HER-2 targeted bispecific composition and method for its preparation and use.

Abstract

Disclosed herein are polypeptides comprising a bispecific antibody construct covalently linked to an extended recombinant polypeptide comprising a barcode fragment releasable from the polypeptide upon digestion by a protease, and a release segment that can be proteolytically cleaved, wherein said cleavage releases the bispecific antibody construct from the extended recombinant polypeptide, as well as methods of making and using such polypeptides. The present invention addresses a long-standing unmet need to