), the XTEN comprising a barcode fragment (BAR) releasable from the polypeptide upon digestion with a protease; (b) a first antigen-binding fragment (AF1) that specifically binds to Cluster of Differentiation 3 T-cell receptor (CD3), wherein AF1 comprises light chain complementarity-determining regions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3) and heavy chain complementarity-determining regions 1 (CDR-H1), 2 (CDR-H2), and 3 (CDR-H3). and a second antigen-binding fragment (AF2) that specifically binds to human epidermal growth factor receptor 2 (HER2), wherein the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10; and (c) a release segment (RS) located between the XTEN and the bispecific antibody construct, wherein the XTEN (i) comprises at least 100, or at least 150 amino acids, and (ii) at least 90% of its amino acid residues are selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or ribonucleotides. or proline (P); (iii) comprising at least four different amino acids identified herein by G, A, S, T, E, or P; and (iv) the XTEN is characterized in that it is formed from a plurality of non-overlapping sequence motifs, each of which is 9 to 14 amino acids in length; the plurality of non-overlapping sequence motifs comprises: (1) a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least twice in the XTEN; and (2) a non-overlapping sequence motif that occurs only once within the XTEN; the barcode fragment (BAR) comprises at least a portion of a non-overlapping sequence motif that occurs only once within the XTEN; the barcode fragment (BAR) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by a protease; and the barcode fragment (BAR) does not include the N-terminal or C-terminal amino acids of the polypeptide.The polypeptides can be expressed as fusion proteins, which, in their uncleaved state, can have a structural arrangement identified herein from N- to C-terminus as AF1-AF2-RS-XTEN, AF2-AF1-RS-XTEN, XTEN-RS-AF1-AF2, or XTEN-RS-AF2-AF1.

[0113] In some embodiments of the polypeptides of the present disclosure, the XTEN is a first extended recombinant polypeptide (XTEN1); the multiple non-overlapping sequence motifs from which the XTEN1 is formed are a first multiple non-overlapping sequence motifs; the BAR is a first barcode fragment (BAR1); and the RS is a first release segment (RS1). In some embodiments, the polypeptide further comprises: (d) a second extended recombinant polypeptide (XTEN2), the XTEN2 comprising a second barcode fragment (BAR2) releasable from the polypeptide upon digestion with a protease; and (e) a second release segment (RS2) located between the second XTEN (XTEN2) and the bispecific antibody construct (BsAb); the XTEN2 (i) comprises at least 100, or at least 150 amino acids; (ii) at least 90% of its amino acid residues are selected from the group consisting of glycine (G), alanine (A), (iii) characterized in that the second barcode fragment (BAR2) is characterized by being identified herein as serine (S), threonine (T), glutamate (E), or proline (P); and (iii) containing at least four different amino acids identified herein as G, A, S, T, E, or P, wherein the second barcode fragment (BAR2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by a protease; the second barcode fragment (BAR2) does not contain the N- or C-terminal amino acids of the polypeptide. XTEN1 may be located at the N-terminus of the bispecific antibody construct (BsAb), and XTEN2 may be located at the C-terminus of the bispecific antibody construct (BsAb). Alternatively, XTEN1 may be located at the C-terminus of the bispecific antibody construct (BsAb), and XTEN2 may be located at the N-terminus of the bispecific antibody construct (BsAb).In some embodiments of the polypeptide, (iv) XTEN2 can be formed from a second plurality of non-overlapping sequence motifs, each 9 to 14 amino acids in length, the second plurality of non-overlapping sequence motifs comprising: (1) a second set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of the second set of non-overlapping sequence motifs is repeated at least twice in the second XTEN; and (2) a non-overlapping sequence motif that occurs only once in the second XTEN; and the second barcode fragment (BAR2) comprises at least a portion of the non-overlapping sequence motif that occurs only once in the second XTEN. The polypeptides are expressed as fusion proteins which, in their uncleaved state, can have, from N- to C-terminus, a structural arrangement identified herein by XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN1-RS1-diabodyRS2-XTEN2, or XTEN2-RS2-diabodyRS1-XTEN1. The diabody comprises the light chain variable region (VL) of AF1. I ), the heavy chain variable region of AF1 (VH I ), the light chain variable region of AF2 (VL II ), and the heavy chain variable region of AF2 (VH II ).

[0114] In some embodiments of the polypeptides of the present disclosure, (a) the first extended recombinant polypeptide (XTEN1) may comprise an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3a, and (b) the bispecific antibody construct (BsAb) may comprise: (I) light chain complementarity determining region 1 (CDR-L1), 2 ( (II) a first antigen-binding fragment (AF1) comprising heavy chain complementarity-determining regions 1 (CDR-H1), 2 (CDR-H2) and 3 (CDR-L3) and heavy chain complementarity-determining regions 1 (CDR-H1), 2 (CDR-H2) and 3 (CDR-H3), wherein CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; and (II) a light chain variable region (VL) identified herein by SEQ ID NOs: 778 to 783. II ) and heavy chain variable regions (VH) identified herein by SEQ ID NOs: 878-883 II (c) a first release segment (RS1) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 7001-7626; (d) a second extended recombinant polypeptide (XTEN2) comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3a; (e) a second release segment (RS1) comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3a. XTEN1-RS1-AF2-AF1-RS2-XTEN2; XTEN1-RS1-AF1-AF2-RS2-XTEN2; XTEN2-RS2-AF2-AF1-RS1-XTEN1; or XTEN2-RS2-AF1-AF2-RS1-XTEN1-RS1-XTEN1.

[0115] Extended Recombinant Polypeptides (XTEN) Chain length and amino acid composition In some embodiments, the XTEN comprises at least 100, or at least 150 amino acids. In some embodiments, the XTEN is 100 to 3,000, or 150 to 3,000 amino acids in length. In some embodiments, the XTEN is 100 to 1,000, or 150 to 1,000 amino acids in length. In some embodiments, the XTEN is at least (about) 100, at least (about) 150, at least (about) 200, at least (about) 250, at least (about) 300, at least (about) 350, at least (about) 400, at least (about) 450, at least (about) 500, at least (about) 550, at least (about) 600, at least (about) 650, at least (about) 700, at least (about) 750, at least (about) 800, at least at least (about) 850, at least (about) 900, at least (about) 950, at least (about) 1,000, at least (about) 1,100, at least (about) 1,200, at least (about) 1,300, at least (about) 1,400, at least (about) 1,500, at least (about) 1,600, at least (about) 1,700, at least (about) 1,800, at least (about) 1,900, or at least (about) 2,000 amino acids. In some embodiments, the XTEN has a length of at most (about) 100, at most (about) 150, at most (about) 200, at most (about) 250, at most (about) 300, at most (about) 350, at most (about) 400, at most (about) 450, at most (about) 500, at most (about) 550, at most (about) 600, at most (about) 650, at most (about) 700, at most (about) 750, at most (about) 800, at most (about) 900, at most (about) 1000, at most (about) 1100, at most (about) 1200, at most (about) 1300, at most (about) 1400, at most (about) 1500, at most (about) 1600, at most (about) 1700, at most (about) 1800, at most (about) 1900, at most (about) 2100, at most (about) 2200, at most (about) 2300, at most (about) 2400, at most (about) 2500, at most (about) 2600, at most (about) 2700, at most (about) 2800, at most (about) 2900, at most (about) 3000, at most (about) 3500, at most (about) 3600, at most (about) 3700, at most (about) 3800, at most (about) 3900, at most (about) 4000, at most (about) 4500, at most (about) 4000, at most (about) 4500, at most (about) 500, at most (about) 550, At most (approximately) 850, at most (approximately) 900, at most (approximately) 950, at most (approximately) 1,000, at most (approximately) 1,100, at most (approximately) 1,200, at most (approximately) 1,300, at most (approximately) 1,400, at most (approximately) 1,500, at most (approximately) 1,600, at most (approximately) 1,700, at most (approximately) 1,800, at most (approximately) 1,900, or at most (approximately) 2,000 amino acids.In some embodiments, the XTEN can be of length (about) 100, (about) 150, (about) 200, (about) 250, (about) 300, (about) 350, (about) 400, (about) 450, (about) 500, (about) 550, (about) 600, (about) 650, (about) 700, (about) 750, (about) 800, (about) 850, (about) 900, (about) 950, (about) 1000, (about) 1050, (about) 1050, (about) 1100, (about) 1200, (about) 1300, (about) 1400, (about) 1500, (about) 1600, (about) 1700, (about) 1800, (about) 1900, (about) 2100, (about) 2200, (about) 2300, (about) 2400, (about) 2500, (about) 2600, (about) 2700, (about) 2800, (about) 2900, (about) 3000, (about) 3100, (about) 3200, (about) 3300, (about) 3400, (about) 3500, (about) 3600, (about) 3700, (about) 3800, (about) 3900, (about) 4000, (about) 4500, (about) 4500, (about) 5000, (about) 5500, (about) 6000, (about) 6500, (about) 7000, (about) (about) 1,900, or (about) 2,000 amino acids, or a length ranging between any two of the foregoing. In some embodiments, at least 90% of the amino acid residues of the XTEN are identified herein by 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 are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). In some embodiments, the XTEN comprises at least four different amino acids that are G, A, S, T, E, or P, assigned substantially randomly relative to any other non-overlapping sequence motifs that make up the XTEN polypeptide. In some embodiments, XTEN (e.g., XTEN1, XTEN2, etc.) are characterized in that they (i) contain at least 100, or at least 150 amino acids, (ii) at least 90% of the amino acid residues of the XTEN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P), and (iii) contain at least four different amino acids from G, A, S, T, E, or P, assigned substantially randomly to any other non-overlapping sequence motifs that make up the XTEN polypeptide. Those of skill in the art will understand that, as used herein, the term "glutamate" is synonymous with "glutamic acid" and refers to glutamic acid residues whether or not the side chain carboxyl is deprotonated.In some embodiments, the XTEN-containing fusion polypeptide comprises a first XTEN and a second XTEN, and the sum of the total number of amino acids in the first XTEN and the total number of amino acids in the second XTEN 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.

[0116] Non-overlapping sequence motifs In some embodiments, the XTEN comprises or is formed from multiple non-overlapping sequence motifs. In some embodiments, at least one of the non-overlapping sequence motifs is recurring (or repeated at least twice in the XTEN) and at least one other of the non-overlapping sequence motifs is non-recurring (or occurs only once in the XTEN). In some embodiments, the multiple non-overlapping sequence motifs comprise: (a) a set of (recurring) non-overlapping sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least twice in the XTEN; and (b) non-overlapping (non-recurring) sequence motifs that occur (or occur only once) in the XTEN. In some embodiments, each non-overlapping sequence motif is 9-14 (or 10-14, or 11-13) 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 comprises a set of non-overlapping (recurring) sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs (1) is repeated at least twice in the XTEN and (2) is between 9 and 14 amino acids in length. In some embodiments, the set of (recurring) non-overlapping sequence motifs comprises the 12-mer sequence motifs identified in Table 1 herein by SEQ ID NOs: 179-200 and 1715-1722. In some embodiments, the set of (recurring) non-overlapping sequence motifs comprises the 12-mer sequence motifs identified in Table 1 herein by SEQ ID NOs: 186-189. In some embodiments, the set of (recurring) 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. Table 1. Exemplary 12-mer sequence motifs for construction of XTENs [Table 1-1] [Table 1-2] *Indicates individual motif sequences that, when used together in various permutations, become a "family of sequences."

[0117] Barcode fragment In some embodiments, a polypeptide comprises a barcode fragment (e.g., a first, second, or third barcode fragment of an XTEN) that is releasable from the polypeptide upon digestion with a protease. In some embodiments, the barcode fragment (1) is a portion of an XTEN that includes at least a portion of a sequence motif that occurs (or is found) only once (non-recurring, non-overlapping) within the XTEN; or (2) is distinct in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon complete digestion of the polypeptide with a protease. Those skilled in the art will understand that the term "barcode fragment" (or "barcode" or "barcode sequence") can refer to either the portion of the XTEN that is cleavably fused within the polypeptide or the resulting peptide fragment that is released from the polypeptide.

[0118] In some embodiments, a barcode fragment does not include the N-terminal or C-terminal amino acid of a polypeptide. As described more fully below or elsewhere herein, in some embodiments, a barcode fragment is releasable (configured to be released) upon Glu-C digestion of the fusion polypeptide. In some embodiments, a barcode fragment does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in the XTEN. In some embodiments, a barcode fragment has a glutamic acid at its C-terminus. Those skilled in the art will understand that when cleavably fused within an XTEN, the C-terminus of a barcode fragment can refer to the "last" (or most C-terminal) amino acid residue within the barcode fragment, even if other "non-barcode" amino acid residues are located C-terminal to the barcode fragment within the same XTEN. In some embodiments, a barcode fragment has an N-terminal amino acid immediately preceded by 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 a (second) glutamic acid residue at a position other than the C-terminus of the barcode fragment, unless the glutamic acid is immediately followed by a proline. In some embodiments, the barcode fragment is located a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10 to 150, or 10 to 125 amino acids. In some embodiments, the barcode fragment is located within, or at, 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 of the N-terminus of the polypeptide, or at a location within a range between any of the foregoing. In some embodiments, the barcode fragment is located within 200 amino acids, within 150 amino acids, within 100 amino acids, or within 50 amino acids of the N-terminus of the polypeptide.In some embodiments, the barcode fragment is located between 10 and 200 amino acids, between 30 and 200 amino acids, between 40 and 150 amino acids, or between 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 range therebetween. 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 amino acids, between 30 and 200 amino acids, between 40 and 150 amino acids, or between 50 and 100 amino acids from the C-terminus of the polypeptide. In some embodiments, the barcode fragment (BAR) is characterized in that it (i) does not contain a glutamic acid, if present, that is immediately adjacent to another glutamic acid in the XTEN; (ii) has a glutamic acid at its C-terminus; (iii) has an N-terminal amino acid immediately preceded by a glutamic acid residue; and (iv) is located at a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10 to 150 amino acids in length, or 10 to 125 amino acids in length. In some embodiments, the barcode fragment (i) does not include the N-terminal or C-terminal amino acid of the polypeptide, (ii) does not include a glutamic acid in the XTEN that is immediately adjacent to another glutamic acid, (iii) has a glutamic acid at its C-terminus, (iv) has an N-terminal amino acid that is immediately preceded by a glutamic acid residue, and (v) is located a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10-150, or 10-125 amino acids in length. In some embodiments, the glutamic acid residue that precedes the N-terminal amino acid is not immediately adjacent to another glutamic acid residue.In some embodiments, a barcode fragment does not contain a glutamic acid residue at a position other than the C-terminus of the barcode fragment, unless the glutamic acid is immediately followed by a proline. Those skilled in the art will understand that the term "distance," as used herein, can refer to the number of amino acid residues from the N-terminus of a polypeptide to the most N-terminal amino acid residue of a barcode fragment, or the number of amino acid residues from the C-terminus of a polypeptide to the most C-terminal amino acid residue of a barcode fragment. In some embodiments, for a barcoded XTEN fused to a biologically active polypeptide, at least one barcode fragment (or at least two barcode fragments, or three barcode fragments) contained in the barcoded XTEN is located at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids from the biologically active polypeptide. In some embodiments, the barcode fragment is at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids in length. In some embodiments, the barcode fragment is at least 4 amino acids in length. In some embodiments, the barcode fragment is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids in length, or within a range between any of the foregoing values. In some embodiments, the barcode fragment is between 4 and 20 amino acids in length, between 5 and 15 amino acids in length, between 6 and 12 amino acids in length, or between 7 and 10 amino acids in length. In some embodiments, the barcode fragment comprises an amino acid sequence identified in Table 2 herein by SEQ ID NOs: 68-77. Table 2. Exemplary barcode fragments that can be released upon Glu-C digestion [Table 2]

[0119] In some embodiments of the polypeptides of the present disclosure, the XTEN has a length defined by a proximal end and a distal end, where (1) the proximal end is located closer to the bispecific antibody construct (BsAb) than the distal end, and (2) the barcode fragment (BAR) can be located within a region of the XTEN spanning between 5% and 50%, between 7% and 40%, or between 10% and 30% of the length of the XTEN, as measured from the distal end.

[0120] In some embodiments of polypeptides of the present disclosure, the XTEN further comprises one or more additional barcode fragments, each of which differs in sequence and molecular weight from all other peptide fragments releasable from the polypeptide upon complete digestion of the polypeptide by the protease. In some embodiments, the barcoded XTEN comprises only one barcode fragment. In some embodiments, the barcoded XTEN comprises a set of barcode fragments, including a first barcode fragment, such as those described above or elsewhere herein. In some embodiments, the set of barcode fragments comprises a second barcode fragment (or additional barcode fragments), such as those described above or elsewhere herein. In some embodiments, the set of barcode fragments comprises a third barcode fragment, such as those described above or elsewhere herein. The set of barcode fragments fused within an N-terminal XTEN may be referred to as the N-terminal set of barcodes (the "N-terminal set"). The set of fused barcode fragments within a C-terminal XTEN may be referred to as a C-terminal set of barcodes ("C-terminal set"). In some embodiments, the N-terminal set comprises a first barcode fragment and a second barcode fragment. In some embodiments, the N-terminal set further comprises a third barcode fragment. In some embodiments, the C-terminal set comprises a first barcode fragment and a second barcode fragment. In some embodiments, the C-terminal set further comprises a third barcode fragment. In some embodiments, the second barcode fragment is located N-terminal to the first barcode fragment of the same set. In some embodiments, the second barcode fragment is located C-terminal to the first barcode fragment of the same set. In some embodiments, the third barcode fragment is located N-terminal to both the first barcode fragment and the second barcode fragment. In some embodiments, the third barcode fragment is located C-terminal to both the first barcode fragment and the second barcode fragment. In some embodiments, the third barcode fragment is located between the first barcode fragment and the second barcode fragment.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 of the set of barcode fragments (1) is part of a second XTEN and (2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by a protease.

[0121] Exemplary barcoded XTEN The amino acid sequences of 13 exemplary barcoded XTENs containing one barcode (e.g., SEQ ID NOs: 8002-8003, 8005-8009, and 8013), or two barcodes (e.g., SEQ ID NOs: 8001, 8004, and 8012), or three barcodes (e.g., SEQ ID NO: 8011) are shown in Table 3a. Of these 13 exemplary barcoded XTENs, six (SEQ ID NOs: 8001-8003, 8008-8009, and 8011) will be fused to the C-terminus of a biologically active protein, and seven (SEQ ID NOs: 8004-8007, 8010, and 8012-8013) will be fused to the N-terminus of a biologically active protein. In some embodiments, the XTEN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 8001-8020 in Table 3a. Table 3a. Exemplary barcoded XTEN [Table 3a-1] [Table 3a-2] [Table 3a-3] [Table 3a-4] [Table 3a-5] Table 3a-6

[0122] In some embodiments, barcoded XTENs can be obtained by adding one or more mutations to a generic XTEN, such as any listed in Table 3b, according to one or more of the following criteria: minimizing sequence changes in the XTEN, minimizing changes in amino acid composition in the XTEN, substantially maintaining the net charge of the XTEN, substantially maintaining (or improving) the low immunogenicity of the XTEN, and substantially maintaining (or improving) the pharmacokinetic properties of the XTEN. In some embodiments, the XTEN sequence has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs:601-659 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 to any of SEQ ID NOs:601-659 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, fewer than 2 mutations) of the corresponding sequence from Table 3b. In some embodiments, the one or more mutations comprise a deletion of, an insertion of, a substitution with, or a substitution of a glutamic acid residue, or any combination thereof. In some embodiments in which the XTEN sequence differs from, but has at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity to, any one of SEQ ID NOs:601-659 listed in Table 3b, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the differences between the XTEN sequence and the corresponding sequence in Table 3b include deletions of, insertions of, substitutions with, or substitutions of glutamic acid residues, or any combination thereof.In some embodiments, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the differences between an XTEN sequence and the corresponding sequence in Table 3b include substitutions with glutamic acid residues, substitutions of glutamic acid residues, or both. The term "substitution with a first amino acid," as used herein, refers to the replacement of a second amino acid residue with a first amino acid residue, thereby resulting in the second amino acid residue occupying the substitution position in the resulting sequence. For example, "substitution with glutamic acid" refers to the replacement of a non-glutamic acid residue (e.g., serine (S)) with a glutamic acid residue (E). The term "substitution of a first amino acid," as used herein, refers to the replacement of a first amino acid residue with a second amino acid residue, thus resulting in the first amino acid residue occupying the substitution position in the resulting sequence. For example, "substitution of glutamic acid" refers to the replacement of a glutamic acid residue with a non-glutamic acid residue (e.g., serine (S)). Table 3b. Exemplary generic XTENs for engineering into barcoded XTENs [Table 3b-1] [Table 3b-2] [Table 3b-3] [Table 3b-4] [Table 3b-5] [Table 3b-6] [Table 3b-7] [Table 3b-8] [Table 3b-9] [Table 3b-10] [Table 3b-11]

[0123] In some embodiments, for construction of barcoded XTEN sequences, amino acid mutations are made to XTENs of intermediate length relative to those in Table 3b, and to XTENs of longer length than those in Table 3b, for example, those in which one or more 12-mer motifs from Table 1 are added to the N- or C-terminus of the generic XTENs of Table 3b.

[0124] Additional examples of generic XTEN sequences that can be used in accordance with the present disclosure are described in U.S. Patent Application Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1, or International Patent Application Publication Nos. These methods are disclosed in WO2010091122A1, WO2010144502A2, WO2010144508A1, WO2011028228A1, WO2011028229A1, WO2011028344A2, WO2014 / 011819A2, or WO2015 / 023891.

[0125] In some embodiments, a barcoded XTEN fused within a polypeptide chain adjacent to the N-terminus of the polypeptide chain (an "N-terminal XTEN") can be attached to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus to facilitate purification of the fusion protein. In some embodiments, a barcoded XTEN fused within a polypeptide chain at the C-terminus of the polypeptide chain (a "C-terminal XTEN") can include or have attached to it the sequence EPEA at the C-terminus to facilitate purification of the fusion protein. In some embodiments, the fusion polypeptide comprises both an N-terminally barcoded XTEN and a C-terminally barcoded XTEN, wherein the N-terminally barcoded XTEN is attached at its N-terminus to a His tag of the sequence HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49), and the C-terminally barcoded XTEN is attached at its C-terminus to the sequence EPEA, thereby facilitating purification of the fusion polypeptide to a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% by chromatographic methods known in the art, including, but not limited to, IMAC chromatography, C-tagXL affinity matrix, and other such methods, including, but not limited to, those described in the Examples section below.

[0126] Protease digestion The barcode fragments described above or elsewhere herein can be cleavably fused within an XTEN and can be (configured to be) releasable from the XTEN upon digestion of the polypeptide by a protease. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease cleaves C-terminal to glutamic acid residues that are not followed by proline. Those skilled in the art will understand that barcoded XTENs (XTENs containing barcode fragments 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 upon Glu-C digestion. Thus, if Glu-C protease is used for barcode release, the barcoded XTEN or barcode fragment may not contain any Glu-Glu (EE) sequences. It will also be understood by those skilled in the art that the dipeptide Glu-Pro (EP) sequence, if present in the fusion polypeptide, cannot be cleaved by Glu-C protease during the barcode release process.

[0127] Configuration of the BPXTEN structure In some embodiments, a BPXTEN fusion protein comprises a single BP and a single XTEN. Such a BPXTEN may have at least the following configurational permutations, listed from N- to C-terminus: BP-XTEN, XTEN-BP, BP-S-XTEN, and XTEN-S-BP.

[0128] In some embodiments, BPXTEN comprises a C-terminal XTEN and, optionally, a spacer sequence (S) (e.g., as described herein, e.g., in Table C) between the XTEN and the BP. Such BPXTEN has Formula I (depicted from N-terminus to C-terminus): (BP)-(S) x -(XTEN) (I) where BP is a biologically active protein as described herein below; S is a spacer sequence having between 1 and about 50 amino acid residues (e.g., those described herein, e.g., in Table C), which may optionally include a BP release segment (as described more fully herein below); x is either 0 or 1; and XTEN can be any of those described herein above or elsewhere herein.

[0129] In some embodiments, BPXTEN comprises an N-terminal XTEN and, optionally, a spacer sequence (S) (e.g., as described herein, e.g., in Table C) between the XTEN and the BP. Such BPXTEN has Formula II (depicted from N-terminus to C-terminus): (XTEN)-(S) x -(BP) (II) where BP is a biologically active protein as described herein below; S is a spacer sequence (as described herein, e.g., in Table C) having between 1 and about 50 amino acid residues, which may optionally include a BP release segment (as described more fully herein below); x is either 0 or 1; and XTEN can be any of those described herein above or elsewhere herein.

[0130] In some embodiments, the BPXTEN comprises both an N-terminal XTEN and a C-terminal XTEN. Such a BPXTEN (e.g., XPAT in Figures 1-2) has the formula III: (XTEN)-(S) y -(BP)-(S) z -(XTEN) (III) where BP is a biologically active protein as described herein below; S is a spacer sequence (as described herein, e.g., in Table C) having between 1 and about 50 amino acid residues, which may optionally include a BP release segment (as described more fully herein below); y is either 0 or 1; z is either 0 or 1; and XTEN can be any described herein above or elsewhere herein.

[0131] Bioactive Polypeptides

[0132] Biologically active proteins (BPs) fused to XTEN (as described herein above or elsewhere herein), particularly those disclosed herein below, including the sequences identified herein by those in Tables 6a-6f, along with their corresponding nucleic acid and amino acid sequences, 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 may be a wild-type polynucleotide sequence (e.g., either full-length or mature) encoding a given BP, or in some cases, the sequence may be a variant of the wild-type polynucleotide sequence (e.g., a polynucleotide encoding a wild-type biologically active protein), where the DNA sequence of the polynucleotide has been optimized, for example, for expression in a particular species, or may be a polynucleotide encoding a variant of the wild-type protein, e.g., a site-directed mutant or allelic variant. It is well within the capabilities of one of ordinary skill in the art to use wild-type or consensus cDNA sequences or codon-optimized variants of BP to generate BPXTEN constructs contemplated by the present invention using methods known in the art and / or in conjunction with the guidance and methods provided herein.

[0133] BPs for inclusion in BPXTEN (a fusion polypeptide comprising at least one BP and at least one XTEN) can include any protein of biological, therapeutic, prophylactic, or diagnostic interest, or any protein useful in mediating biological activity or preventing or ameliorating a disease, disorder, or condition when administered to a subject. Of particular interest are BPs that seek increased pharmacokinetic parameters, increased solubility, improved stability, masked activity, or some other enhanced pharmaceutical property, or BPs whose extended terminal half-life would improve efficacy, safety, or result in reduced dosing frequency and / or improved patient compliance. Thus, BPXTEN fusion protein compositions are prepared with various objectives in mind, including improving the therapeutic efficacy of a biologically active compound, for example, by increasing in vivo exposure or extending the length of time that BPXTEN remains within the therapeutic window when administered to a subject, compared to a BP not linked to an XTEN.

[0134] The BP may be a native, full-length protein, or may be a fragment or sequence variant of a biologically active protein that retains at least a portion of the biological activity of the native protein.

[0135] In one embodiment, the BP incorporated into the subject compositions can be a recombinant polypeptide having a sequence corresponding to a protein found in nature. In some embodiments, the BP can be a sequence variant, fragment, homolog, or mimic of a native sequence that retains at least a portion of the biological activity of the native BP. In a non-limiting example, the BP can be a sequence that exhibits at least about 80% sequence identity, 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 to a protein sequence identified herein. In a further non-limiting example, the BP is a bispecific sequence comprising a first binding domain and a second binding domain, wherein the first binding domain has specific binding affinity for a tumor-specific marker or antigen of a target cell and has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 17 The second binding domain exhibiting 8%, 99%, or 100% sequence identity and having specific binding affinity for effector cells can be a bispecific sequence exhibiting at least about 80% sequence identity, 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, to the VL and VH sequences of a pair of anti-target cell antibodies identified herein by Table 6a. In one embodiment, a BPXTEN fusion protein can comprise a single BP molecule linked to XTEN. In some embodiments, BPXTEN can include a first BP and a second molecule of the same BP, resulting in a fusion protein containing two BPs linked to one or more XTENs (e.g., two molecules of glucagon, or two molecules of hGH).

[0136] Generally, a BP will exhibit binding specificity for a given target (or a given number of targets) or another desired biological characteristic when used in vivo or in an in vitro assay. For example, the BP can be an agonist, receptor, ligand, antagonist, enzyme, antibody (e.g., monospecific or bispecific), or hormone. Of particular interest are BPs used in, or known to be useful for, diseases or disorders where the native BP has a relatively short terminal half-life and improved pharmacokinetic parameters (which can be released on demand from the fusion protein by cleavage of the spacer sequence) allow for less frequent administration or enhanced pharmacological effect. Also of interest are BPs with a narrow therapeutic window between the minimum effective dose or blood concentration (Cmin) and the maximum tolerated dose or blood concentration (Cmax). In such cases, linking the BP to a fusion protein containing a selected XTEN sequence can result in improved properties, thus making them useful as therapeutic or prophylactic agents compared to BPs not linked to XTEN.

[0137] The BPs encompassed by the compositions of the present invention may be useful in treating a variety of therapies or disease categories, including, but not limited to, glucose and insulin disorders, metabolic disorders, cardiovascular diseases, coagulation and bleeding disorders, growth disorders or conditions, endocrine disorders, ocular diseases, renal diseases, liver diseases, tumorigenic conditions, inflammatory conditions, autoimmune conditions, and the like.

[0138] "Anti-CD3" refers to monoclonal antibodies against the T cell surface protein CD3, its species and sequence variants, and fragments, including OKT3 (also known as muromonab) and humanized anti-CD3 monoclonal antibody (hOKT31(Ala-Ala)) (KC Herold et al., New England Journal of Medicine 346: 1692-1698, 2002). Anti-CD3 prevents T cell activation and proliferation by binding to the T cell receptor complex, which is present on all differentiated T cells. The anti-CD3-containing fusion proteins of the present invention may be particularly useful for delaying incipient type 1 diabetes, including the use of anti-CD3 as a therapeutic effector, as well as the use of anti-CD3 as a targeting moiety for a second therapeutic BP in a BPXTEN composition. The variable region sequence of anti-CD3 and the production of anti-CD3 are described in U.S. Patent Nos. 5,885,573 and 6,491,916.

[0139] The BPs of the subject compositions are not limited to naturally occurring, full-length polypeptides, but also include recombinant versions and biologically and / or pharmacologically active variants or fragments thereof. For example, it will be understood that various amino acid substitutions can be made in a BP to create variants without departing from the spirit of the present invention with respect to the biological activity or pharmacological properties of the BP. Examples of conservative substitutions of amino acids in a polypeptide sequence are shown in Table 5. However, in embodiments of BPXTEN where the BP has less than 100% sequence identity with a particular sequence disclosed herein, the present invention contemplates the substitution of a given amino acid residue of a given BP, which may be located anywhere within the sequence of the BP, including adjacent amino acid residues, with any of the other 19 naturally occurring L-amino acids. If any one substitution results in an undesirable change in biological activity, one of the alternative amino acids can be utilized, and constructs can be evaluated by the methods described herein, or using any of the teachings and guidelines for conservative and non-conservative mutations set forth, for example, in U.S. Pat. No. 5,364,934 (the contents of which are incorporated herein by reference in their entirety), or using methods generally known to those of skill in the art. In addition, variants can also include, for example, polypeptides in which one or more amino acid residues have been added or deleted at the N- or C-terminus of the full-length native amino acid sequence of the BP, which retain at least a portion of the biological activity of the native peptide.

[0140] Table 5: Exemplary Conservative Amino Acid Substitutions [Table 5]

[0141] In some embodiments, the BP incorporated into the BPXTEN fusion protein may have a sequence that exhibits at least about 80% sequence identity to a sequence, 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. In some embodiments, the BP incorporated into BPXTEN is a bispecific sequence comprising a first binding domain and a second binding domain, wherein the first binding domain has specific binding affinity for a tumor-specific marker or antigen of a target cell and has at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 1 The second binding domain exhibiting 97%, 98%, 99%, or 100% sequence identity and having specific binding affinity for effector cells may be a bispecific sequence exhibiting at least about 80% sequence identity, 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, to the VL and VH sequences of a pair of anti-target cell antibodies identified herein by Table 6a. The BPs of the foregoing embodiments can be evaluated for activity using the assays or measured or determined parameters described herein, and 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 sequence will be considered suitable for incorporation into the subject BPXTEN.BPs found to retain a suitable level of activity can be linked to one or more XTEN polypeptides described herein above or elsewhere herein. In one embodiment, BPs found to retain a suitable level of activity can be linked to one or more XTEN polypeptides having at least about 80% sequence identity (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) to a sequence from Tables 3a-3b to form a chimeric fusion protein.

[0142] T Cell Engager Additional structural configurational formulas of BPXTEN relate to Xtenylated protease-activated T cell engagers ("XPATs" or "XPATs") (e.g., bispecific T cell engagers) in which BP is a bispecific antibody. In some embodiments, the XPAT composition comprises: (1) a first portion comprising a first binding domain and a second binding domain; and (2) a second portion comprising a release segment, and (3) a third portion comprising a bulking portion. In some embodiments, the XPAT composition has the configuration of Formula Ia (depicted from N-terminus to C-terminus): (First part)-(Second part)-(Third part)(Ia) wherein the first portion is bispecific, comprising two scFvs, wherein the first binding domain has specific binding affinity for a tumor-specific marker or antigen of a target cell and the second binding domain has specific binding affinity for an effector cell; the second portion comprises a release segment (RS) that can be cleaved by a mammalian protease (as explained more fully herein below, the protease can be activated by being tumor-specific or antigen-specific); and the third portion is a bulking moiety. In the foregoing embodiments, the binding domains of the first portion may be in the order (VL-VH)i-(VL-VH)2 (where "i" and "2" represent the first and second binding domains, respectively), or (VL-VH)i-(VH-VL)2, or (VH-VL)i-(VL-VH)2, or (VH-VL)i-(VH-VL)2 (where the paired binding domains are linked by a polypeptide linker (as described more fully herein below)). In one embodiment, the first portions VL and VH are set forth in Tables 6a-6f; RS is identified herein by the group of sequences set forth in Tables 8a-8b (as described more fully below); and the bulking moiety is XTEN, an albumin binding domain, albumin, an IgG binding domain, a polypeptide consisting of proline, serine, and alanine, a fatty acid, an Fc domain, polyethylene glycol (PEG), PLGA, or hydroxyethyl starch. If desired, the bulking moiety is an XTEN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Tables 3a-3b. In the foregoing embodiments, the composition is a recombinant fusion protein. In some embodiments, the moieties are linked by chemical conjugation.

[0143] In some embodiments, the XPAT composition has the configuration of Formula IIa (depicted N-terminus to C-terminus): (Third Part)-(Second Part)-(First Part)(IIa) wherein the first portion is bispecific, comprising two scFvs, wherein a first binding domain has specific binding affinity for a tumor-specific marker or antigen of a target cell and a second binding domain has specific binding affinity for an effector cell; the second portion comprises a release segment (RS) that can be cleaved by a mammalian protease; and the third portion is a bulking portion. In the foregoing embodiment, the binding domains of the first portion can be in the order (VL-VH)1-(VL-VH)2 (where "1" and "2" represent the first and second binding domains, respectively), or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VH-VL)2 (where the paired binding domains are linked by a polypeptide linker as described herein). In one embodiment, the first portions VL and VH are identified in Tables 6a-6f; RS is identified herein as a group of sequences set forth in Tables 8a-8b; and the bulking moiety is XTEN, an albumin-binding domain, albumin, an IgG-binding domain, a polypeptide consisting of proline, serine, and alanine, a fatty acid, or an Fc domain. If desired, the bulking moiety is XTEN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Tables 3a-3b. In the foregoing embodiments, the composition is a recombinant fusion protein. In some embodiments, the moieties are linked by chemical conjugation.

[0144] In some embodiments, the XPAT composition has the configuration of Formula IIIa (depicted from N-terminus to C-terminus): (5th part)-(4th part)-(1st part)-(2nd part)-(3rd part) (IIIa) wherein the first portion is bispecific, comprising two scFvs, wherein the first binding domain has specific binding affinity for a tumor-specific marker or antigen of a target cell and the second binding domain has specific binding affinity for an effector cell; the second portion comprises a release segment (RS) that can be cleaved by a mammalian protease; the third portion is a bulking moiety; the fourth portion comprises a release segment (RS) that can be cleaved by a mammalian protease, which may be the same as or different from the second portion; and the fifth portion is a bulking moiety, which may be the same as or different from the third portion. In the foregoing embodiments, the binding domains of the first portion can be in the order (VL-VH)1-(VL-VH)2 (where "1" and "2" represent the first and second binding domains, respectively), or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2 (where the paired binding domains are linked by a polypeptide linker as described herein). In the foregoing embodiments, the RS is identical to a sequence set forth in Tables 8a-8b. In the foregoing embodiments, the bulking moiety is XTEN, an albumin binding domain, albumin, an IgG binding domain, a polypeptide consisting of proline, serine, and alanine, a fatty acid, or an Fc domain. If desired, the bulking moiety is an XTEN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by the sequences set forth in Tables 3a-3b. In the foregoing embodiments, the composition is a recombinant fusion protein. In some embodiments, the moieties are linked by chemical conjugation.

[0145] Based on their design and specific components, the subject compositions advantageously provide bispecific therapeutics that, when cleaved by proteases found associated with target tissues or tissues rendered unhealthy by disease, have greater selectivity, longer half-lives, leading to lower toxicity and fewer side effects; thus, the subject compositions have an improved therapeutic index compared to bispecific antibody compositions known in the art. Such compositions are useful for the treatment of certain diseases, including, but not limited to, cancer. Those skilled in the art will understand that the compositions of the invention achieve this reduction in nonspecific interactions through a combination of mechanisms, including steric hindrance due to the placement of the binding domains on bulky XTEN molecules, and steric hindrance, whereby the flexible, unstructured character of the long, flexible XTEN polypeptide, tethered to the composition, allows it to oscillate and rotate around the binding domains, thereby providing a barrier between the composition and the tissue or cell, as well as a reduction in the ability of the intact composition to enter cells or tissues due to its large molecular weight relative to the size of the individual binding domains (both resulting in the actual molecular weight of the XTEN and due to the large hydrodynamic radius of the unstructured XTEN). However, the compositions are designed so that when in proximity to target tissues or cells that possess or secrete proteases capable of cleaving the RS, or when internalized into the target cells or tissues upon binding of the binding domains to a ligand, the bispecific binding domains will be liberated from the bulk of the XTEN by the action of the protease, thereby removing the steric hindrance barrier and allowing them to more freely exert their pharmacological effects. The subject compositions are used to treat a variety of conditions in which selective delivery of a therapeutic bispecific antibody composition to a cell, tissue, or organ is desirable, hi one embodiment, the target tissue is a cancer, which may be a leukemia, lymphoma, or organ or system tumor.

[0146] Binding domain The present disclosure contemplates 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-domain antibody molecules (scFv), and diabodies, that can bind to a ligand or receptor associated with an effector cell and can bind to an antigen of a diseased tissue or cell, such as a cancer, tumor, or other malignant tissue. In some embodiments, the antigen-binding fragment (AF) (e.g., the first antigen-binding fragment (AF1) or the second antigen-binding fragment (AF2)) can (each independently) be a chimeric or humanized antigen-binding fragment. The antigen-binding fragment (AF) (e.g., the first antigen-binding fragment (AF1) or the second antigen-binding fragment (AF2)) can (each independently) be an Fv, Fab, Fab', Fab'-SH, linear antibody, or single-chain variable fragment (scFv). The two antigen-binding fragments (e.g., the first and second antigen-binding fragments) can be configured as (Fab')2 or single-chain diabodies. In some embodiments, the bispecific comprises a first binding domain with binding specificity for a target cell marker and a second binding domain with binding specificity for an effector cell antigen. In some embodiments, the first and second binding domains can be non-antibody scaffolds, such as anticalins, adnectins, phenomers, affilins, affibodies, centilins, or DARPins. In other embodiments, the tumor cell-targeted binding domain is a variable domain of a T cell receptor engineered to bind to an MHC carrying a peptide fragment of a protein overexpressed by tumor cells. In some embodiments, the XPAT composition is designed to provide a broad therapeutic window, taking into account the location of the target tissue protease and the presence of that protease in healthy tissues intended to be untargeted, and the presence of the target ligand in healthy tissues, but the greater presence of that ligand in unhealthy target tissues. "Therapeutic range" refers to the maximum difference between the minimum effective dose and the maximum tolerated dose for a given therapeutic composition.To facilitate achieving a broad therapeutic window, the binding domain of a first portion of the composition is shielded by the proximity of a bulking moiety (e.g., XTEN), such that the binding affinity of the intact composition for one or both ligands is reduced compared to a composition cleaved with a mammalian protease, thereby freeing the first portion from the shielding effect of the bulking moiety.

[0147] With regard to single-chain binding domains, it is well established that Fvs are the smallest antibody fragments containing a complete antigen recognition and binding site and consist of a dimer of one noncovalently associated heavy-chain variable domain (VH) and one light-chain variable domain (VL). Within each VH and VL chain, there are three complementarity-determining regions (CDRs) that interact to define an antigen-binding site on the surface of the VH-VL dimer, and these six CDRs of the binding domain confer antigen-binding specificity to the antibody or single-chain binding domain. In some cases, scFvs are produced, each with three, four, or five CHRs within each binding domain. The framework sequences flanking the CDRs have a tertiary structure that is essentially conserved in native immunoglobulins across species, and the framework residues (FRs) serve to hold the CDRs in their proper orientation. The constant domains are not required for binding function but may help stabilize the VH-VL interaction. In some embodiments, the domains of a polypeptide binding site can be VH-VL, VH-VH, or VL-VL domain pairs from either the same or different immunoglobulins, although it is generally preferred to create a single-chain binding domain using the respective 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 present invention, and the order of a given domain can usually be reversed without any loss of function, although of course the VH and VL domains are positioned such that the antigen-binding site can fold correctly. Thus, the single-chain binding domains of the bispecific scFv embodiments of the subject compositions may be in the order (VL-VH)1-(VL-VH)2 (where "1" and "2" represent the first and second binding domains, respectively), or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2 (where the paired binding domains are linked by a polypeptide linker, as described herein below).

[0148] Therefore, the binding domain arrangement in the exemplary bispecific single-chain antibodies disclosed herein can be such that the first binding domain is located C-terminal to the second binding domain. The V chain arrangement can be VH(target cell surface antigen)-VL(target cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), VH(target cell surface antigen)-VL(target cell surface antigen)-VH(effector cell antigen)-VL(effector cell antigen), VL(target cell surface antigen)-VH(target cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), or VL(target cell surface antigen)-VH(target cell surface antigen)-VH(effector cell antigen)-VL(effector cell antigen). For an arrangement in which the second binding domain is located N-terminal to the first binding domain, the following orders are possible: VH(effector cell antigen)-VL(effector cell antigen)-VL(target cell surface antigen)-VH(target cell surface antigen), VH(effector cell antigen)-VL(effector cell antigen)-VH(target cell surface antigen)-VL(target cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen)-VL(target cell surface antigen)-VH(target cell surface antigen), or VL(effector cell antigen)-VH(effector cell antigen)-VH(target cell surface antigen)-VL(target cell surface antigen). As used herein, "N-terminal to" or "C-terminal to" and grammatical variations refer to relative positions within the primary amino acid sequence rather than to the absolute N- or C-terminus of the bispecific single chain antibody. Thus, as a non-limiting example, a first binding domain "located C-terminal to a second binding domain" means that the first binding domain is located carboxyl-terminal to the second binding domain within the bispecific single chain antibody, and does not exclude the possibility that an additional sequence, e.g., a His tag, or another compound, e.g., a radioisotope, may be located at the C-terminus of the bispecific single chain antibody.

[0149] In one embodiment, the chimeric polypeptide assembly composition comprises a first portion comprising a first binding domain and a second binding domain, each of which is an scFv, each scFv comprising one VL and one VH. In some embodiments, the chimeric polypeptide assembly composition comprises a first portion comprising a first binding domain and a second binding domain, each of which is in a diabody configuration, each domain comprising one VL domain and one VH.

[0150] An scFv embodiment of an XPAT composition of the invention comprises a first binding domain and a second binding domain, wherein the VL and VH domains are derived from a monoclonal antibody having binding specificity for a tumor-specific marker or antigen of a target cell and an effector cell antigen, respectively. In other cases, the first and second binding domains each comprise six CDRs derived from a monoclonal antibody having binding specificity for a target cell marker, e.g., a tumor-specific marker, and an effector cell antigen, respectively. In other embodiments, the first and second binding domains of the first portion of the subject composition may have three, four, or five CHRs within each binding domain. In other embodiments, embodiments of the 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, each of which is derived from a monoclonal antibody capable of binding to a tumor-specific marker or antigen of a target cell and an effector cell antigen, respectively. In one embodiment, the present invention provides a chimeric polypeptide assembly composition in which the second binding domain comprises VH and VL regions derived from a monoclonal antibody capable of binding to human CD3. In some embodiments, the present invention provides a chimeric polypeptide assembly composition in which the second binding domain of an scFv comprises VH and VL regions, and 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 to, or is identical to, the VL and VH sequences of a pair of anti-CD3 antibodies identified in Table 6a. In some embodiments, the second domain embodiments of the present invention comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-H3 regions, each of which regions is derived from a monoclonal antibody identified herein as an antibody set forth in Table 6a. In the foregoing embodiments, the VH and / or VL domains may be configured as an scFv, a diabody, a single domain antibody, or a single domain camelid antibody.

[0151] In other embodiments, the second domain of the subject compositions is derived from an anti-CD3 antibody identified herein as an antibody set forth in Table 6a. In one such embodiment, the second binding domain of the subject compositions comprises paired VL and VH region sequences of an anti-CD3 antibody identified herein as the group of antibodies set forth in Table 6a. In some embodiments, the invention provides chimeric polypeptide assembly compositions in which the second binding domain comprises VH and VL regions, each VH and VL region exhibiting at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to, or identical to, the paired VL and VH sequences of the huUCHT1 anti-CD3 antibody of Table 6a. In the foregoing embodiments, the VH and / or VL domains may be configured as an scFv, a portion of a diabody, a single-domain antibody, or a single-domain camelid antibody.

[0152] In other embodiments, the scFv of the first domain of the composition is derived from an anti-tumor cell antibody identified as an antibody listed in Table 6f. In some embodiments, the present invention provides chimeric polypeptide assembly compositions in which the first binding domain comprises a VH and a VL region, and 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 to, or is identical to, the VL and VH sequences of a pair of anti-tumor cell antibodies identified in Table 6f. In some of the foregoing embodiments, the first domain of the described composition comprises the VL and VH region sequences of a pair of anti-tumor cell antibodies disclosed herein. In the foregoing embodiments, the VH and / or VL domains may be configured as an scFv, a portion of a diabody, a single-domain antibody, or a single-domain camelid antibody.

[0153] In some embodiments, a chimeric polypeptide assembly composition comprises a first portion comprising a first binding domain and a second binding domain, wherein the binding domains are in a diabody configuration, and each of the binding domains comprises one VL domain and one VH domain. In one embodiment, a diabody embodiment of the present invention comprises a first binding domain and a second binding domain, wherein the VL and VH domains are derived from a monoclonal antibody having binding specificity for a tumor-specific marker or antigen of a target cell and for an effector cell antigen, respectively. In some embodiments, a diabody embodiment of the present invention comprises a first binding domain and a second binding domain, each of which 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, each of which is derived from a monoclonal antibody capable of binding to a tumor-specific marker or antigen of a target cell and to an effector cell antigen, respectively. Diabody embodiments of the present invention comprise a first binding domain and a second binding domain, the VL and VH domains of which are derived from monoclonal antibodies with binding specificity for a tumor-specific marker or target cell antigen and for an effector cell antigen, respectively. In some embodiments, diabody embodiments of the present invention comprise a first binding domain and a second binding domain, each of which 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, each of which is derived from a monoclonal antibody capable of binding to a tumor-specific marker or target cell antigen and to an effector cell antigen, respectively. In one embodiment, the present invention provides a chimeric polypeptide assembly composition in which the second binding domain of the diabody comprises paired VH and VL regions derived from a monoclonal antibody capable of binding to human CD3.In some embodiments, the present invention provides chimeric polypeptide assembly compositions in which the second binding domain of a diabody comprises a VH and a VL region, and 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 to, or is identical to, the VL and VH sequences of a pair of anti-CD3 antibodies identified in Table 6a. In some embodiments, the present invention provides chimeric polypeptide assembly compositions in which the second binding domain of a diabody comprises a VH and a VL region, and 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 to, or is identical to, the VL and VH sequences of a huUCHT1 antibody in Table 6a. In other embodiments, the second binding domain of the diabody of the composition is derived from an anti-CD3 antibody described herein. In some embodiments, the present invention provides chimeric polypeptide assembly compositions in which the first binding domain of the diabody comprises a VH and a VL region, and 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 to, or is identical to, the VL and VH sequences of an anti-tumor cell antibody identified in Table 6f. In other embodiments, the first domain of the diabody of the composition is derived from an anti-tumor cell antibody described herein.

[0154] Methods for measuring the binding affinity and / or other biological activities of the subject compositions of the present invention can be those disclosed herein or methods generally known in the art. For example, K dThe binding affinity of a binding pair (e.g., antibody and antigen), as indicated by *, can be determined using a variety of suitable assays, including, but not limited to, radioactive binding assays, non-radioactive binding assays, such as fluorescence resonance energy transfer and surface plasmon resonance (SPR, Biacore), as well as enzyme-linked immunosorbent assays (ELISAs), equilibrium exclusion assays (KinExA®), or those described in the Examples. An increase or decrease in binding affinity, for example, an increase or decrease in binding affinity of a chimeric polypeptide assembly that has been cleaved to remove a bulking moiety compared to a chimeric polypeptide assembly with the bulking moiety attached, can be determined by measuring the binding affinity of the chimeric polypeptide assembly to its target binding partner with or without the bulking moiety.

[0155] The half-life of a subject chimeric assembly can be measured by a variety of suitable methods. For example, the half-life of a substance can be determined by administering the substance to a subject and periodically withdrawing a biological sample (e.g., a bodily fluid, such as blood, plasma, or ascites) to determine the concentration and / or amount of the substance in the sample over time. The concentration of the substance in the biological sample can be determined using a variety of suitable methods, including enzyme-linked immunosorbent assay (ELISA), immunoblot, and chromatographic techniques, including high-pressure liquid chromatography and fast protein-lipid chromatography. In some cases, the substance can be labeled with a detectable tag, e.g., a radioactive or fluorescent tag, that can be used to determine the concentration of the substance in a sample (e.g., a blood or plasma sample). Various pharmacokinetic parameters are then determined from the results; this determination can be made using a software package, such as SoftMax Pro software, or by manual calculations known in the art.

[0156] Additionally, the physicochemical properties of the chimeric polypeptide assembly compositions can be measured to confirm retention of solubility, structure, and stability. Assays of the subject compositions can be performed to determine the binding dissociation constant (K d , K. on and K. off ), the half-life of dissociation of the ligand-receptor complex, and the activity of the binding domain to inhibit the biological activity of the captured ligand compared to the free ligand (IC 50 This allows the determination of the binding characteristics of the binding domain to the ligand, including the IC value. 50 " refers to the concentration required to inhibit half of the maximal biological response of a ligand agonist, and is generally determined by competitive binding assays. 50 " refers to the concentration required to achieve half of the maximal biological response of an active agent, generally determined by ELISA or cell-based assays (including the methods of the Examples described herein).

[0157] Anti-CD3 binding domain In some embodiments, the present invention provides chimeric polypeptide assembly compositions comprising a first portion binding domain having binding affinity for T cells. In one embodiment, the second portion binding domain comprises a VL and a VH derived from a monoclonal antibody that binds to CD3. In some embodiments, the binding domain comprises a VL and a VH derived from a monoclonal antibody against CD3 epsilon and / or CD3 delta. Illustrative, non-limiting examples of VL and VH sequences of monoclonal antibodies against CD3 are provided in Table 6a. In one embodiment, the present invention provides chimeric polypeptide assemblies comprising a binding domain having binding affinity for CD3, comprising the anti-CD3 VL and VH sequences set forth in Table 6a. In some embodiments, the present invention provides chimeric polypeptide assemblies comprising a first portion binding domain having binding affinity for CD3 epsilon, comprising the anti-CD3 epsilon VL and VH sequences set forth in Table 6a. In some embodiments, the present invention provides a chimeric polypeptide assembly composition wherein the second binding domain of the scFv of the first portion comprises a VH and a VL region, and 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 to, or is identical to, a pair of VL and VH sequences of the huUCHT1 anti-CD3 antibody in Table 6a. In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a binding domain having binding affinity for CD3, comprising a CDR-L1 region, a CDR-L2 region, a CDR-L3 region, a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region, each derived from a respective anti-CD3 VL and VH sequence listed in Table 6a.In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a binding domain having binding affinity for CD3, the binding domain comprising a CDR-L1 region, a CDR-L2 region, a CDR-L3 region, a CDR-H1 region, a CDR-H2 region and a CDR-H3 region, wherein the CDR sequences are RASQDIRNYLN (SEQ ID NO: 50), YTSRLESQQGNTLPWT (SEQ ID NO: 78), GYSFTGYTMN (SEQ ID NO: 79), LINPYKGVST (SEQ ID NO: 80), and SGYYGDSDWYFDV (SEQ ID NO: 81).

[0158] The CD3 complex is a group of cell surface molecules associated with the T cell antigen receptor (TCR) that functions in the cell surface expression of the TCR and in the signaling cascade that occurs upon binding of a peptide:MHC ligand to the TCR. Typically, upon binding of an antigen to the T cell receptor, CD3 sends a signal across the cell membrane to the cytoplasm within the T cell. This results in T cell activation, which causes the T cell to rapidly divide and produce new T cells primed to attack the specific antigen exposed to the TCR. The CD3 complex is composed of the CD3 epsilon molecule along with four other membrane-bound polypeptides (CD3-gamma, -delta, and / or -zeta). In humans, CD3-epsilon is encoded by the CD3E gene on chromosome 11. The intracellular domain of each CD3 chain contains an immunoreceptor tyrosine-based activation motif (ITAM), which serves as a nucleation point for the intracellular signaling machinery upon T cell receptor engagement.

[0159] Some therapeutic strategies target TCR signaling to modulate T cell immunity, especially anti-human CD3 monoclonal antibodies (mAbs), which are widely used clinically in immunosuppressive regimens. The CD3-specific murine mAb OKT3 was the first mAb approved for human use (Sgro, C. Side-effects of a monoclonal antibody, muromonab CD3 / orthoclone OKT3: bibliographic review. Toxicology 105:23-29, 1995), and has been widely used clinically as an immunosuppressant in transplantation (Chatenoud, Clin. Transplant 7:422-430, (1993); Chatenoud, 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 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)). Notably, Wong's work demonstrated that target killing can be achieved by cross-linking CD3 T cells with target cells, and that neither FcR-mediated ADCC nor complement fixation is required for bivalent anti-CD3 MABs to lyse target cells.

[0160] OKT3 exhibits both mitogenic and T cell killing activity in a time-dependent manner, and after the initial activation of T cells leading to cytokine release, further administration of OKT3 subsequently blocks all known T cell functions. It is this subsequent blockade of T cell function that has led OKT3 to find broad application as an immunosuppressant in therapeutic regimens for reducing or even eliminating allograft tissue rejection. Other antibodies specific for the CD3 molecule are disclosed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50, WO2005 / 118635 and WO2007 / 033230 describe anti-human monoclonal CD3 epsilon antibodies, U.S. Pat. No. 5,821,337 describes the VL and VH sequences of the murine 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), and U.S. Patent Application Publication No. 20120034228 discloses binding domains capable of binding to epitopes of the human and non-chimpanzee primate CD3 epsilon chain. Table 6a: Anti-CD3 monoclonal antibodies and sequences [Table 6a-1] [Table 6a-2] [Table 6a-3] *Underlined sequences are CDRs within VL and VH, if present

[0161] CD3 cell antigen-binding fragment In some embodiments, the present disclosure relates to an antigen-binding fragment (AF1) having specific binding affinity for an effector cell antigen, which can be incorporated into any of the subject composition embodiments described herein. In some cases, the effector cell antigen is expressed on the surface of an effector cell that is a plasma cell, T cell, B cell, cytokine-induced killer cell (CIK cell), mast cell, dendritic cell, regulatory T cell (RegT cell), helper T cell, myeloid cell, or NK cell.

[0162] Various AF1s that bind to effector cell antigens are particularly useful for pairing with antigen-binding fragments that have binding affinity for the HER2 antigen associated with diseased cells or tissues in the form of a composition to cause cell death of the diseased cells or tissues. Binding specificity can be determined by the complementarity-determining regions (CDRs), e.g., light chain CDRs or heavy chain CDRs. In many cases, binding specificity is determined by the light chain CDRs and heavy chain CDRs. A given combination of heavy chain CDRs and light chain CDRs provides a given binding pocket that confers greater affinity and / or specificity to the effector cell antigen compared to other reference antigens. The resulting bispecific composition, which has a first antigen-binding fragment (AF1) against HER2 linked by a short, flexible peptide linker to a second antigen-binding fragment (AF2) that has binding affinity for the effector cell antigen, is bispecific because each antigen-binding fragment has a specific binding affinity for its respective ligand. It will be appreciated that in such compositions, AF2 directed against HER2 in diseased tissue and AF1 directed to an effector cell marker are used in combination to bring effector cells into close proximity to cells of the diseased tissue to effect cytolysis of the cells of the diseased tissue. Furthermore, AF1 and AF2 are incorporated into a specially designed polypeptide comprising a cleavable release segment and XTEN, which polypeptide is specially designed to confer prodrug characteristics to the composition that are activated when in proximity to diseased tissue having a protease capable of cleaving the release segment at one or more locations in the release segment sequence, thereby releasing the fused AF1 and AF2.

[0163] In one embodiment, AF1 of the subject compositions has binding affinity for an effector cell antigen expressed on the surface of T cells. In some embodiments, AF1 of the subject compositions has binding affinity for CD3. In some embodiments, AF1 of the subject compositions has binding affinity for members of the CD3 complex, including all known CD3 subunits of the CD3 complex, e.g., CD3 epsilon, CD3 delta, CD3 gamma, and CD3 zeta, either individually or in any combination. In some embodiments, AF1 has binding affinity for CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta.

[0164] The antigen-binding fragments contemplated by the present disclosure can 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 engineered antibodies or fragments thereof. Methods for generating antibodies against a given target marker are well known in the art. For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be produced by recombinant DNA methods (U.S. Patent No. 4,816,567). The structures of antibodies and their fragments, antibody heavy and light chain variable regions (VH and VL), 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.

[0165] It will be understood that the use of the term "antigen-binding fragment" for the composition embodiments disclosed herein is intended to include portions or fragments of antibodies that retain the ability to bind to an antigen that is the ligand of the corresponding intact antibody. In such embodiments, the antigen-binding fragment may be, but is not limited to, CDRs and intervening framework regions, variable or hypervariable regions of the antibody light and / or heavy chains (VL, VH), variable fragments (Fv), Fab' fragments, F(ab')2 fragments, Fab fragments, single-chain antibodies (scAbs), VHH camelid antibodies, single-chain variable fragments (scFv), linear antibodies, single-domain antibodies, complementarity-determining regions (CDRs), domain antibodies (dAbs), single-domain heavy chain immunoglobulins of the BHH or BNAR type, single-domain light chain immunoglobulins, or other polypeptides known in the art that contain fragments of antibodies capable of binding to antigen. The antigen-binding fragments having CDR-H and CDR-L can be configured in the following orientation from N-terminus to C-terminus: (CDR-H)-(CDR-L) or (CDR-H)-(CDR-L). The VL and VH of the two antigen-binding fragments can also be configured in a single-chain diabody configuration, i.e., the VL and VH of AF1 and AF2 can be configured using a linker of appropriate length to allow for configuration as a diabody.

[0166] The various CD3-binding AF1s of the present disclosure have been specifically modified to enhance their stability in the polypeptide embodiments described herein. Protein aggregation of antibodies remains a significant challenge in their developability and a major area of ​​focus in antibody production. Antibody aggregation can be triggered by partial unfolding of their domains, leading to association between monomers and subsequent nucleation and aggregate growth. The aggregation tendency of antibodies and antibody-based proteins can be influenced by external experimental conditions, but is strongly dependent on intrinsic antibody properties determined by their sequence and structure. While it is well known that proteins are only marginally 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 that the resulting aggregates can be extremely stable and long-lived. Reduced aggregation tendency has also been shown to be accompanied by increased expression titers, indicating that reducing protein aggregation can be beneficial throughout the development process and provide a more efficient route to clinical studies. For therapeutic proteins, aggregates are a significant risk factor for adverse immune responses in patients and can form through a variety of mechanisms. Controlling aggregation can improve protein stability, manufacturability, attrition rate, safety, formulation, potency, immunogenicity, and solubility. Intrinsic protein properties, such as size, hydrophobicity, electrostatics, and charge distribution, play an important role in protein solubility. Poor solubility of therapeutic proteins due to surface hydrophobicity has been shown to make formulation development more challenging and can result in poor biodistribution, undesirable pharmacokinetic behavior, and in vivo immunogenicity. Reducing the overall surface hydrophobicity of candidate monoclonal antibodies can also provide benefits and cost savings related to purification and administration regimens. Individual amino acids can be identified as contributing to antibody aggregation potential through structural analysis and can be located in CDRs and framework regions. Residues, in particular, can be predicted to be at high risk of causing hydrophobicity problems in a given antibody.In one embodiment, the present disclosure provides an AF1 having the ability to specifically bind to CD3, wherein the AF1 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 isoleucine, leucine, or methionine. In some embodiments, the CD3 AF1 has at least two amino acid substitutions of a hydrophobic amino acid in one or more framework regions, wherein the hydrophobic amino acid is isoleucine, leucine, or methionine.

[0167] The isoelectric point (pI) is the pH at which an antibody or antibody fragment has no net charge. If the pH is lower than the pI of an antibody or antibody fragment, it will have a net positive charge. A greater positive charge tends to correlate with increased blood clearance and tissue retention, and generally a shorter half-life. If the pH is higher than the pI of an antibody or antibody fragment, it will have a negative charge. A negative charge generally results in reduced tissue uptake and a longer half-life. This charge can be manipulated by mutations to framework residues. These considerations were reflected in the design of the sequence of AF1 in the embodiments described herein, in which individual amino acid substitutions were made to the parent antibody used as a starting point. The isoelectric point of a polypeptide can be determined mathematically (e.g., by calculation) or experimentally in an in vitro assay. The isoelectric point (pI) is the pH at which a protein has a net charge of zero and can be calculated using the charges associated with specific amino acids in the protein sequence. An estimate of the charge, called the acid dissociation constant or pKa value, is used to calculate the pI. The pI can be determined in vitro by methods such as capillary isoelectric focusing (see Datta-Mannan, A., et al. The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies. mAbs 7:1084 (2015); Li, B., et al. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecular charge. mAbs 6, 1255-1264 (2014)) or other methods known in the art. In some embodiments, the isoelectric points of AF1 and AF2 are designed to be within a specific range of each other, thereby promoting stability.

[0168] In one embodiment, the present disclosure provides an antigen-binding fragment (e.g., AF1 or AF2) for use in any of the polypeptide embodiments described herein, comprising a CDR-L and a CDR-H (see Table 6b), which (a) specifically binds to the Cluster of Differentiation 3 T-cell receptor (CD3), and (b) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively. In some embodiments, CDR-H1 and CDR-H2 of the antigen-binding fragment (AF) may comprise the amino acid sequences of SEQ ID NOs: 8 and 9, respectively. In some embodiments, the present disclosure provides an antigen-binding fragment (e.g., AF1 or AF2) for use in any of the polypeptide embodiments described herein, the antigen-binding fragment (e.g., AF1 or AF2) comprising a CDR-L and a CDR-H, and (a) specifically binds to Cluster of Differentiation 3 T-cell receptor (CD3), and (b) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively. The antigen-binding fragment (e.g., AF1 or AF2) may comprise a CDR-L, wherein the CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 1 or 2, CDR-L2 having the amino acid sequence of SEQ ID NO: 4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, wherein the peptide comprises an antigen-binding fragment (AF) (e.g., AF1 or AF2) comprising CDR-L1, CDR-L2, and CDR-L3, CDR-L1 of the AF may comprise the amino acid sequence of SEQ ID NO: 1 or 2, CDR-L2 of the AF may comprise the amino acid sequence of SEQ ID NO: 4 or 5, and said CDR-L3 of the AF may comprise the amino acid sequence of SEQ ID NO: 6. In some embodiments, wherein the peptide comprises an antigen-binding fragment (AF) (e.g., AF1 or AF2) comprising CDR-L1, CDR-L2, and CDR-L3, CDR-L1 of the AF may comprise the amino acid sequence of SEQ ID NO: 1, CDR-L2 of the AF may comprise the amino acid sequence of SEQ ID NO: 4 or 5, and said CDR-L3 of the AF may comprise the amino acid sequence of SEQ ID NO: 6.In some embodiments, wherein the peptide comprises an antigen-binding fragment (AF) (e.g., AF1 or AF2) comprising CDR-L1, CDR-L2, and CDR-L3, CDR-L1 of the AF may comprise the amino acid sequence of SEQ ID NO: 2, CDR-L2 of the AF may comprise the amino acid sequence of SEQ ID NO: 4 or 5, and said CDR-L3 of the AF may comprise the amino acid sequence of SEQ ID NO: 6. In some embodiments, wherein the peptide comprises an antigen-binding fragment (AF) (e.g., AF1 or AF2) comprising CDR-L1, CDR-L2, and CDR-L3, CDR-L1 of the AF may comprise the amino acid sequence of SEQ ID NO: 1, CDR-L2 of the AF may comprise the amino acid sequence of SEQ ID NO: 4, and said CDR-L3 of the AF may comprise the amino acid sequence of SEQ ID NO: 6. In some embodiments, the peptide comprises an antigen-binding fragment (AF) (e.g., AF1 or AF2) comprising CDR-L1, CDR-L2, and CDR-L3, CDR-L1 of the AF may comprise the amino acid sequence of SEQ ID NO: 2, CDR-L2 of the AF may comprise the amino acid sequence of SEQ ID NO: 5, and said CDR-L3 of the AF may comprise the amino acid sequence of SEQ ID NO: 6.

[0169] In some embodiments, the aforementioned antigen-binding fragment (AF) (e.g., AF1 or AF2) embodiments in the immediately preceding paragraph further comprise a light chain framework region (FR-L) and a heavy chain framework region (FR-H) (see Table 6c), wherein the antigen-binding fragment (AF) comprises FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 51; FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 52; , 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequence of any one of SEQ ID NOs: 53 to 56; FR-L3 which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 59; and FR-L4 which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 59. The antigen-binding fragment (AF) is an FR-L1 fragment that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 51; an FR-L2 fragment that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 52; L2, FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 53, and FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 59.The antigen-binding fragment (AF) is an FR-L1 fragment that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 51; an FR-L2 fragment that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 52; L2, FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 54, and FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 59. The antigen-binding fragment (AF) is an FR-L1 fragment that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 51; an FR-L2 fragment that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 52; L2, FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 55, and FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 59.The antigen-binding fragment (AF) is an FR-L1 fragment that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 51; an FR-L2 fragment that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 52; L2, FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 56, and FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 59. The antigen-binding fragment (AF) includes FR-H1 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of any one of SEQ ID NOs: 60 to 63, and FR-H2 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 64. FR-H2, FR-H3 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 65 or 66, and FR-H4 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 67.The antigen-binding fragment (AF) is FR-H1, which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 60; and FR-H2, which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 64. , FR-H3 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 65, and FR-H4 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 67. The antigen-binding fragment (AF) is selected from the group consisting of FR-H1, FR-H2, FR-H3, FR-H4, FR-H5, FR-H6, ​​FR-H7, FR-H8, FR-H9, FR-H10, FR-H11, FR-H12, FR-H13, FR-H14, FR-H15, FR-H16, FR-H17, FR-H18, FR-H19 ... H2, FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 65, and FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 67.An antigen-binding fragment (AF) (e.g., AF1 or AF2) for use in any of the polypeptide embodiments described herein may comprise a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 51, FR-L1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 52, FR-L2 exhibiting or being identical to the amino acid sequence of any one of SEQ ID NOs: 53 to 56, FR-L3 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L4 exhibiting or being identical to the amino acid sequence of any one of SEQ ID NOs: 53 to 56, FR-L5 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L6 exhibiting or being identical to the amino acid sequence of any one of SEQ ID NOs: 53 to 56, FR-L7 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L8 exhibiting or being identical to the amino acid sequence of any one of SEQ ID NOs: 53 to 56, FR-L9 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L10 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L11 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L12 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L13 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L14 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L15 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L16 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L17 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L18 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L19 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 59, FR-L20 exhibiting or being identical to the amino acid sequence of SEQ FR-L4 exhibiting 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto to the amino acid sequence of SEQ ID NO: 60 or 61; FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto to the amino acid sequence of SEQ ID NO: 64; FR-H2 exhibiting sequence identity to or being identical to the amino acid sequence of SEQ ID NO:65; FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or being identical to the amino acid sequence of SEQ ID NO:65; and FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or being identical to the amino acid sequence of SEQ ID NO:67.AF is selected from the group consisting of FR-L1, FR-L2, FR-L3, FR-L4, FR-L5, FR-L6, FR-L7, FR-L8, FR-L9, FR-L10, FR-L11, FR-L12, FR-L13, FR-L14, FR-L15, FR-L16, FR-L17, FR-L18, FR-L19, FR-L19, FR-L110, FR-L111, FR-L121, FR-L13, FR-L14, FR-L15, FR-L16, FR-L17, FR-L18, FR-L19 ... FR-L3 exhibiting 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto to the amino acid sequence of SEQ ID NO: 59; FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto to the amino acid sequence of SEQ ID NO: 60; FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 65; FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 65; and FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 67.AF is FR-L1, which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 51; and FR-L2, which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 52. FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 54; FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 59; FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO:61; FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO:64; and FR-H4 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 67.AF is FR-L1, which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 51; and FR-L2, which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 52. FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 55; FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 59; FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO:61; FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO:64; and FR-H4 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 67.AF is FR-L1, which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 51; and FR-L2, which has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 52. FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 56; FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 59; FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO:61; FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO:64; and FR-H4 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 67.

[0170] In some embodiments, the present disclosure provides an antigen-binding fragment (AF) (e.g., AF1 or AF2) for use in any of the polypeptide embodiments described herein, comprising a variable heavy chain (VH) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 102 or SEQ ID NO: 105 in Table 6d. In some embodiments, the present disclosure provides an AF for use in any of the polypeptide embodiments described herein, comprising a variable light chain (VL) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of any one of SEQ ID NOs: 101, 103, 104, 106, or 107 in Table 6d. In some embodiments, the present disclosure provides an antigen-binding fragment (e.g., AF1 or AF2) for use in any of the polypeptide embodiments described herein, which may comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of any one of SEQ ID NOs:201-205 in Table 6e.

[0171] In some embodiments, the present disclosure provides antigen-binding fragments (e.g., AF1 or AF2) that bind to the CD3 protein complex with enhanced stability compared to CD3-binding antibodies or antigen-binding fragments known in the art. Furthermore, the CD3 antigen-binding fragments of the present disclosure are designed to confer a greater degree of stability to the chimeric bispecific antigen-binding fragment compositions into which they are incorporated, resulting in improved expression and recovery of the fusion protein, increased shelf life, and enhanced stability when administered to a subject. In one approach, the CD3 AFs of the present disclosure are designed to have a greater degree of thermal stability compared to certain CD3-binding antibodies and antigen-binding fragments known in the art. As a result, the CD3 AFs utilized as components of the chimeric bispecific antigen-binding fragment compositions into which they are incorporated exhibit favorable pharmaceutical properties, including high thermal stability and low aggregation tendency, resulting in improved expression and recovery during manufacturing and storage, and promoting extended serum half-life. Biophysical properties such as thermal stability are often limited by antibody variable domains, whose intrinsic properties are significantly different. High thermal stability is often associated with other desirable properties, including high expression levels and reduced aggregation (Buchanan A, et al. Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression. MAbs 2013; 5:255). Thermal stability is measured by the "melting temperature" (T m The melting temperature of each heterodimer indicates its thermal stability. mIn vitro assays for determining the melting point of a heterodimer are known in the art, including the methods described in the Examples below. The melting point of a 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 a heterodimer can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9), or as described in the Examples below.

[0172] In some embodiments of the polypeptides of the present disclosure, the antigen-binding fragment (e.g., AF1 or AF2) has a melting temperature (T m ) or a higher melting temperature of the test bispecific antigen-binding construct compared to that of the control bispecific antigen-binding construct when the first antigen-binding fragment is incorporated into the test bispecific antigen-binding construct. m High T compared to m The test bispecific antigen-binding construct comprises a first antigen-binding fragment and a reference antigen-binding fragment that binds to an antigen other than CD3, and the control bispecific antigen-binding construct comprises an anti-CD3-binding fragment consisting of the sequence of SEQ ID NO: 206 (see Table 6e) and a reference antigen-binding fragment, as evidenced by the melting temperature (T m ) is the T of an anti-CD3 binding fragment consisting of the sequence of SEQ ID NO: 206 (see Table 6e). m It may be at least 2°C higher, at least 3°C ​​higher, or at least 4°C higher, or at least 5°C higher.

[0173] Thermal denaturation curves of anti-CD3 bispecific antibodies comprising CD3-binding fragments and anti-CD3 binding fragments of the present disclosure and reference binding demonstrate that the constructs of the present disclosure are more resistant to thermal denaturation than an antigen-binding fragment consisting of the sequence set forth in SEQ ID NO: 781 (see Table 6f) or a control bispecific antibody, where the control bispecific antigen-binding fragment comprises SEQ ID NO: 781 (see Table 6f) and a reference antigen-binding fragment that binds to a HER2 embodiment described herein. In one embodiment, the polypeptide of any of the embodiments of the subject compositions described herein comprises an anti-CD3 AF of an embodiment described herein, and the T of AF m is the T of an antigen-binding fragment consisting of the sequence of SEQ ID NO: 781 (see Table 6f), as determined by an increase in melting temperature in an in vitro assay. m 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.

[0174] In some embodiments, the polypeptide of any of the embodiments of the subject compositions described herein comprises an antigen-binding fragment (AF) that specifically binds to human or cynomolgus monkey (cyno) CD3. The antigen-binding fragment (AF) can specifically bind to human CD3. The antigen-binding fragment (AF) can bind to a CD3 complex subunit identified herein as the CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta unit of CD3. The antigen-binding fragment (AF) can bind to the CD3 epsilon fragment of CD3. The antigen-binding fragment (AF) has a dissociation constant (K) of between about 10 nM and about 400 nM, or between about 50 nM and about 350 nM, or between about 100 nM and 300 nM, as determined in an in vitro antigen-binding assay involving human or cyno CD3 antigen. dIn some embodiments, the polypeptide of any of the embodiments of the subject compositions described herein can specifically bind to human or cyno CD3 with a dissociation constant (K) of less 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 less than about 400 nM, as determined in an in vitro antigen binding assay. d ) containing an antigen-binding fragment (AF) that specifically binds to human or cyno CD3. For clarity, a K of 400 d The antigen-binding fragment (AF) has a K d In some embodiments, the polypeptide of any of the embodiments of the subject compositions described herein binds to its ligand more weakly in an in vitro antigen binding assay than one having a dissociation constant (K d ), that specifically binds to human or cyno CD3 with at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold weaker binding affinity than an antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 781 (see Table 6f). In some embodiments, the present disclosure provides antigen-binding fragments (AF) that specifically bind to human or cyno CD3 with at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold weaker binding affinity in in vitro antigen-binding assays, as determined by K dBispecific polypeptides are provided comprising antigen-binding fragments (AF) (anti-CD3 AF) that exhibit binding affinity for CD3 at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold weaker than that of the anti-HER2 AF embodiments described herein incorporated into the subject polypeptide, as determined by ELISA. The binding affinity of the subject compositions for the target ligand can be assayed using a binding or competition assay, such as a Biacore assay using chip-bound receptors or binding proteins as described in U.S. Pat. No. 5,534,617, or an ELISA assay, an 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 Scatchard analysis as described in van Zoelen, et al., Trends Pharmacol Sciences (1998) 19)12):487, or other methods known in the art.

[0175] In a related aspect, the present disclosure provides antigen-binding fragments (AFs) incorporated into chimeric bispecific polypeptide compositions that bind to CD3 (anti-CD3 AFs) and are designed to have an isoelectric point (pI) that confers enhanced stability to the disclosed compositions relative to corresponding compositions comprising antibodies or antigen-binding fragments that bind to CD3 known in the art. In one embodiment, the polypeptide of any of the subject composition embodiments described herein comprises an AF (anti-CD3 AF) that binds to CD3, wherein the anti-CD3 AF exhibits a pI that is between 6.0 and 6.6, inclusive. In some embodiments, the polypeptide of any of the subject composition embodiments described herein comprises an AF (anti-CD3 AF) that binds to CD3, wherein the anti-CD3 AF exhibits a pI that is 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 (e.g., consisting of the sequence set forth in SEQ ID NO: 206 (see Table 6e)). In some embodiments, a polypeptide of any of the embodiments of the subject compositions described herein comprises an AF that binds CD3 (anti-CD3 AF) fused to another AF that binds the HER2 antigen (anti-HER2 AF), where the anti-CD3 AF exhibits a pI that is 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 AF that binds the HER2 antigen or an epitope thereof. In some embodiments, a polypeptide of any of the embodiments of the subject compositions described herein comprises an AF that binds CD3 (anti-CD3 AF) fused to an AF that binds the HER2 antigen (anti-HER2 AF), where the AF exhibits a pI that is 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 anti-CD3 AF.It is specifically contemplated that such a design, in which the pIs of the two antigen-binding fragments are within such a range, will confer a high degree of stability to the chimeric bispecific antigen-binding fragment composition into which they are incorporated, resulting in improved expression and enhanced recovery of the soluble, non-aggregated form of the fusion protein, increased shelf life of the formulated chimeric bispecific polypeptide composition, and enhanced stability when the composition is administered to a subject. In other words, having two AFs (anti-CD3 AF and anti-HER2 AF) within a relatively narrow pI range may allow for the selection of a buffer or other solution in which both AFs (anti-CD3 AF and anti-HER2 AF) are stable, thereby potentially promoting the overall stability of the composition. The antigen-binding fragment (AF) may exhibit an isoelectric point (pI) of less than or equal to 6.6. The antigen-binding fragment (AF) may exhibit an isoelectric point (pI) between 6.0 and 6.6, inclusive. The antigen-binding fragment (AF) may exhibit an isoelectric point (pi) that is 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 consisting of the sequence set forth in SEQ ID NO: 206 (see Table 6e). The antigen-binding fragment (AF) may exhibit a dissociation constant (K) of between about 10 nM and about 400 nM. d The antigen-binding fragment (AF) can specifically bind to human or cyno CD3 with a dissociation constant (K) of less than about 10 nM, or less than about 50 nM, or less than about 100 nM, or less than about 150 nM, or less than about 200 nM, or less than about 250 nM, or less than about 300 nM, or less than about 350 nM, or less than about 400 nM (e.g., as determined in an in vitro antigen-binding assay involving human or cyno CD3 antigen). d) (e.g., as determined in an in vitro antigen binding assay). The antigen-binding fragment (AF) may exhibit binding affinity for CD3 that is at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold weaker (e.g., as determined in an in vitro antigen binding assay) than that of an antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 206 (see Table 6e) (e.g., as determined in an in vitro antigen binding assay). d ) is determined by).

[0176] In certain embodiments, the VL and VH of the antigen-binding fragment are fused by a relatively long linker of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids that possess flexible characteristics when joined together. In one embodiment, the VL and VH of any of the scFv embodiments described herein are linked by a relatively long linker of hydrophilic amino acids having the sequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 82), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 83), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 84), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 85). In some embodiments, AF1 and AF2 are linked together by a short linker of hydrophilic amino acids having 3, 4, 5, 6, or 7 amino acids. In one embodiment, the short linker sequence is identified herein as the sequence SGGGGS (SEQ ID NO: 86), GGGGS (SEQ ID NO: 87), GGSGGS (SEQ ID NO: 88), GGS, or GSP. In some embodiments, the present disclosure provides a composition comprising a single-chain diabody, wherein, after folding, the first domain (VL or VH) pairs with the last domain (VH or VL) to form one scFv, and the middle two domains pair to form the other scFv, where the first and second domains, and 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 longer linkers. As will be recognized by those skilled in the art, the selection of short and long linkers is intended to prevent mispairing of adjacent variable domains, thereby facilitating the formation of a single-chain diabody configuration comprising the VL and VH of the first and second antigen-binding fragments. Table 6b. Exemplary CD3 CDR sequences [Table 6b] Table 6c. Exemplary CD3 FR sequences [Table 6c-1] [Table 6c-2] Table 6d: Exemplary CD3 VL and VH sequences [Table 6d] Table 6e: Exemplary CD3 scFv sequences [Table 6e-1] [Table 6e-2] [Table 6e-3]

[0177] Anti-HER2 binding domain In some embodiments, the present invention provides chimeric polypeptide assembly compositions comprising a first partial binding domain having binding affinity for tumor-specific marker HER-2 and a second binding domain that binds to an effector cell antigen, such as the CD3 antigen. In one embodiment, the binding domain comprises a VL and a VH derived from a monoclonal antibody against HER-2. Monoclonal antibodies against HER-2 are known in the art. Illustrative, non-limiting examples of VL and VH sequences are shown in Table 6f. In one embodiment, the present invention provides chimeric polypeptide assembly compositions comprising a first partial binding domain having binding affinity for tumor-specific marker HER-2, comprising the anti-HER-2 VL and VH sequences listed in Table 6f. In some embodiments, the present invention provides chimeric polypeptide assembly compositions comprising a first partial binding domain having binding affinity for tumor-specific marker HER-2, comprising a CDR-L1 region, a CDR-L2 region, a CDR-L3 region, a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region, each derived from a respective VL and VH sequence listed in Table 6f. Preferably, in embodiments, binding is determined in an in vitro binding assay and is greater than 10 -10 Greater than 10 -7 K to M d In some embodiments, the polypeptide comprises an antigen-binding fragment that specifically binds to HER2 (anti-HER2 AF), the anti-HER2 AF (e.g., AF1 or AF2) comprises: (1) a heavy chain variable region (VH) comprising the amino acid sequence set forth as SEQ ID NOs: 778-783 in Table 6f; II ), and (2) a light chain variable region (VL) comprising the amino acid sequences shown as SEQ ID NOs: 878 to 883 in Table 6f. II) . It is specifically contemplated that a chimeric polypeptide assembly composition may comprise any one of the foregoing binding domains or sequence variants thereof (so long as the variant exhibits binding specificity for the described antigen). In one embodiment, sequence variants will be created by substitution of amino acids in the VL or VH sequence with different amino acids. In deletion variants, one or more amino acid residues in the VL or VH sequences described herein are removed. Thus, deletion variants include all fragments of the binding domain polypeptide sequence. In substitution variants, one or more amino acid residues in the VL or VH (or CDR) polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature, and this type of conservative substitution is well known in the art. Furthermore, it is specifically contemplated that a composition comprising the first and second binding domains disclosed herein can be utilized in any of the methods disclosed herein. Table 6f. Anti-HER2 monoclonal antibodies and sequences [Table 6f-1] [Table 6f-2] *Underlined and bolded sequences are CDRs within VL and VH, if present Table A: Intramolecular long linkers [Table A] Table B: Intramolecular short linkers [Table B]

[0178] In some embodiments of the polypeptides of the present disclosure, the light chain variable region (VL) and heavy chain variable region (VH) pair of the antigen-binding fragment may be linked by a linker or a long linker (e.g., composed of hydrophilic amino acids). Such linkers linking the light chain variable region (VL) and heavy chain variable region (VH) of the antigen-binding fragment (e.g., the first antigen-binding fragment (AF1), the second antigen-binding fragment (AF2)) (each independently) comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table A. Such linkers linking the light chain variable region (VL) and heavy chain variable region (VH) of the antigen-binding fragment (e.g., the first antigen-binding fragment (AF1), the second antigen-binding fragment (AF2)) (each independently) may comprise an amino acid sequence identical to a sequence set forth in Table A. In some embodiments of the polypeptides of the present disclosure, two antigen-binding fragments (e.g., a first and a second antigen-binding fragment) may be fused together by a peptide linker or a short linker. Such a peptide linker linking two antigen-binding fragments (e.g., a first and a second antigen-binding fragment) may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table B. Such a peptide linker linking two antigen-binding fragments (e.g., a first and a second antigen-binding fragment) may comprise an amino acid sequence identical to a sequence set forth in Table B. In some cases, the first antigen-binding fragment is a single-chain variable fragment (scFv). In some cases, the second antigen-binding fragment is a single-chain variable fragment (scFv). The two single-chain variable fragments of the first and second antigen-binding fragments may be linked together by a peptide linker. In some embodiments of the polypeptides of the present disclosure, the linker used to link the VL and VH of a first antigen-binding fragment and / or the linker used to link the VL and VH of a second antigen-binding fragment may be L7 of Table A. In such embodiments, the peptide linker used to link the two antigen-binding fragments may be S-1 or S-2 of Table B.In some embodiments, the present disclosure provides polypeptides comprising single-chain diabodies, wherein, after folding, the first domain (VL or VH) pairs with the last domain (VH or VL) to form one scFv, and the middle two domains pair to form the other scFv, where the first and second domains, and the third and last domains, are fused together by short linkers of hydrophilic amino acids identified herein by the sequences set forth in Table B, and the second and third variable domains are fused by a long linker identified in Table A. As will be recognized by one of skill in the art, the selection of short and long linkers is to prevent mispairing of adjacent variable domains, thereby facilitating the formation of a single-chain diabody configuration comprising the VL and VH of the first and second binding moieties. Table C: Exemplary spacers between the release segment and the bispecific antibody construct [Table C-1] [Table C-2]

[0179] In some embodiments of polypeptides of the present disclosure, a release segment (RS) (e.g., a first release segment (RS1), a second release segment (RS2), etc.) may be fused to a bispecific antibody construct (BsAb) via a spacer. Such spacers (each independently) comprise at least four amino acids that are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). A peptide of the present disclosure may comprise a first release segment fused to a bispecific antibody construct via a first spacer and a second release segment fused to a bispecific antibody construct via a second spacer. The spacers (e.g., a first spacer, a second spacer, etc.) (each independently) comprise an amino acid sequence having at least (about) 80%, at least (about) 90%, or 100% sequence identity to a sequence set forth in Table C. The spacers (eg, the first spacer, the second spacer, etc.) (each independently) comprise an amino acid sequence identical to a sequence set forth in Table C.

[0180] unstructured 3D structure Typically, the XTEN component of a fusion protein is designed to behave similarly to a denatured peptide sequence under physiological conditions, despite the extended length of the polymer. Denatured describes the state of a peptide in solution, characterized by a large conformational freedom of the peptide backbone. Most peptides and proteins adopt a denatured conformation in the presence of high concentrations of denaturants or at elevated temperatures. Peptides in denatured conformations are characterized, for example, by a characteristic circular dichroism (CD) spectrum and an absence of long-range interactions as determined by NMR. "Denatured conformation" and "unstructured conformation" are used interchangeably herein. In some cases, the invention provides XTEN sequences that can resemble denatured sequences that are largely devoid of secondary structure under physiological conditions. In other cases, the XTEN sequence may be substantially devoid of secondary structure under physiological conditions. "Largely devoid," as used in this context, means that less than 50% of the XTEN amino acid residues of the XTEN sequence contribute to secondary structure, as measured or determined by the means described herein. "Substantially devoid," as used in this context, means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the XTEN amino acid residues of the XTEN sequence do not contribute to secondary structure, as measured or determined by the means described herein.

[0181] Various methods have been established in the art to identify the presence or absence of secondary and tertiary structures in a given polypeptide. In particular, XTEN secondary structure can be measured spectrophotometrically, for example, by circular dichroism spectroscopy in the "far UV" region of the spectrum (190-250 nm). Secondary structure elements such as alpha helices and beta sheets each produce CD spectra with characteristic shapes and sizes. Secondary structure can also be predicted for polypeptide sequences via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, PY, et al. (1974) Biochemistry, 13: 222-45) and the Garnier-Osguthorpe-Robson ("GOR") algorithm (Garnier J, Gibrat JF, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553), as described, for example, in U.S. Patent Application Publication No. 20030228309A1. For a given sequence, the algorithm can predict the presence of some or no secondary structure, expressed, for example, as the total number and / or percentage of residues in the sequence that form alpha helices or beta sheets, or the percentage of residues in the sequence that are predicted to form random coils (lacking secondary structure).

[0182] In some cases, the XTEN sequences used in the fusion protein compositions of the invention may have an alpha-helix percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In other cases, the XTEN sequences of the fusion protein compositions may have a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In some cases, the XTEN sequences of the fusion protein compositions may have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by the Chou-Fasman algorithm. In preferred embodiments, the XTEN sequences of the fusion protein compositions will have an alpha-helix percentage of less than about 2% and a beta-sheet percentage of less than about 2%. In other cases, the XTEN sequences of the fusion protein compositions may have a high degree of random coil percentage as determined by the GOR algorithm. In some embodiments, the XTEN sequence may have at least about 80%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% random coil as determined by the GOR algorithm.

[0183] Net Charge In other cases, XTEN polypeptides can have unstructured characteristics imparted by incorporating amino acid residues with a net charge and / or reducing the proportion of hydrophobic amino acids in the XTEN sequence. The overall net charge and net charge density can be controlled by modifying the content of charged amino acids in the XTEN sequence. In some cases, the net charge density of the XTEN of the composition can be greater than +0.1 charges / residue or less than -0.1 charges / residue. In other cases, the net charge of the XTEN can be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or higher.

[0184] Because most human or animal tissues and surfaces have a net negative charge, XTEN sequences are designed to have a net negative charge to minimize nonspecific interactions between XTEN-containing compositions and various surfaces, such as blood vessels, healthy tissues, or various receptors. Without being bound by theory, XTENs individually have a high net negative charge, allowing them to adopt an open conformation due to electrostatic repulsion between the individual amino acids of the XTEN polypeptide distributed throughout the sequence of the XTEN polypeptide. This distribution of net negative charges in XTENs with extended sequence length can lead to an unstructured conformation, which in turn can effectively increase the hydrodynamic radius. Thus, in one embodiment, the invention provides XTENs whose XTEN sequences contain about 8, 10, 15, 20, 25, or even about 30% glutamic acid. XTENs in the compositions of the invention generally have no or a low content of positively charged amino acids. In some cases, XTEN may have less than about 10% of amino acid residues with a positive charge, or less than about 7%, or less than about 5%, or less than about 2% of amino acid residues with a positive charge. However, the present invention contemplates constructs that can incorporate a limited number of positively charged amino acids, such as lysine, into XTEN to allow conjugation between the epsilon amine of lysine and a reactive group on a peptide, a linker bridge, or a reactive group on a drug or small molecule conjugated to the XTEN backbone. As described above, fusion proteins can be constructed that include XTEN, a biologically active protein, and a chemotherapeutic agent useful in treating metabolic diseases or disorders, where the maximum number of drug molecules incorporated into the XTEN component is determined by the number of lysines or other amino acids (e.g., cysteine) with reactive side chains incorporated into the XTEN.

[0185] In some cases, the XTEN sequence may contain charged residues separated by other residues, such as serine or glycine, which may result in better expression or purification behavior. Based on net charge, the XTEN of the subject compositions may have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In preferred embodiments, the XTEN will have an isoelectric point between 1.5 and 4.5. In these embodiments, the XTEN incorporated into the BPXTEN fusion protein compositions of the invention will have a net negative charge under physiological conditions, which may contribute to an unstructured conformation and reduced binding of the XTEN component to mammalian proteins and tissues.

[0186] Because hydrophobic amino acids can impart structure to a polypeptide, the present invention provides for a hydrophobic amino acid content within the XTEN that typically results in a hydrophobic amino acid content of less than 5%, or less than 2%, or less than 1%. In one embodiment, the methionine and tryptophan amino acid content within the XTEN component of a BPXTEN fusion protein is typically less than 5%, or less than 2%, and most preferably less than 1%. In some embodiments, the XTEN has a sequence with less than 10% positively charged amino acid residues, or less than about 7%, or less than about 5%, or less than about 2% positively charged amino acid residues, the sum of methionine and tryptophan residues is less than 2%, and the sum of asparagine and glutamine residues is less than 10% of the total XTEN sequence.

[0187] Increase in hydrodynamic radius In some embodiments, XTENs have a high hydrodynamic radius and can confer a corresponding increased apparent molecular weight to BPXTEN fusion proteins incorporating the XTEN. Linking XTENs to BP sequences can result in BPXTEN compositions that can have an increased hydrodynamic radius, increased apparent molecular weight, and increased apparent molecular weight coefficient compared to BPs not linked to XTENs. For example, in therapeutic applications where extended half-life is desired, compositions incorporating XTENs with large hydrodynamic radii into fusion proteins containing one or more BPs can effectively expand the hydrodynamic radius of the composition beyond the glomerular pore size of approximately 3-5 nm (corresponding to an apparent molecular weight of approximately 70 kDa) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv. Drug Deliv. Rev. 55:1261-1277), which can result in reduced renal clearance of circulating proteins. The hydrodynamic radius of a protein is determined not only by its molecular weight, but also by its structure, including shape and compactness.Without being bound by any particular theory, XTEN may adopt an open conformation due to electrostatic repulsion between the individual charges of the peptide or the inherent flexibility conferred by specific amino acids in the sequence, which lack the possibility of conferring secondary structure.The open, extended, unstructured conformation of XTEN polypeptide may have a proportionally larger hydrodynamic radius compared to polypeptides with comparable sequence length and / or molecular weight and secondary and / or tertiary structure, such as typical globular proteins.Methods for determining hydrodynamic radius are well known in the art, such as by using size exclusion chromatography (SEC), as described in U.S. Patent Nos. 6,406,632 and 7,294,513.Addition of XTEN with increased length proportionally increases the hydrodynamic radius parameters, apparent molecular weight, and apparent molecular weight coefficient, thereby allowing BPXTEN to be tailored to a desired characteristic cutoff apparent molecular weight or hydrodynamic radius. Thus, in certain embodiments, BPXTEN fusion proteins can be configured with XTEN to enable the fusion protein to have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or at least about 15 nm. In the foregoing embodiments, the large hydrodynamic radius imparted by the XTEN in the BPXTEN fusion protein can result in reduced renal clearance of the resulting fusion protein, leading to a corresponding increase in terminal half-life, increased mean retention time, and / or reduced renal clearance rate.

[0188] In some embodiments, XTEN of selected length and sequence may be selectively incorporated into BPXTEN to create fusion proteins having an apparent molecular weight under physiological conditions of at least about 150 kDa, or at least about 300 kDa, or at least about 400 kDa, or at least about 500 kDa, or at least about 600 kDa, or at least about 700 kDa, or at least about 800 kDa, or at least about 900 kDa, or at least about 1000 kDa, or at least about 1200 kDa, or at least about 1500 kDa, or at least about 1800 kDa, or at least about 2000 kDa, or at least about 2300 kDa or more. In some embodiments, XTEN of selected length and sequence may be selectively linked to a BP that results in a BPXTEN fusion protein having, under physiological conditions, an apparent molecular weight factor of at least 3, alternatively at least 4, alternatively at least 5, alternatively at least 6, alternatively at least 7, alternatively at least 8, alternatively at least 9, alternatively at least 10, alternatively at least 15, or an apparent molecular weight factor of at least 20 or greater. In some embodiments, the BPXTEN fusion protein has, under physiological conditions, an apparent molecular weight factor relative to the actual molecular weight of the fusion protein that is about 4 to about 20, or about 6 to about 15, or about 8 to about 12, or about 9 to about 10. In some embodiments, the (fusion) polypeptide exhibits an apparent molecular weight factor of greater than about 6 under physiological conditions.

[0189] Increased terminal half-life In some embodiments, the (fusion) polypeptide has a terminal half-life that is at least 2-fold longer, or at least 3-fold longer, or at least 4-fold longer, or at least 5-fold longer than a biologically active polypeptide that is not linked to any XTEN. In some embodiments, the (fusion) polypeptide has a terminal half-life that is at least 2-fold longer than a biologically active polypeptide that is not linked to any XTEN.

[0190] Administration of a therapeutically effective dose of any of the embodiments of the BPXTEN fusion proteins described herein to a subject in need thereof may result in at least a two-fold, or at least a three-fold, or at least a four-fold, or at least a five-fold, or greater increase in the time spent within the therapeutic window for the fusion protein compared to the corresponding BP not linked to XTEN and administered to a subject at an equivalent dose.

[0191] Low immunogenicity In some embodiments, the present invention provides compositions in which the XTEN sequence has a low degree of immunogenicity or is substantially non-immunogenic. The low immunogenicity of XTEN can be contributed to by several factors, such as a substantially non-repetitive sequence, an unstructured conformation, a high degree of solubility, a low degree or lack of self-aggregation, a low degree or lack of proteolytic sites within the sequence, and a low degree or lack of epitopes within the XTEN sequence.

[0192] Those skilled in the art will generally understand that polypeptides having short, highly repetitive amino acid sequences (e.g., a 200 amino acid long sequence containing an average of 20 or more repeats of a limited set of 3- or 4-mers) and / or having consecutively repeated amino acid residues (e.g., a 5- or 6-mer sequence with identical amino acid residues) have a tendency to aggregate or form higher order structures or contact points leading to crystalline or pseudo-crystalline structures.

[0193] In some embodiments, the XTEN sequence is substantially non-repetitive, such that (1) the XTEN sequence does not have three consecutive amino acids of the same amino acid type unless the amino acid is serine, in which case no more than three consecutive amino acids may be serine residues; and (2) the XTEN does not contain a three-amino acid sequence (3-mer) that occurs more than 16 times, more than 14 times, more than 12 times, or more than 10 times within the sequence of a 200 amino acid long XTEN. One of skill in the art will appreciate that such substantially non-repetitive sequences have a reduced tendency to aggregate, thereby enabling the design of XTEN with longer sequences that have a relatively low frequency of charged amino acids that are more likely to aggregate if the sequence or amino acid residues are otherwise more repetitive.

[0194] Conformational epitopes are formed by regions on the protein surface composed of multiple, noncontiguous amino acid sequences of a protein antigen. Correct protein folding converts these sequences into well-defined, stable spatial locations, or epitopes, that can be recognized as "foreign" by the host's humoral immune system, resulting in the production of antibodies or the elicitation of a cell-mediated immune response against the protein. In the latter case, an individual's immune response to a protein is heavily influenced by T cell epitope recognition, which is a function of the peptide-binding specificity of that individual's HLA-DR allotype. Engagement of MHC class II peptide complexes by cognate T cell receptors on the surface of T cells, along with cross-linking of certain other coreceptors, such as CD4 molecules, can induce an activated state within the T cell. Activation leads to the release of cytokines that further activate other lymphocytes, such as B cells, resulting in the production of antibodies or the activation of killer T cells as a complete cellular immune response.

[0195] The ability of a peptide to bind to a given MHC class II molecule for presentation on the surface of an APC (antigen-presenting cell) depends on several factors, particularly its primary sequence. In one embodiment, low immunogenicity can be achieved by designing an XTEN sequence that resists antigen processing in antigen-presenting cells and / or selecting a sequence that does not bind well to MHC receptors. The present invention provides BPXTEN fusion proteins having substantially non-repetitive XTEN polypeptides designed to reduce binding to MHC II receptors and avoid the formation of epitopes to which T cell receptors or antibodies bind, resulting in low immunogenicity. Avoidance of immunogenicity is, in part, a direct result of the conformational flexibility of the XTEN sequence, i.e., the lack of secondary structure due to the selection and order of amino acid residues. Of particular interest are sequences that have a low tendency to adopt tightly folded conformations that may result in conformational epitopes, for example, in aqueous solution or under physiological conditions. Administration of fusion proteins containing XTEN using conventional therapeutic practices and administration generally does not result in the formation of neutralizing antibodies against the XTEN sequence and may also reduce the immunogenicity of the BP fusion partner in the BPXTEN composition.

[0196] In one embodiment, the XTEN sequence utilized in the subject fusion protein may be substantially free of epitopes recognized by human T cells. Eliminating such epitopes to generate less immunogenic proteins has been previously disclosed; see, for example, WO98 / 52976, WO02 / 079232, and WO00 / 3317, which are incorporated herein by reference. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Of particular interest are peptide sequences that can oligomerize without generating T cell epitopes or non-human sequences. This can be achieved by testing tandem repeats of these sequences for the presence of T cell epitopes and the occurrence of non-human 6- to 15-mer, particularly 9-mer, sequences, and then modifying the design of the XTEN sequence to eliminate or disrupt the epitope sequences. In some cases, XTEN sequences are substantially non-immunogenic due to a limited number of epitopes of the XTEN predicted to bind to MHC receptors. A reduction in the number of epitopes capable of binding to MHC receptors concomitantly reduces the potential for T cell activation and T cell helper function, reduces B cell activation or upregulation, and reduces antibody production. Low predicted T cell epitopes can be determined by epitope prediction algorithms such as TEPITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61), as shown, for example, in Example 74 of International Patent Application Publication No. WO 2010 / 144502 A2, which is incorporated herein by reference in its entirety. The TEPITOPE score for a given peptide frame within a protein is calculated by the K of binding of that peptide frame with a number of the most common human MHC alleles, as disclosed in Sturniolo, T., et al. (1999) Nature Biotechnology 17:555. d (dissociation constant, affinity, off-rate). The score should be at least 20 log(10e) between about 10 and about -10.10 K d ~10e -10 K d The TEPITOPE score ranges from about -5 to about -6, or about -7, or about -8, or about -9 or greater, and can be reduced by avoiding hydrophobic amino acids, such as M, I, L, V, or F, which may serve as anchor residues during peptide presentation on the MHC. In some embodiments, the XTEN component incorporated into BPXTEN does not have a predicted T cell epitope with a TEPITOPE score of about -5 or greater, or about -6 or greater, or about -7 or greater, or about -8 or greater, or about -9 or greater. As used herein, a score of "-9 or greater" encompasses TEPITOPE scores from 10 to -9, inclusive, but does not include a score of -10, since -10 is less than -9.

[0197] In some embodiments, XTEN sequences of the invention, including those incorporated into a subject BPXTEN fusion protein, can be made substantially non-immunogenic by limiting known proteolytic sites from the XTEN sequence, thereby reducing XTEN processing into small peptides capable of binding to MHC II receptors. In some embodiments, XTEN sequences can be made substantially non-immunogenic by using sequences that are substantially devoid of secondary structure, conferring resistance to many proteases due to their high structural entropy. Thus, by reducing the TEPITOPE score and eliminating known proteolytic sites from the XTEN, XTEN compositions, including the XTEN of the BPXTEN fusion protein composition, can be substantially unable to bind to mammalian receptors, including those of the immune system. In one embodiment, the XTEN of the BPXTEN fusion protein has a K d or a K of greater than 500 nM for mammalian cell surface or circulating polypeptide receptors d , or a K greater than 1 μM d may have

[0198] Furthermore, the substantially non-repetitive sequence and lack of a corresponding epitope in such embodiments of XTEN may limit the ability of B cells to bind to or be activated by the XTEN. Although XTEN can contact many different B cells over its extended sequence, each individual B cell can only make one or a few contacts with an individual XTEN. As a result, XTEN may typically have a much lower tendency to stimulate B cell proliferation and thus an immune response. In one embodiment, BPXTEN may have reduced immunogenicity compared to the corresponding unfused BP. In one embodiment, administration of up to three parenteral doses of BPXTEN to a mammal can result in anti-BPXTEN IgG that is detectable at a serum dilution of 1:100 but not at a dilution of 1:1000. In some embodiments, administration of up to three parenteral doses of BPXTEN to a mammal can result in anti-BP IgG that is detectable at a serum dilution of 1:100 but not at a dilution of 1:1000. In some embodiments, administration of up to three parenteral doses of BPXTEN to a mammal may result in anti-XTEN IgG that is detectable at a serum dilution of 1:100, but not at a dilution of 1:1000. In the foregoing embodiments, the mammal may be a mouse, rat, rabbit, or cynomolgus monkey.

[0199] An additional feature of certain embodiments of XTEN having substantially non-repetitive sequences compared to less non-repetitive sequences (such as those with three consecutive amino acids that are identical) may be that the non-repetitive XTEN may form weaker contacts (e.g., monovalent interactions) with antibodies, thereby reducing the likelihood of immune clearance and potentially resulting in longer retention of the BPXTEN composition in the circulation.

[0200] In some embodiments, the (fusion) polypeptide is less immunogenic than a biologically active polypeptide that is not linked to any XTEN, where immunogenicity is confirmed by measuring the production of IgG antibodies that selectively bind to the biologically active polypeptide after administration of an equivalent dose to a subject.

[0201] Spacer and BP release segment In some embodiments, at least a portion of the biological activity of each BP is retained by intact BPXTEN. In some embodiments, the BP components either become biologically active or have increased biological activity when released from the XTEN by cleavage of a selective cleavage sequence incorporated within a spacer sequence in BPXTEN, as described more fully herein below.

[0202] Any of a number of spacer sequences are optional in the fusion proteins encompassed by the present invention. Spacers may be provided to enhance expression of the fusion protein from host cells or to reduce steric hindrance so that the BP component can assume its desired tertiary structure and / or properly interact with its target molecule. For information on spacers and methods for identifying desired spacers, see, for example, George, et al. (2003) Protein Engineering 15:871-879, specifically incorporated herein by reference. In one embodiment, the spacer comprises one or more peptide sequences that are between 1 and 50 amino acid residues in length, or about 1-25 residues in length, or about 1-10 residues in length. The spacer sequence, excluding the cleavage site, may comprise any of the 20 naturally occurring L-amino acids, and preferably comprises hydrophilic amino acids that do not cause steric hindrance, which may include, but are not limited to, glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). In some embodiments, the spacer may be polyglycine or polyalanine, or may be a mixture of a combination of glycine and alanine residues. The spacer polypeptide, excluding the cleavage sequence, is substantially devoid of secondary structure. In one embodiment, one or both spacer sequences in a BPXTEN fusion protein composition may each further contain a cleavage sequence, which may be the same or different, that can be acted upon by a protease to release the BP from the fusion protein.

[0203] In some cases, the incorporation of a cleavage sequence into BPXTEN is designed to allow the release of an active or more active BP upon release from XTEN. The cleavage sequence is located sufficiently close to the BP sequence, generally within 18, 12, 6, or 2 amino acids from the end of the BP sequence, so that any remaining residues attached to the BP after cleavage do not significantly interfere with the activity of the BP (e.g., receptor binding) and provide sufficient access for the protease to further cleave the cleavage sequence. In some embodiments, the cleavage site is a sequence that can be cleaved by a protease endogenous to a mammalian subject, so that BPXTEN can be cleaved after administration to the subject. In such cases, BPXTEN can function as a prodrug or a circulatory depot for the BP. Examples of cleavage sites contemplated by the present invention include, but are not limited to, polypeptide sequences cleavable by mammalian endogenous proteases, such as FXIa, FXIIa, kallikrein, FVIIa, FIXa, FXa, FIIa (thrombin), elastase-2, granzyme B, MMP-12, MMP-13, MMP-17, or MMP-20, or by non-mammalian proteases, such as TEV, enterokinase, PreScission™ protease (rhinovirus 3C protease), or sortase A. Sequences known to be cleaved by the aforementioned proteases are known in the art. Exemplary cleavage sequences and cut sites within the sequences, along with sequence variants, are listed in Table 7a. For example, thrombin (activated coagulation factor II) acts on the sequence LTPRSLLV (SEQ ID NO: 222), which is cleaved after arginine at position 4 of the sequence [Rawlings ND, et al. (2008) Nucleic Acids Res., 36: D320]. Active FIIa is produced by cleavage of FII by FXa in the presence of phospholipids and calcium, and is downstream from factor IX in the coagulation pathway. Once activated, its natural role in coagulation is to cleave fibrinogen, which then initiates clot formation.FIIa activity is tightly controlled and only occurs when coagulation is required for proper hemostasis.However, because coagulation is an ongoing process in mammals, due to the incorporation of LTPRSLLV (SEQ ID NO: 222) sequence between the BP and XTEN of BPXTEN, the XTEN domain is removed from the adjacent BP upon the activation of either extrinsic or intrinsic coagulation pathways when coagulation is physiologically required, thereby releasing BP over time.Similarly, the incorporation of other sequences into BPXTEN that are acted upon by endogenous proteases provides sustained release of BP, which in some cases can provide a higher degree of activity for BP from the "prodrug" form of BPXTEN.

[0204] In some cases, only two or three amino acids flanking either side of the cut site (four to six amino acids total) will be incorporated into the cleavage sequence. In other cases, the known cleavage sequence can have one or more deletions or insertions, or substitutions of one, two, or three amino acids for any one, two, or three amino acids of the known sequence, where the deletions, insertions, or substitutions result in reduced or enhanced susceptibility to proteases, rather than absent susceptibility, resulting in the ability to tailor the release rate of the BP from the XTEN. Exemplary substitutions are shown in Table 7a. Table 7a: Protease cleavage sequences for BP release [Table 7a] ↓ indicates the cleavage site; NA: Not Applicable; * A list of multiple amino acids before, between, or after a slash indicates alternative amino acids that may be substituted in that place; "-" indicates that any amino acid may be substituted with the corresponding amino acid shown in the middle column.

[0205] In some embodiments, the BPXTEN fusion protein may include a spacer sequence that may further include one or more cleavage sequences configured to release the BP from the fusion protein when acted upon by a protease. In some embodiments, the one or more cleavage sequences may have at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%) sequence identity to a sequence from Table 7a.

[0206] In some embodiments, the present disclosure provides BP release segment peptides (or release segments (RS)) that are substrates for one or more mammalian proteases associated with or produced by diseased tissue or cells found near diseased tissue. Such proteases include classes of proteases such as metalloproteinases, cysteine ​​proteases, aspartic acid proteases, and serine proteases, including, but not limited to, those in Table 7b. RSs are useful, among other things, for incorporation into a subject recombinant polypeptide to confer a prodrug format that can be activated by cleavage of the RS by a mammalian protease. As described herein, an RS is incorporated into a subject recombinant polypeptide composition, linking the incorporated binding moiety to an XTEN (this configuration is described more fully below), such that upon cleavage of the RS by the action of one or more proteases for which the RS is a substrate, the binding moiety and XTEN are released from the composition, and the binding moiety is no longer protected by the XTEN and regains full ability to bind their ligand. In these recombinant polypeptide compositions comprising a first and a second antibody fragment, the compositions are referred to herein as activatable antibody compositions (AAC). Table 7b: Target tissue proteases [Table 7b-1] [Table 7b-2]

[0207] In one embodiment, the disclosure provides an activatable recombinant polypeptide comprising a first release segment (RS1) sequence that, when optimally aligned, has at least 88%, or at least 94%, or 100% sequence identity to a sequence identified herein by the sequence shown in Table 8a, where RS1 is a substrate for one or more mammalian proteases. In other embodiments, the disclosure provides an activatable recombinant polypeptide comprising RS1 and second release segment (RS2) sequences that, when optimally aligned, have at least 88%, or at least 94%, or 100% sequence identity, respectively, to a sequence identified herein by the sequence shown in Table 8a, where RS1 and RS2 are each substrates for one or more mammalian proteases. In some embodiments, the present disclosure provides an activatable recombinant polypeptide comprising a first RS (RS1) sequence that, when optimally aligned, has at least 90%, at least 93%, at least 97%, or 100% identity to a sequence identified herein by the sequence shown in Table 8b, where the RS is a substrate for one or more mammalian proteases. In other embodiments, the present disclosure provides an activatable recombinant polypeptide comprising an RS1 and a second release segment (RS2) sequence that, when optimally aligned, have at least 88%, or at least 94%, or 100% sequence identity, respectively, to a sequence identified herein by the sequence shown in Table 8b, where RS1 and RS2 are each substrates for one or more mammalian proteases (e.g., at one, two, or three cleavage sites within each release segment sequence). In embodiments of an activatable recombinant polypeptide comprising an RS1 and an RS2, the two release segments may be identical or may be different in sequence.

[0208] The present disclosure contemplates release segments that are substrates for one, two, or three different classes of proteases: metalloproteinases, cysteine ​​proteases, aspartic acid proteases, or serine proteases, including those in Table 7b. In particular aspects, the RS serves as a substrate for proteases found intimately associated with or coexisting with diseased tissues or cells, such as, but not limited to, tumors, cancer cells, and inflamed tissues; upon cleavage of the RS, binding moieties otherwise protected by the XTEN of a subject recombinant polypeptide composition (and thus having low binding affinity for their respective ligands) are released from the composition and regain their full ability to bind to ligands on target and / or effector cells. In some embodiments, the RS of a subject recombinant polypeptide composition comprises an amino acid sequence that is a substrate for a cellular protease located within a targeted cell, including, but not limited to, a protease in Table 7b. In another particular feature of a subject recombinant polypeptide composition, an RS that is a substrate for two or three classes of proteases is designed with a sequence that can be cleaved at different locations in the RS sequence by different proteases. Thus, an RS that is a substrate for two, three, or more classes of proteases has two, three, or multiple different cleavage sites in the RS sequence, yet cleavage by a single protease nevertheless results in release of the binding moiety and XTEN from the recombinant polypeptide composition that includes the RS.

[0209] In one embodiment, a RS of the present disclosure for incorporation into a subject recombinant polypeptide composition is selected from the group consisting of meprin, neprilysin (CD10), PSMA, BMP-1, A disintegrin and metalloproteinase (ADAM), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM with thrombospondin motifs (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloproteinases (MTPs), and ribosomal proteinases (RIs). matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3 (MMP-3, stromelysin 1), matrix metalloproteinase-7 (MMP-7, matrilysin 1), matrix metalloproteinase-8 (MMP-8, collagenase 2), matrix metalloproteinase-9 (MMP-9, gelatinase B), matrix metalloproteinase-10 (MMP-10, stromelysin 2), matrix metalloproteinase-11 (MMP-11 , stromelysin 3), matrix metalloproteinase-12 (MMP-12, macrophage elastase), matrix metalloproteinase-13 (MMP-13, collagenase 3), matrix metalloproteinase-14 (MMP-14, MT1-MMP), matrix metalloproteinase-15 (MMP-15, MT2-MMP), matrix metalloproteinase-19 (MMP-19), matrix metalloproteinase-23 (MMP-23, CA-MMP), matrix metalloproteinase-24 (MMP- 24, MT5-MMP), matrix metalloproteinase-26 (MMP-26, matrilysin 2), matrix metalloproteinase-27 (MMP-27, CMMP), legumain, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X, cathepsin D, cathepsin E, secretase, urokinase (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, prostate-specific antigen (PSA, KLK3), human neutrophil elastase (HNE), elastase, tryptase,The RS is a substrate for one or more proteases, including, but not limited to, type II transmembrane serine protease (TTSP), DESC1, hepsin (HPN), matriptase, matriptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast activation protein (FAP), kallikrein-related peptidase (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14. In one embodiment, the RS is a substrate for ADAM17. In one embodiment, the RS is a substrate for BMP-1. In one embodiment, the RS is a substrate for cathepsin. In one embodiment, the RS is a substrate for HtrA1. In one embodiment, the RS is a substrate for legumain. In one embodiment, the RS is a substrate for MMP-1. In one embodiment, RS is a substrate for MMP-2. In one embodiment, RS is a substrate for MMP-7. In one embodiment, RS is a substrate for MMP-9. In one embodiment, RS is a substrate for MMP-11. In one embodiment, RS is a substrate for MMP-14. In one embodiment, RS is a substrate for uPA. In one embodiment, RS is a substrate for matriptase. In one embodiment, RS is a substrate for MT-SP1. In one embodiment, RS is a substrate for neutrophil elastase. In one embodiment, RS is a substrate for thrombin. In one embodiment, RS is a substrate for TMPRSS3. In one embodiment, RS is a substrate for TMPRSS4. In one embodiment, RS of a subject recombinant polypeptide composition is a substrate for at least two proteases, including, but not limited to, legumain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In some embodiments, the RS of a subject recombinant polypeptide composition is a substrate for legumain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. Table 8a: BP release segment sequences [Table 8a-1] [Table 8a-2] [Table 8a-3] Table 8b: Emission segment sequence [Table 8b-1] [Table 8b-2] [Table 8b-3] [Table 8b-4] [Table 8b-5] [Table 8b-6] [Table 8b-7] [Table 8b-8] [Table 8b-9] [Table 8b-10] [Table 8b-11]

[0210] In some embodiments, RSs for incorporation into a subject recombinant polypeptide can be designed to be selectively sensitive to the various proteases for which they are substrates, thereby having different cleavage rates and cleavage efficiencies. Because a given protease may be found at different concentrations in diseased tissues, including, but not limited to, tumors, blood cancers, or inflamed tissues or sites of inflammation, compared to healthy tissues or the circulation, the present disclosure provides RSs with individual amino acid sequences engineered to have higher or lower cleavage efficiencies for a given protease to ensure that the recombinant polypeptide is preferentially converted from a prodrug form to an active form (i.e., by separation and release of the binding moiety and XTEN from the recombinant polypeptide after cleavage of the RS) when in proximity to target cells or tissues and the proteases coexisting therewith, compared to the cleavage rate of the RS in healthy tissues or the circulation, such that the released antibody fragment-binding moiety has a higher ability to bind to the ligand in diseased tissues compared to the prodrug form that remains in the circulation. Such selective design can improve the therapeutic index of the resulting composition and reduce side effects relative to conventional therapeutic agents that do not incorporate such site-specific activation.

[0211] As used herein, cleavage efficiency is defined as the log2 ratio of the percentage of test substrates containing cleaved RS to the percentage of control substrate AC1611 cleaved when each is subjected to a protease enzyme in a biochemical assay (described further in the Examples) in which the reaction is performed with an initial substrate concentration of 6 μM; the reactions are incubated at 37°C for 2 hours, then stopped by adding EDTA; the amounts of digestion products and uncleaved substrate are analyzed by non-reducing SDS-PAGE to establish the ratio of percentage cleavage. Cleavage efficiency is calculated as follows:

number

[0212] In some embodiments, the disclosure provides an AAC comprising a plurality of RSs, each RS sequence identified herein by a group of sequences set forth in Table 8a, and the RSs are linked to one another by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In one embodiment, the AAC comprises a first RS and a second RS different from the first RS, each RS sequence identified herein by a sequence set forth in Table 8a, and the RSs are linked to one another by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In some embodiments, the AAC comprises a first RS, a second RS different from the first RS, and a third RS different from the first and second RSs, each sequence identified herein by a sequence set forth in Table 8a, and the first, second, and third RSs are linked to one another by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. It is specifically contemplated that multiple RSs of an AAC can be linked to form a sequence that can be cleaved by multiple proteases with different cleavage rates or cleavage efficiencies. In some embodiments, the present disclosure provides an AAC comprising RS1 and RS2 identified herein by the sequences set forth in Tables 8a-8b, and XTEN1 and XTEN2, such as those described above or elsewhere herein, where RS1 is fused between XTEN1 and the binding moiety and RS2 is fused between XTEN2 and the binding moiety. Such compositions are contemplated to be more readily cleaved by diseased target tissues that express multiple proteases compared to healthy tissues or normal circulation, resulting in the resulting fragments bearing the binding moiety penetrating the target tissue, e.g., tumors, more easily and having enhanced ability to bind and ligate to target cells and effector cells (or just target cells in the case of AACs designed with a single binding moiety).

[0213] The RS of the present disclosure is useful for inclusion in a recombinant polypeptide as a therapeutic agent for the treatment of cancer, autoimmune diseases, inflammatory diseases, and other conditions where localization of the recombinant polypeptide's activity is desirable. The subject compositions address unmet needs and are superior in one or more aspects compared to conventional antibody or bispecific antibody therapeutics that are active upon injection, including enhanced terminal half-life, targeted delivery, and improved therapeutic ratios with reduced toxicity to healthy tissues.

[0214] In some embodiments, the (fusion) polypeptide comprises a first release segment (RS1) positioned between the (first) XTEN and the biologically active polypeptide. In some embodiments, the polypeptide further comprises a second release segment (RS2) positioned between the biologically active polypeptide and the second XTEN. In some embodiments, RS1 and RS2 are identical in sequence. In some embodiments, RS1 and RS2 are not identical in sequence. 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 to a sequence identified herein by a sequence in Tables 8a-8b, or a subset thereof. In some embodiments, RS2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by a sequence in Tables 8a-8b, or a subset thereof. In some embodiments, RS1 and RS2 are substrates for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence, respectively.

[0215] reference fragment

[0216] In some embodiments, the (fusion) polypeptide further comprises one or more reference fragments releasable from the polypeptide upon digestion with a protease. In some embodiments, the one or more reference fragments each comprise a biologically active portion of the polypeptide. In some embodiments, the one or more reference fragments is a single reference fragment that differs in sequence and molecular weight from all other peptide fragments releasable from the polypeptide upon digestion of the polypeptide with a protease.

[0217] Exemplary Polypeptides

[0218] In some embodiments of the compositions of the present disclosure, the polypeptide is a recombinant polypeptide comprising an amino acid sequence having at least (about) 80% sequence identity to a sequence set forth in Table D (consisting of SEQ ID NOS: 12-47), or a subset thereof. The polypeptide may comprise an amino acid sequence having 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 (about) 100% sequence identity to a sequence set forth in Table D (SEQ ID NOS: 12-47), or a subset thereof. A polypeptide may comprise an amino acid sequence having 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 (about) 100% sequence identity to a sequence set forth in Table D (SEQ ID NOS: 12-47), or a subset thereof. A polypeptide may comprise an amino acid sequence identical to a sequence set forth in Table D (SEQ ID NOS: 12-47), or a subset thereof. It is specifically contemplated that compositions of the present disclosure may comprise sequence variants of the amino acid sequences set forth in Table D, such as including a linker sequence inserted or including a purification tag sequence attached thereto, so long as the variant exhibits substantially similar or identical biological activity(ies) and / or mechanism of activity. Table D: Exemplary amino acid sequences of polypeptides [Table D-1] [Table D-2] [Table D-3] [Table D-4] [Table D-5] [Table D-6] [Table D-7] [Table D-8] [Table D-9] [Table D-10] [Table D-11] [Table D-12] [Table D-13] [Table D-14] [Table D-15] [Table D-16] [Table D-17] [Table D-18]

[0219] Polypeptide Mixture The disclosure herein also includes a mixture comprising a plurality of polypeptides of varying lengths, the mixture comprising a first set of polypeptides and a second set of polypeptides. In some embodiments, each polypeptide in the first set of polypeptides comprises a barcode fragment (a) releasable from the polypeptide by digestion with a protease and (b) having a sequence and molecular weight that is different from the sequences and molecular weights of all other fragments releasable from the first set of polypeptides. In some embodiments, the second set of polypeptides lacks the barcode fragment (e.g., by cleavage) of the first set of polypeptides. In some embodiments, both the first set of polypeptides and the second set of polypeptides each comprise a reference fragment that is (a) common to the first set of polypeptides and the second set of polypeptides and (b) releasable by digestion with a protease. In some embodiments, the ratio of the first set of polypeptides to the polypeptide comprising the reference fragment is greater than 0.70. In some embodiments, the ratio of the first set of polypeptides to the polypeptide comprising the reference fragment is greater than 0.80, 0.90, 0.95, or 0.98. In some embodiments, the reference fragment occurs only once in each polypeptide in the first set of polypeptides and the second set of polypeptides. In some embodiments, the protease is a protease that cleaves C-terminal to glutamic acid residues. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, the polypeptides of various lengths include polypeptides comprising at least one extended recombinant polypeptide (XTEN), such as any described herein above or elsewhere herein. In some embodiments, the first set of polypeptides comprises a full-length polypeptide, and the barcode fragment is a portion of the full-length polypeptide. In some embodiments, the full-length polypeptide is a (fusion) polypeptide, such as any described herein above or elsewhere herein. In some embodiments, the barcode fragment lacks (does not include) both the N-terminal and C-terminal amino acids of the full-length polypeptide.In some embodiments, the mixture of polypeptides of various lengths differ from each other by N-terminal truncation, C-terminal truncation, or both N- and C-terminal truncation of the full-length polypeptide. In some embodiments, the first set of polypeptides and the second set of polypeptides may differ in one or more pharmacological properties. Non-limiting exemplary properties include:

[0220] Methods for characterizing polypeptides The present disclosure includes a method for assessing the relative abundance of a first set of polypeptides in a mixture containing polypeptides of varying lengths relative to a second set of polypeptides in the mixture, wherein (1) each polypeptide in the first set of polypeptides shares a barcode fragment that occurs only once among the polypeptides, and (2) each polypeptide in the second set of polypeptides lacks the barcode fragment shared by the polypeptides in the first set, and each individual polypeptide in both the first polypeptide and the second set of polypeptides each comprises a reference fragment. The method may include contacting the mixture with a protease to produce a plurality of proteolytic fragments resulting from cleavage of the first set of polypeptides and the second set of polypeptides, the plurality of proteolytic fragments comprising a plurality of reference fragments and a plurality of barcode fragments. The method may further include determining the ratio of the amount of the barcode fragment to the amount of the reference fragment, thereby assessing the relative abundance of the first set of polypeptides relative to the second set of polypeptides. In some embodiments, the barcode fragment occurs only once in each polypeptide in the first set of polypeptides. In some embodiments, the reference fragment occurs only once in each polypeptide in the first set of polypeptides and the second set of polypeptides. In some embodiments, the plurality of proteolytic fragments comprises a plurality of reference fragments and a plurality of barcode fragments. In some embodiments, the protease cleaves the first and second sets of polypeptides (or polypeptides of various lengths) C-terminal to glutamic acid residues that are not followed by proline residues. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, determining the ratio of the amount of barcode fragments to the amount of reference fragments comprises identifying the barcode fragments and the reference fragments from the mixture after contacting with the protease. In some embodiments, the barcode fragments and the reference fragments are identified based on their respective masses. In some embodiments, the barcode fragments and the reference fragments are identified by mass spectrometry.In some embodiments, the barcode fragments and reference fragments are identified by liquid chromatography-mass spectrometry (LC-MS). In some embodiments, determining the ratio of the barcode fragments to the reference fragments comprises isobaric labeling. In some embodiments, determining the ratio of the barcode fragments to the reference fragments comprises spiking the mixture with one or both of the isotopically labeled reference fragments and the isotopically labeled barcode fragments. In some embodiments, the polypeptides of various lengths comprise polypeptides comprising at least one extended recombinant polypeptide (XTEN) described hereinabove or elsewhere herein. In some embodiments, the XTEN is characterized in that it (i) comprises at least 100 or at least 150 amino acids, (ii) at least 90% of the amino acid residues of the XTEN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P), and (iii) comprises at least four different amino acids that are G, A, S, T, E, or P. In some embodiments, the barcode fragment, if present, is part of an XTEN. In some embodiments, the mixture of polypeptides of various lengths comprises the polypeptides described herein above or elsewhere herein. In some embodiments, the polypeptides of various lengths include full-length polypeptides and truncated fragments thereof. In some embodiments, the polypeptides of various lengths essentially consist of full-length polypeptides and truncated fragments thereof. In some embodiments, the mixture of polypeptides of various lengths differs from each other by N-terminal truncation, C-terminal truncation, or both N- and C-terminal truncation of the full-length polypeptide. In some embodiments, the full-length polypeptide is a polypeptide described herein above or elsewhere herein. In some embodiments, the ratio of the amount of the barcode fragment to the reference fragment is greater than 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, or 0.99.

[0221] Quantification based on isobaric labeling of peptides In some embodiments, isobaric labeling can be used to determine the ratio of barcode fragments to reference fragments.Those skilled in the art will understand that isobaric labeling is a mass spectrometry strategy used in quantitative proteomics, where peptides or proteins (or parts thereof) are labeled with various chemical groups that are isobaric (same mass) but differ in the distribution of heavy isotopes around their structure.These tags are generally called tandem mass tags, and are designed so that during high-energy collision-induced dissociation (CID) during tandem mass spectrometry, the mass tag is cleaved at a specific linker region, thereby obtaining reporter ions of different masses.Those skilled in the art will understand that one of the most common isobaric tags is an amine-reactive tag.

[0222] The increased ability to detect and quantify cleavage products (e.g., by isobaric labeling) can generate knowledge that can aid in the design of manufacturing processes, including purification steps, to minimize the presence of unwanted variants in the purified drug substance / product.

[0223] Recombinant production The disclosure herein includes nucleic acids, which may comprise a polynucleotide (or polynucleotide sequence) encoding a (fusion) polypeptide such as any described herein above or anywhere else herein, or the nucleic acid may comprise the reverse complement of such a polynucleotide (or polynucleotide sequence).

[0224] The disclosure herein includes expression vectors comprising a polynucleotide sequence such as any described in the preceding paragraph, and a regulatory sequence operably linked to the polynucleotide sequence.

[0225] The disclosure herein includes host cells comprising an expression vector such as any described in the preceding paragraph. In some embodiments, the host cell is a prokaryote. In some embodiments, the host cell is E. coli. In some embodiments, the host cell is a mammalian cell.

[0226] In some embodiments, the present disclosure provides a method for producing a subject composition. In one embodiment, the method comprises culturing a host cell containing a nucleic acid construct encoding any of the polypeptides or XTEN-containing compositions described herein under conditions that promote the expression of the polypeptide or BPXTEN fusion polypeptide, followed by recovering the polypeptide or BPXTEN fusion polypeptide using standard purification methods (e.g., column chromatography, HPLC, etc.), wherein at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the binding fragments of the expressed polypeptide or BPXTEN fusion polypeptide are correctly folded. In some embodiments of the method, the expressed polypeptide or BPXTEN fusion polypeptide is recovered such that at least 90%, or at least 95%, or at least 97%, or at least 99% of the polypeptide or BPXTEN fusion polypeptide is recovered in a monomeric, soluble form.

[0227] In some embodiments, the present disclosure provides expression vectors encoding constructs useful in methods for producing polypeptides and BPXTEN fusion polypeptides at high fermentation expression levels of functional proteins using E. coli or mammalian host cells, as well as methods for producing cytotoxic polypeptide construct compositions at high expression levels. In one embodiment, the method includes the steps of: 1) preparing a polynucleotide encoding a polypeptide of any of the embodiments disclosed herein; 2) cloning the polynucleotide into an expression vector, which may be a plasmid or other vector, under the control of transcription and translation sequences suitable for high-level protein expression in a biological system; 3) transforming a suitable host cell with the expression vector; and 4) culturing the host cell in a conventional nutrient medium under conditions suitable for expression of the polypeptide composition. If desired, the host cell is E. coli. By this method, expression of the polypeptide results in a fermentation titer of at least 0.05 g / L, or at least 0.1 g / L, or at least 0.2 g / L, or at least 0.3 g / L, or at least 0.5 g / L, or at least 0.6 g / L, or at least 0.7 g / L, or at least 0.8 g / L, or at least 0.9 g / L, or at least 1 g / L, or at least 2 g / L, or at least 3 g / L, or at least 4 g / L, or at least 5 g / L of host cell expression product, and at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the expressed protein is correctly folded. As used herein, the term "correctly folded" means that the antigen-binding fragment component of the composition has the ability to specifically bind to its target ligand.In some embodiments, the disclosure provides a method for producing a polypeptide or a BPXTEN fusion polypeptide, comprising culturing a host cell comprising a vector encoding a polypeptide comprising a polypeptide or a BPXTEN fusion polypeptide under conditions effective to express in a fermentation reaction a concentration of polypeptide product greater than about 10 milligrams per gram dry weight of host cells (mg / g), or at least about 250 mg / g, or about 300 mg / g, or about 350 mg / g, or about 400 mg / g, or about 450 mg / g, or about 500 mg / g of polypeptide when the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, wherein the antigen-binding fragment of the expressed protein is correctly folded. In some embodiments, the disclosure provides a method for producing a polypeptide or BPXTEN fusion polypeptide, comprising culturing a host cell comprising a vector encoding the composition under conditions effective to express in a fermentation reaction a concentration of polypeptide product greater than about 10 milligrams per gram dry weight of host cells (mg / g), or at least about 250 mg / g, or about 300 mg / g, or about 350 mg / g, or about 400 mg / g, or about 450 mg / g, or about 500 mg / g of polypeptide when the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, wherein the expressed polypeptide product is soluble.

[0228] Pharmaceutical Composition The present disclosure includes pharmaceutical compositions comprising any (fusion) polypeptide, such as those described herein above or elsewhere herein, and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is formulated for intradermal, subcutaneous, oral, intravenous, intraarterial, intraperitoneal, intraperitoneal, intravitreal, intrathecal, or intramuscular administration. In some embodiments, the pharmaceutical composition is in liquid form or frozen. In some embodiments, the pharmaceutical composition is a device implanted in the eye or another body part. In some embodiments, the pharmaceutical composition is in a pre-filled syringe for single injection. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder that is reconstituted before administration.

[0229] In some embodiments, the dose is administered intradermally, subcutaneously, orally, intravenously, intravitreally (or otherwise intraocularly), intraarterially, intraabdominally, intraperitoneally, intrathecally, or intramuscularly. In some embodiments, the pharmaceutical composition is administered using a device implanted in the eye or other body part. In some embodiments, the subject is a mouse, rat, monkey, or human.

[0230] The pharmaceutical composition may be administered for therapy by any suitable route. Additionally, the pharmaceutical composition may contain other pharmaceutically active compounds or multiple compounds of the present invention.

[0231] In some embodiments, the pharmaceutical composition may be administered subcutaneously, orally, intramuscularly, or intravenously. In one embodiment, the pharmaceutical composition is administered at a therapeutically effective dose. In some of the above cases, the therapeutically effective dose results in an increase in the time spent within the therapeutic range for the fusion protein compared to the corresponding BP of the fusion protein not linked to XTEN and administered to a subject at an equivalent dose. The increase in time spent within the therapeutic range may be at least 3 times greater than the corresponding BP not linked to XTEN, or at least 4 times, or 5 times, or 6 times, or 7 times, or 8 times, or 9 times, or at least 10 times, or at least 20 times greater than the corresponding BP not linked to XTEN.

[0232] In some embodiments, the present invention provides a method of treating a disease, disorder, or condition, comprising administering to a subject a pharmaceutical composition using multiple consecutive doses of the pharmaceutical composition, the doses being administered using a therapeutically effective dose regimen. In one such embodiment, the therapeutically effective dose regimen is a therapeutically effective dose regimen that is greater than or equal to at least two consecutive Cs for blood levels of the fusion protein compared to a corresponding BP of the fusion protein not linked to XTEN and administered to the subject using an equivalent dose regimen. max Peak and / or C mm It may result in an increase in time between troughs of at least 3-fold, or at least 4-fold, or 5-fold, or 6-fold, or 7-fold, or 8-fold, or 9-fold, or at least 10-fold, or at least 20-fold. In some of the foregoing embodiments, administration of the fusion protein results in a comparable improvement in at least one measured parameter using less frequent administration or a lower total molar dose of the fusion protein of the pharmaceutical composition compared to the corresponding biologically active protein component not linked to the XTEN(s) and administered to the subject using a therapeutically effective regimen to the subject.

[0233] In one embodiment, pharmaceutical composition is administered subcutaneously.In this embodiment, composition can be provided as a lyophilized powder that is reconstituted before administration.Composition can be provided in liquid form or frozen state that can be directly administered to patients.In one embodiment, composition is provided as a liquid in a pre-filled syringe, so that patients can easily self-administer composition.

[0234] The extended-release formulation useful in the present invention may be an oral formulation comprising a matrix and a coating composition. Suitable matrix materials include waxes (e.g., carnauba, beeswax, paraffin wax, ceresin, shellac wax, fatty acids, and fatty alcohols), oils, hydrogenated oils or fats (e.g., hydrogenated rapeseed oil, castor oil, beef tallow, palm oil, and soybean oil), and polymers (e.g., hydroxypropyl cellulose, polyvinylpyrrolidone, hydroxypropylmethylcellulose, and polyethylene glycol). Other suitable matrix tableting materials include microcrystalline cellulose, powdered cellulose, hydroxypropyl cellulose, and ethyl cellulose, including other carriers and fillers. Tablets may also contain granules, coated powders, or pellets. Tablets may also be multi-layered. Multi-layer tablets are particularly preferred when the active ingredients have significantly different pharmacokinetic profiles. Optionally, the finished tablet may be coated or uncoated.

[0235] The coating composition may contain an insoluble matrix polymer and / or a water-soluble material. The water-soluble material may be a polymer such as polyethylene glycol, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, or a monomeric material such as a sugar (e.g., lactose, sucrose, fructose, mannitol, etc.), a salt (e.g., sodium chloride, potassium chloride, etc.), an organic acid (e.g., fumaric acid, succinic acid, lactic acid, and tartaric acid), or a mixture thereof. If necessary, an enteric polymer may be incorporated into the coating composition. Suitable enteric polymers include hydroxypropyl methylcellulose, succinic acetate, hydroxypropyl methylcellulose, phthalic acid, polyvinyl acetate phthalate, cellulose acetate phthalate, cellulose acetate trimellitate, shellac, zein, and carboxy-containing polymethacrylates. The coating composition may be plasticized by adding a suitable plasticizer, such as diethyl phthalate, citric acid esters, polyethylene glycol, glycerol, acetylated glycerides, acetylated citric acid esters, dibutyl sebacate, and castor oil. The coating composition may contain a filler, which may be an insoluble material such as silicon dioxide, titanium dioxide, talc, kaolin, alumina, starch, powdered cellulose, MCC, or polacrilin potassium. The coating composition may be applied as a solution or latex in an organic solvent, an aqueous solvent, or a mixture thereof. Solvents such as water, lower alcohols, lower chlorinated hydrocarbons, ketones, or mixtures thereof may also be used.

[0236] The BPXTEN polypeptides of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the polypeptides are combined in a mixture with a pharmaceutically acceptable carrier vehicle, such as an aqueous solution or buffer, a pharmaceutically acceptable suspension, or an emulsion. Examples of non-aqueous solvents include propylethylene glycol, polyethylene glycol, and vegetable oils. Therapeutic formulations can be prepared in the form of lyophilized formulations or aqueous solutions, as described in Remington's Pharmaceutical Sciences 16. th As described in the "Osol, A. Ed. Edition, 1980," the active ingredient having the desired purity is prepared for storage by mixing with physiologically acceptable carriers, excipients, or stabilizers as needed. The compositions of the present invention can be formulated using a variety of excipients. Suitable excipients include microcrystalline cellulose (e.g., Avicel PH 102, Avicel PH101), polymethacrylate, poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride) (e.g., Eudragit RS-30D), hydroxypropyl methylcellulose (Methocel K10OM, Premium CR Methocel K10OM, Methocel E5, Opadry®), magnesium stearate, talc, triethyl citrate, ethylcellulose dispersion (Surelease®), and protamine sulfate. The slow-release agent may also contain a carrier, and the carrier may contain, for example, a solvent, a dispersion medium, a coating, an antibacterial agent and an antifungal agent, an isotonic agent, and an absorption retardant.These slow-release agents can also use pharmaceutically acceptable salts, for example, inorganic salts such as hydrochloride, hydrobromide, phosphate, or sulfate, and organic acid salts such as acetate, propionate, malonate, or benzoate.The composition can also contain liquids such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifiers, or pH buffers.Liposomes can also be used as carriers.

[0237] In some embodiments, the compositions of the present invention are encapsulated in liposomes, which have proven useful in delivering beneficial agents in a controlled manner over a long period of time. Liposomes are closed bilayer membranes containing a trapped aqueous volume. Liposomes may be unilamellar vesicles with a single membrane bilayer or multilamellar vesicles with multiple membrane bilayers, each separated from the next by an aqueous phase. The resulting membrane bilayer structure is such that the hydrophobic (non-polar) tails of the lipids are oriented toward the center of the bilayer, while the hydrophilic (polar) heads are oriented toward the aqueous phase. In one embodiment, liposomes can be coated with a flexible water-soluble polymer that avoids uptake by organs of the mononuclear phagocyte system, primarily the liver and spleen. Suitable hydrophilic polymers for surrounding liposomes include, without limitation, PEG, polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene glycol, polyaspartamide, and hydrophilic peptide sequences described in U.S. Pat. Nos. 6,316,024; 6,126,966;...

Claims

1. A polypeptide having an N-terminal amino acid and a C-terminal amino acid, (a) an extended recombinant polypeptide (XTEN), XTEN comprising a barcode fragment (BAR) that is releasable from the polypeptide upon digestion with a protease; (b) a bispecific antibody construct (BsAb), a first antigen-binding fragment (AF1) that specifically binds to the Cluster of Differentiation 3 T-cell receptor (CD3) and comprises light chain complementarity determining regions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3) and heavy chain complementarity determining regions 1 (CDR-H1), 2 (CDR-H2), and 3 (CDR-H3), wherein the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10; and a second antigen-binding fragment (AF2) that specifically binds to human epidermal growth factor receptor 2 (HER2); and a bispecific antibody construct comprising: (c) a release segment (RS) located between the XTEN and the bispecific antibody construct. Including, The XTEN is (i) containing at least 100, or at least 150 amino acids; (ii) at least 90% of its amino acid residues are identified herein by glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P); (iii) comprises at least four different amino acids identified herein by G, A, S, T, E, or P; and (iv) the XTEN is formed from multiple non-overlapping sequence motifs, each of which is 9-14 amino acids in length. It is characterized by the plurality of non-overlapping sequence motifs (1) a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least twice in the XTEN; and (2) a non-overlapping sequence motif that appears only once within the XTEN; Including, the barcode fragment (BAR) comprises at least a portion of the non-overlapping sequence motif that occurs only once within the XTEN; said barcode fragment (BAR) differs in sequence and molecular weight from all other peptide fragments that are releasable from said polypeptide upon complete digestion of said polypeptide by said protease; A polypeptide, wherein the barcode fragment (BAR) does not include the N-terminal amino acid or the C-terminal amino acid of the polypeptide.

2. 2. The polypeptide of claim 1, wherein the sets of non-overlapping sequence motifs are each independently identified herein by SEQ ID NOs: 179-200 and 1715-1722.

3. 3. The polypeptide of claim 1 or 2, wherein the set of non-overlapping sequence motifs are each independently identified herein by SEQ ID NOs: 186-189.

4. 4. The polypeptide of claim 3, wherein the set of non-overlapping sequence motifs comprises at least two, at least three, or all four of the sequence motifs SEQ ID NOs: 186-189.

5. 5. The polypeptide of any one of claims 1 to 4, wherein the XTEN comprises a length of 100 to 3,000, 150 to 3,000, 100 to 1,000, or 150 to 1,000 amino acid residues.

6. 6. The polypeptide of any one of claims 1 to 5, wherein the XTEN comprises 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 acid residues.

7. 7. The polypeptide of any one of claims 1-6, wherein at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P).

8. 8. The polypeptide of any one of claims 1 to 7, wherein the XTEN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a sequence listed in Table 3a.

9. 9. The polypeptide of any one of claims 1 to 8, wherein the barcode fragment (BAR) does not contain a glutamic acid that is immediately adjacent to another glutamic acid, if present, in the XTEN.

10. 10. The polypeptide of claim 1, wherein the barcode fragment (BAR) has a glutamic acid at its C-terminus.

11. 11. The polypeptide of claim 1, wherein the barcode fragment (BAR) has an N-terminal amino acid immediately preceded by a glutamic acid residue.

12. 12. The polypeptide of any one of claims 1 to 11, wherein the barcode fragment (BAR) is located at a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being between 10 and 150 amino acids in length, or between 10 and 125 amino acids in length.

13. The barcode fragment (BAR) (i) the XTEN does not contain a glutamic acid, if present, that is immediately adjacent to another glutamic acid; (ii) having a glutamic acid at its C-terminus; (iii) having an N-terminal amino acid immediately preceded by a glutamic acid residue; and (iv) located at a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10 to 150 amino acids in length, or 10 to 125 amino acids in length.

13. A polypeptide according to any one of claims 1 to 12, characterized in that:

14. 14. The polypeptide of any one of claims 11 to 13, wherein the glutamic acid residue preceding the N-terminal amino acid of the barcode fragment (BAR) is not immediately adjacent to another glutamic acid residue.

15. 15. The polypeptide of any one of claims 1 to 14, wherein the barcode fragment (BAR) does not contain a second glutamic acid residue at a position other than the C-terminus of the barcode fragment, unless the second glutamic acid is immediately followed by a proline.

16. 16. The polypeptide of any one of claims 1 to 15, wherein the XTEN is located at the N-terminus of the bispecific antibody construct (BsAb) and the barcode fragment (BAR) is located within 200 amino acids, within 150 amino acids, within 100 amino acids, or within 50 amino acids of the N-terminus of the polypeptide.

17. 17. The polypeptide of any one of claims 1 to 16, wherein the XTEN is located at the N-terminus of the bispecific antibody construct (BsAb) and the barcode fragment (BAR1) is located between 10 and 200 amino acids, between 30 and 200 amino acids, between 40 and 150 amino acids, or between 50 and 100 amino acids from the N-terminus of the protein.

18. 16. The polypeptide of any one of Claims 1-15, wherein the XTEN is located at the C-terminus of the bispecific antibody construct (BsAb) and the barcode fragment (BAR) is located within 200 amino acids, within 150 amino acids, within 100 amino acids, or within 50 amino acids of the C-terminus of the polypeptide.

19. 19. The polypeptide of any one of claims 1-15 and 18, wherein the XTEN is located at the C-terminus of the bispecific antibody construct (BsAb) and the barcode fragment (BAR) is located between 10 and 200 amino acids, between 30 and 200 amino acids, between 40 and 150 amino acids, or between 50 and 100 amino acids from the C-terminus of the protein.

20. 20. The polypeptide of any one of claims 1 to 19, wherein the barcode fragment (BAR) is at least 4 amino acids in length.

21. 21. The polypeptide of claim 20, wherein the barcode fragment (BAR) is between 4 and 20 amino acids, between 5 and 15 amino acids, between 6 and 12 amino acids, or between 7 and 10 amino acids in length.

22. 22. The polypeptide of any one of claims 1 to 21, wherein the barcode fragment (BAR) comprises an amino acid sequence set forth in Table 2.

23. 23. The polypeptide of any one of claims 1-22, wherein the XTEN has a length defined by a proximal end and a distal end, (1) the proximal end is located closer to the bispecific antibody construct (BsAb) than the distal end, and (2) the barcode fragment (BAR) is located within a region of the XTEN that spans between 5% and 50%, between 7% and 40%, or between 10% and 30% of the length of the XTEN, as measured from the distal end.

24. 24. The polypeptide of any one of Claims 1-23, wherein the XTEN further comprises one or more additional barcode fragments, each of the one or more additional barcode fragments differing in sequence and molecular weight from all other peptide fragments releasable from the polypeptide upon complete digestion of the polypeptide by the protease.

25. 25. The polypeptide of any one of claims 1 to 24, wherein the release segment (RS) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 7001-7626.

26. 26. The polypeptide of any one of claims 1 to 25, wherein the protease cleaves C-terminally of glutamic acid residues that are not followed by proline.

27. 27. The polypeptide of claim 26, wherein the protease is Glu-C protease.

28. 28. The polypeptide of any one of claims 1 to 27, wherein the polypeptide is expressed as a fusion protein, wherein the fusion protein, in its uncleaved state, has a structural arrangement identified herein from N-terminus to C-terminus as AF1-AF2-RS-XTEN, AF2-AF1-RS-XTEN, XTEN-RS-AF1-AF2, or XTEN-RS-AF2-AF1.

29. 29. The polypeptide of claim 1 , wherein the release segment (RS) is fused to the bispecific antibody construct (BsAb) via a spacer.

30. 30. The polypeptide of claim 29, wherein the spacer comprises at least four amino acids that are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P).

31. 30. The polypeptide of claim 29, wherein the spacer comprises an amino acid sequence having at least 80%, 90%, or 100% sequence identity to a sequence set forth in Table C.

32. 32. The polypeptide of any one of claims 1 to 31, wherein the CDR-H1 and CDR-H2 of the first antigen-binding fragment (AF1) comprise the amino acid sequences of SEQ ID NOs: 8 and 9, respectively.

33. the CDR-L1 of the AF1 comprises the amino acid sequence of SEQ ID NO: 1 or 2; the CDR-L2 of the AF1 comprises the amino acid sequence of SEQ ID NO: 4 or 5; the CDR-L3 of the AF1 comprises the amino acid sequence of SEQ ID NO: 6; 33. A polypeptide according to any one of claims 1 to 32.

34. the CDR-L1 of the AF1 comprises the amino acid sequence of SEQ ID NO: 1; the CDR-L2 of the AF1 comprises the amino acid sequence of SEQ ID NO: 4 or 5; the CDR-L3 of the AF1 comprises the amino acid sequence of SEQ ID NO: 6; 33. A polypeptide according to any one of claims 1 to 32.

35. the CDR-L1 of the AF1 comprises the amino acid sequence of SEQ ID NO:2; the CDR-L2 of the AF1 comprises the amino acid sequence of SEQ ID NO: 4 or 5; the CDR-L3 of the AF1 comprises the amino acid sequence of SEQ ID NO: 6; 33. A polypeptide according to any one of claims 1 to 32.

36. the CDR-L1 of the AF1 comprises the amino acid sequence of SEQ ID NO: 1; the CDR-L2 of the AF1 comprises the amino acid sequence of SEQ ID NO: 4; the CDR-L3 of the AF1 comprises the amino acid sequence of SEQ ID NO: 6; 33. A polypeptide according to any one of claims 1 to 32.

37. the CDR-L1 of the AF1 comprises the amino acid sequence of SEQ ID NO:2; the CDR-L2 of the AF1 comprises the amino acid sequence of SEQ ID NO:5; the CDR-L3 of the AF1 comprises the amino acid sequence of SEQ ID NO: 6; 33. A polypeptide according to any one of claims 1 to 32.

38. 38. The polypeptide of any one of claims 1 to 37, wherein the first antigen-binding fragment (AF1) comprises four chain variable domain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3) and 4 (FR-H4), each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 60, 64, 65 and 67, respectively.

39. 38. The polypeptide of any one of claims 1 to 37, wherein the first antigen-binding fragment (AF1) comprises four chain variable domain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3) and 4 (FR-H4), each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 61, 64, 65 and 67, respectively.

40. 40. The polypeptide of any one of claims 1 to 39, wherein the first antigen-binding fragment further comprises four light chain variable domain framework regions (FR-L): FR-L1, FR-L2, FR-L3 and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 51, 52, 53 and 59, respectively.

41. 40. The polypeptide of any one of claims 1 to 39, wherein the first antigen-binding fragment further comprises four light chain variable domain framework regions (FR-L): FR-L1, FR-L2, FR-L3 and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 51, 52, 54 and 59, respectively.

42. 40. The polypeptide of any one of claims 1 to 39, wherein the first antigen-binding fragment further comprises four light chain variable domain framework regions (FR-L): FR-L1, FR-L2, FR-L3 and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 51, 52, 55 and 59, respectively.

43. 40. The polypeptide of any one of claims 1 to 39, wherein the first antigen-binding fragment further comprises four light chain variable domain framework regions (FR-L): FR-L1, FR-L2, FR-L3 and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or identical to the amino acid sequences of SEQ ID NOs: 51, 52, 56 and 59, respectively.

44. the first antigen-binding fragment (AF1) further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4); the FR-L1 comprises the amino acid sequence of SEQ ID NO: 51; the FR-L2 comprises the amino acid sequence of SEQ ID NO: 52; the FR-L3 comprises the amino acid sequence of SEQ ID NO: 53, 54, 55 or 56; the FR-L4 comprises the amino acid sequence of SEQ ID NO: 59; the FR-H1 comprises the amino acid sequence of SEQ ID NO: 60 or 61; the FR-H2 comprises the amino acid sequence of SEQ ID NO: 64; the FR-H3 comprises the amino acid sequence of SEQ ID NO: 65; the FR-H4 comprises the amino acid sequence of SEQ ID NO: 67; 40. A polypeptide according to any one of claims 1 to 39.

45. the first antigen-binding fragment (AF1) further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4); the FR-L1 comprises the amino acid sequence of SEQ ID NO: 51; the FR-L2 comprises the amino acid sequence of SEQ ID NO: 52; the FR-L3 comprises the amino acid sequence of SEQ ID NO: 53; the FR-L4 comprises the amino acid sequence of SEQ ID NO: 59; the FR-H1 comprises the amino acid sequence of SEQ ID NO: 60; the FR-H2 comprises the amino acid sequence of SEQ ID NO: 64; the FR-H3 comprises the amino acid sequence of SEQ ID NO: 65; the FR-H4 comprises the amino acid sequence of SEQ ID NO: 67; 40. A polypeptide according to any one of claims 1 to 39.

46. the first antigen-binding fragment (AF1) further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4); the FR-L1 comprises the amino acid sequence of SEQ ID NO: 51; the FR-L2 comprises the amino acid sequence of SEQ ID NO: 52; the FR-L3 comprises the amino acid sequence of SEQ ID NO: 54; the FR-L4 comprises the amino acid sequence of SEQ ID NO: 59; the FR-H1 comprises the amino acid sequence of SEQ ID NO: 61; the FR-H2 comprises the amino acid sequence of SEQ ID NO: 64; the FR-H3 comprises the amino acid sequence of SEQ ID NO: 65; the FR-H4 comprises the amino acid sequence of SEQ ID NO: 67; 40. A polypeptide according to any one of claims 1 to 39.

47. the first antigen-binding fragment (AF1) further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4); the FR-L1 comprises the amino acid sequence of SEQ ID NO: 51; the FR-L2 comprises the amino acid sequence of SEQ ID NO: 52; the FR-L3 comprises the amino acid sequence of SEQ ID NO: 55; the FR-L4 comprises the amino acid sequence of SEQ ID NO: 59; the FR-H1 comprises the amino acid sequence of SEQ ID NO: 61; the FR-H2 comprises the amino acid sequence of SEQ ID NO: 64; the FR-H3 comprises the amino acid sequence of SEQ ID NO: 65; the FR-H4 comprises the amino acid sequence of SEQ ID NO: 67; 40. A polypeptide according to any one of claims 1 to 39.

48. the first antigen-binding fragment (AF1) further comprises light chain framework regions 1 (FR-L1), 2 (FR-L2), 3 (FR-L3), and 4 (FR-L4) and heavy chain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4); the FR-L1 comprises the amino acid sequence of SEQ ID NO: 51; the FR-L2 comprises the amino acid sequence of SEQ ID NO: 52; the FR-L3 comprises the amino acid sequence of SEQ ID NO: 56; the FR-L4 comprises the amino acid sequence of SEQ ID NO: 59; the FR-H1 comprises the amino acid sequence of SEQ ID NO: 61; the FR-H2 comprises the amino acid sequence of SEQ ID NO: 64; the FR-H3 comprises the amino acid sequence of SEQ ID NO: 65; the FR-H4 comprises the amino acid sequence of SEQ ID NO: 67; 40. A polypeptide according to any one of claims 1 to 39.

49. the first antigen-binding fragment (AF1) is capable of binding to the IgG1 antigen in an in vitro assay The first antigen-binding fragment has a higher melting temperature (T ｍ );or the T of the test bispecific antigen-binding construct when the first antigen-binding fragment is incorporated into the test bispecific antigen-binding construct, and ｍ High T compared to ｍ 49. The polypeptide of any one of claims 1 to 48, wherein the test bispecific antigen-binding construct comprises the first antigen-binding fragment and a reference antigen-binding fragment that binds to an antigen other than CD3, and the control bispecific antigen-binding construct consists of the anti-CD3-binding fragment consisting of the sequence of SEQ ID NO: 206 and the reference antigen-binding fragment, as evidenced by:

50. the T of the first antigen-binding fragment ｍ the T of the anti-CD3 binding fragment consisting of the sequence of SEQ ID NO: 206 ｍ 50. The polypeptide of claim 49, wherein 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 than the temperature of the polypeptide of claim 49.

51. The first antigen-binding fragment (AF1) comprises a heavy chain variable region (VH Ｉ ) and the VH Ｉ 51. The polypeptide of any one of claims 1 to 50, comprising an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 102 or 105.

52. The first antigen-binding fragment (AF1) comprises a light chain variable region (VL Ｉ ) and the VL Ｉ 52. The polypeptide of any one of claims 1 to 51, wherein said polypeptide comprises or is identical to an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 101, 103, 104, 106 or 107.

53. Said VH Ｉ and the VL Ｉ are linked by a linker comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table A.

54. 54. The polypeptide of any one of claims 1 to 53, wherein the first antigen-binding fragment (AF1) comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of any one of SEQ ID NOs: 201-205.

55. 54. The polypeptide of any one of claims 1 to 53, wherein the first antigen-binding fragment (AF1) specifically binds to human or cynomolgus monkey (cyno) CD3.

56. 56. The polypeptide of claim 55, wherein the first antigen-binding fragment (AF1) specifically binds to human CD3.

57. 57. A polypeptide according to any one of claims 1 to 56, wherein the first antigen-binding fragment (AF1) binds to a CD3 complex subunit identified herein by the CD3 epsilon, CD3 delta, CD3 gamma or CD3 zeta unit of CD3.

58. 58. The polypeptide of claim 57, wherein the first antigen-binding fragment (AF1) binds to the CD3 epsilon fragment of CD3.

59. 59. The polypeptide of any one of claims 1 to 58, wherein the first antigen-binding fragment (AF1) exhibits an isoelectric point (pI) of less than or equal to 6.

6.

60. 60. The polypeptide of claim 59, wherein the first antigen-binding fragment (AF1) exhibits an isoelectric point (pI) of between 6.0 and 6.6, inclusive.

61. 61. The polypeptide of any one of claims 1 to 60, wherein the first antigen-binding fragment (AF1) exhibits an isoelectric point (pI) that is 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 consisting of the sequence set forth in SEQ ID NO:

206.

62. The first antigen-binding fragment (AF1) has a dissociation constant (K) of between about 10 nM and about 400 nM, as determined in an in vitro antigen-binding assay involving human or cyno CD3 antigen. ｄ 62. The polypeptide of any one of claims 1 to 61, which specifically binds to human or cyno CD3 at a constant

63. The first antigen-binding fragment (AF1) has a dissociation constant (K) of less than about 10 nM, or less than about 50 nM, or less than about 100 nM, or less than about 150 nM, or less than about 200 nM, or less than about 250 nM, or less than about 300 nM, or less than about 350 nM, or less than about 400 nM, as determined in an in vitro antigen binding assay. ｄ 63. The polypeptide of any one of claims 1 to 62, which specifically binds to human or cyno CD3 at 1:

1.

64. The first antigen-binding fragment (AF1) has a respective dissociation constant (K ｄ 64. The polypeptide of any one of claims 1 to 63, which exhibits a binding affinity for CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold weaker than the binding affinity of an antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 206, as determined by ELISA.

65. 65. The polypeptide of any one of claims 1 to 64, wherein the first antigen-binding fragment (AF1) is a chimeric or humanized antigen-binding fragment.

66. 66. The polypeptide of any one of claims 1 to 65, wherein the first antigen-binding fragment (AF1) is an Fv, Fab, Fab', Fab'-SH, a linear antibody, or a single-chain variable fragment (scFv).

67. 67. The polypeptide of any one of claims 1 to 66, wherein the second antigen-binding fragment (AF2) is an Fv, Fab, Fab', Fab'-SH, a linear antibody, a single domain antibody, or a single-chain variable fragment (scFv).

68. 67. The polypeptide of any one of claims 1 to 66, wherein the first and second antigen-binding fragments are configured as a (Fab')2 or single-chain diabody.

69. the second antigen-binding fragment (AF2) comprises: A heavy chain variable region (VH) comprising the amino acid sequence identified herein by SEQ ID NOs: 778-783. ＩＩ ); and A light chain variable region (VL) comprising the amino acid sequence identified herein by SEQ ID NOs: 878-883. ＩＩ ) 69. The polypeptide of any one of claims 1 to 68, comprising:

70. Said VH ＩＩ and the VL ＩＩ are linked by a linker comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table A.

71. 71. The polypeptide of any one of claims 1 to 70, wherein the first and second antigen-binding fragments are fused together by a peptide linker.

72. 71. The polypeptide of claim 70, wherein the peptide linker comprises two or three amino acids that are glycine, serine, or proline.

73. 73. The polypeptide of claim 70 or 72, wherein the peptide linker comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table B.

74. the XTEN is a first extended recombinant polypeptide (XTEN1); the plurality of non-overlapping sequence motifs from which the XTEN1 is formed is a first plurality of non-overlapping sequence motifs; the BAR is a first barcode fragment (BAR1); the RS is a first release segment (RS1); the polypeptide (d) a second extended recombinant polypeptide (XTEN2), XTEN2, comprising a second barcode fragment (BAR2) releasable from the polypeptide upon digestion by the protease; and (e) a second release segment (RS2) located between the second XTEN (XTEN2) and the bispecific antibody construct (BsAb). Further comprising: The XTEN2 is (i) containing at least 100, or at least 150 amino acids; (ii) at least 90% of its amino acid residues are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P); and (iii) contain at least four different amino acids that are G, A, S, T, E, or P; It is characterized by said second barcode fragment (BAR2) differs in sequence and molecular weight from all other peptide fragments releasable from said polypeptide upon complete digestion of said polypeptide by said protease; 74. The polypeptide of any one of claims 1 to 73, wherein the second barcode fragment (BAR2) does not include the N-terminal amino acid or the C-terminal amino acid of the polypeptide.

75. 75. The polypeptide of claim 74, wherein the XTEN1 is located at the N-terminus of the bispecific antibody construct and the XTEN2 is located at the C-terminus of the bispecific antibody construct.

76. 75. The polypeptide of claim 74, wherein the XTEN1 is located at the C-terminus of the bispecific antibody construct and the XTEN2 is located at the N-terminus of the bispecific antibody construct.

77. (iv) the XTEN2 is formed from a second plurality of non-overlapping sequence motifs, each of which is 9-14 amino acids in length, the second plurality of non-overlapping sequence motifs comprising: (1) a second set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of the second set of non-overlapping sequence motifs is repeated at least twice in the second XTEN; and (2) a non-overlapping sequence motif that appears only once within the second XTEN; Including, 77. The polypeptide of any one of claims 74-76, wherein the second barcode fragment (BAR2) comprises at least a portion of the non-overlapping sequence motif that occurs only once within the second XTEN.

78. 78. The polypeptide of claim 77, wherein the second set of non-overlapping sequence motifs are each independently identified herein by SEQ ID NOs: 179-200 and 1715-1722.

79. 79. The polypeptide of claim 78, wherein the second set of non-overlapping sequence motifs are each independently identified herein by SEQ ID NOs: 186-189.

80. 80. The polypeptide of claim 79, wherein the second set of non-overlapping sequence motifs comprises at least two, at least three, or all four of the sequence motifs SEQ ID NOs: 186-189.

81. 81. The polypeptide of any one of claims 74-80, wherein the XTEN2 comprises a length of 100 to 3,000, 150 to 3,000, 100 to 1,000, or 150 to 1,000 amino acid residues.

82. 82. The polypeptide of any one of claims 74-81, wherein the XTEN2 comprises 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 acid residues.

83. 83. The polypeptide of any one of claims 74-82, wherein at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN2 are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P).

84. 84. The polypeptide of any one of claims 74-83, wherein the XTEN2 has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to a sequence listed in Table 3a.

85. 85. The polypeptide of any one of claims 74-84, wherein the second barcode fragment (BAR2) does not contain a glutamic acid that is immediately adjacent to another glutamic acid, if present, in the XTEN2.

86. 86. The polypeptide of any one of claims 74 to 85, wherein the second barcode fragment (BAR2) has a glutamic acid at its C-terminus.

87. 87. The polypeptide of any one of claims 74 to 86, wherein the second barcode fragment (BAR2) has an N-terminal amino acid immediately preceded by a glutamic acid residue.

88. 88. The polypeptide of any one of claims 74 to 87, wherein the second barcode fragment (BAR2) is located a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, wherein the distance is between 10 and 150 amino acids in length, or between 10 and 125 amino acids in length.

89. The second barcode fragment (BAR2) comprises: (i) the XTEN2 does not contain a glutamic acid, if present, that is immediately adjacent to another glutamic acid; (ii) having a glutamic acid at its C-terminus; (iii) having an N-terminal amino acid immediately preceded by a glutamic acid residue; and (iv) located at a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, the distance being 10 to 150 amino acids in length, or 10 to 125 amino acids in length.

89. A polypeptide according to any one of claims 74 to 88, characterized in that:

90. 90. The polypeptide of any one of claims 87 to 89, wherein the glutamic acid residue preceding the N-terminal amino acid of the BAR2 is not immediately adjacent to another glutamic acid residue.

91. 91. The polypeptide of any one of claims 74 to 90, wherein the second barcode fragment (BAR2) does not comprise a second glutamic acid residue at a position other than the C-terminus of the second barcode fragment (BAR2), unless the second glutamic acid is immediately followed by a proline.

92. 92. The polypeptide of any one of claims 74-91, wherein the XTEN2 is located at the N-terminus of the bispecific antibody construct (BsAb) and the second barcode fragment (BAR2) is located within 200 amino acids, within 150 amino acids, within 100 amino acids, or within 50 amino acids of the N-terminus of the polypeptide.

93. 93. The polypeptide of any one of claims 74-92, wherein the XTEN2 is located at the N-terminus of the bispecific antibody construct (BsAb) and the second barcode fragment (BAR2) is located between 10 and 200 amino acids, between 30 and 200 amino acids, between 40 and 150 amino acids, or between 50 and 100 amino acids from the N-terminus of the protein.

94. 94. The polypeptide of any one of claims 74-93, wherein the XTEN2 is located at the C-terminus of the bispecific antibody construct (BsAb) and the second barcode fragment (BAR2) is located within 200 amino acids, within 150 amino acids, within 100 amino acids, or within 50 amino acids of the C-terminus of the polypeptide.

95. 95. The polypeptide of any one of claims 74-94, wherein the XTEN2 is located at the C-terminus of the bispecific antibody construct (BsAb) and the second barcode fragment (BAR2) is located between 10 and 200 amino acids, between 30 and 200 amino acids, between 40 and 150 amino acids, or between 50 and 100 amino acids from the C-terminus of the protein.

96. 96. The polypeptide of any one of claims 74 to 95, wherein the second barcode fragment (BAR2) is at least 4 amino acids in length.

97. 97. The polypeptide of claim 96, wherein the second barcode fragment (BAR2) is between 4 and 20 amino acids, between 5 and 15 amino acids, between 6 and 12 amino acids, or between 7 and 10 amino acids in length.

98. 98. The polypeptide of any one of claims 74 to 97, wherein the second barcode fragment (BAR2) comprises an amino acid sequence set forth in Table 2.

99. 99. The polypeptide of any one of claims 74-98, wherein the XTEN2 has a length defined by a proximal end and a distal end, (1) the proximal end of the XTEN2 is located closer to the bispecific antibody construct (BsAb) than the distal end, and (2) the second barcode fragment (BAR2) is located within a region of the XTEN2 that spans between 5% and 50%, between 7% and 40%, or between 10% and 30% of the length of the XTEN2, as measured from the distal end of the XTEN2.

100. 100. The polypeptide of any one of claims 74-99, wherein the XTEN2 further comprises one or more additional barcode fragments, wherein each of the one or more additional barcode fragments of the XTEN2 differs in sequence and molecular weight from all other peptide fragments releasable from the polypeptide upon complete digestion of the polypeptide by the protease.

101. 101. A polypeptide according to any one of claims 74 to 100, wherein the first release segment (RS1) and the second release segment (RS2) are identical in terms of sequence.

102. 101. A polypeptide according to any one of claims 74 to 100, wherein the first release segment (RS1) and the second release segment (RS2) are not identical in terms of sequence.

103. 103. The polypeptide of any one of claims 74 to 102, wherein the second release segment (RS2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 7001-7626.

104. A polypeptide described in any one of claims 74 to 103, wherein the first release segment (RS1) and the second release segment (RS2) are each substrates for cleavage by multiple proteases at one, two or three cleavage sites within each release segment sequence.

105. is expressed as a fusion protein, which in its uncleaved state has, from N-terminus to C-terminus, a structural arrangement identified herein by XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN1-RS1-diabodyRS2-XTEN2, or XTEN2-RS2-diabodyRS1-XTEN1; The diabody comprises a light chain variable region (VL) of the AF1 Ｉ ), the heavy chain variable region (VH Ｉ ), the light chain variable region (VL ＩＩ ), and the heavy chain variable region of AF2 (VH ＩＩ 105. The polypeptide of any one of claims 74 to 104, comprising:

106. 106. A polypeptide according to claims 74 to 105, wherein the spacer of the first release segment (RS1) is a first spacer and the second release segment (RS2) is fused to the bispecific antibody construct (BsAb) via a second spacer.

107. 107. The polypeptide of claim 106, wherein the second spacer comprises at least four amino acids that are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P).

108. 107. The polypeptide of claim 106, wherein the second spacer comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table C.

109. (a) the XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3a; (b) the BsAb is (I) the AF1, which comprises light chain complementarity determining regions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3) and heavy chain complementarity determining regions 1 (CDR-H1), 2 (CDR-H2), and 3 (CDR-H3), wherein the CDR-H1, the CDR-H2, and the CDR-H3 comprise the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; (II) a light chain variable region (VL) identified herein by SEQ ID NOs: 778-783 ＩＩ ) and heavy chain variable regions (VH ＩＩ AF2 including Including; (c) the RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 7001-7626; (d) the XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3a; (e) the RS2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 7001-7626; 75. The polypeptide of claim 74, wherein the polypeptide has a structural arrangement identified herein from N-terminus to C-terminus by XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, or XTEN2-RS2-AF1-AF2-RS1-XTEN1.

110. 110. The polypeptide of any one of claims 1 to 109, having a terminal half-life that is at least 2-fold longer compared to the bispecific antibody construct that is not linked to any XTEN.

111. 111. The polypeptide of any one of Claims 1-110, which is less immunogenic than the bispecific antibody construct that is not linked to any XTEN, as determined by measuring the production of IgG antibodies that selectively bind to the bispecific antibody construct after administration of an equivalent dose to a subject.

112. 112. A polypeptide according to any one of claims 1 to 111, which exhibits an apparent molecular weight coefficient of greater than about 3, greater than about 4, greater than about 5, or greater than about 6 under physiological conditions.

113. 2. The polypeptide of claim 1, comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table D.

114. 114. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 113 and one or more pharmaceutically suitable excipients.

115. 115. The pharmaceutical composition of claim 114, formulated for intradermal, subcutaneous, oral, intravenous, intraarterial, intraabdominal, intraperitoneal, intrathecal or intramuscular administration.

116. 116. The pharmaceutical composition of claim 114 or 115, which is in liquid form or frozen.

117. 117. The pharmaceutical composition of any one of claims 114 to 116, in a pre-filled syringe for single injection.

118. 118. The pharmaceutical composition of claim 117, formulated as a lyophilized powder that is reconstituted prior to administration.

119. The pharmaceutical composition of claim 114, wherein the at least one additional therapeutic agent in the pharmaceutical combination is selected from the group consisting of an antibody, an antibody fragment, an antibody conjugate, a cytotoxic agent, a toxin, a radionuclide, an immunomodulatory agent, a photoactive therapeutic agent, a radiosensitizer, a hormonal agent, an anti-angiogenic agent, and combinations thereof.

120. 120. The pharmaceutical composition of claim 119, wherein the additional therapeutic agent is a PD-1 / PD-L1(2) inhibitor.

121. 121. The pharmaceutical combination of claim 120, wherein the PD-1 / PD-L1(2) inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody.

122. 122. The pharmaceutical combination of claim 121, wherein said PD-1 / PD-L1(2) inhibitor is an anti-PD-1 antibody selected from the group comprising nivolumab (Opdivo, BMS-936558, MDX1106), pembrolizumab (Keytruda, MK-3475, lambrolizumab), pidilizumab (CT-011), PDR-001, JS001, STI-A1110, AMP-224, and AMP-514 (MEDI0680).

123. 122. The pharmaceutical combination of claim 121, wherein said PD-1 / PD-L1(2) inhibitor is an anti-PD-L1 antibody selected from the group comprising atezolizumab (Tecentriq, MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX1105), and LY3300054.

124. 122. The pharmaceutical combination of claim 121, wherein said PD-1 / PD-L1(2) inhibitor is an anti-PD-L2 antibody.

125. 120. The pharmaceutical combination of claim 119, which is a combination pack containing said components separate from each other.

126. 120. The pharmaceutical combination of claim 119, wherein the components are administered simultaneously or sequentially for use in the treatment of the same disease in separate dosage forms.

127. 120. The pharmaceutical combination of claim 119 for use as a medicament for treating a hyperproliferative disorder.

128. 128. The pharmaceutical combination of claim 127, wherein the hyperproliferative disorder is selected from the group consisting of breast cancer, respiratory tract cancer, brain cancer, reproductive cancer, gastrointestinal cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer and distant metastases thereof.

129. 129. Use of a polypeptide according to any one of claims 1 to 112 or a pharmaceutical combination according to any one of claims 119 to 128 in the preparation of a medicament for treating a disease in a subject.

130. 130. The use of claim 129, wherein the disease is cancer.

131. 129. A method of treating a disease in a subject, comprising administering to said subject in need thereof one or more therapeutically effective doses of the pharmaceutical composition of any one of claims 114 to 118 or the pharmaceutical combination of any one of claims 119 to 128.

132. 132. The method of claim 131, wherein the disease is cancer.

133. 133. The method of claim 132, wherein the cancer is selected from the group consisting of glioblastoma, melanoma, cholangiocarcinoma, small cell lung cancer, colorectal cancer, prostate cancer, vaginal cancer, angiosarcoma, non-small cell lung cancer, appendix cancer, squamous cell carcinoma, salivary gland duct carcinoma, adenoid cystic carcinoma, small intestine cancer, and gallbladder cancer.

134. 134. The method of any one of claims 131 to 133, wherein the pharmaceutical composition is administered to the subject as one or more therapeutically effective doses.

135. 135. The method of any one of claims 131 to 134, wherein the pharmaceutical composition is administered to the subject in one or more therapeutically effective doses over an effective administration period.

136. 135. The method of any one of claims 131 to 134, wherein the subject is a mouse, rat, monkey, or human.

137. 113. A nucleic acid comprising: (a) a polynucleotide sequence encoding a polypeptide of any one of claims 1 to 112; or (b) the reverse complement of the polynucleotide sequence of (a).

138. 113. An expression vector comprising a polynucleotide sequence according to claims 1 to 112 and a recombinant regulatory sequence operably linked to said polynucleotide sequence.

139. A host cell comprising the expression vector of claim 138.

140. 140. The host cell of claim 139, which is a prokaryote.

141. 141. The host cell of claim 140, which is E. coli.

142. 140. The host cell of claim 139, which is a mammalian cell.

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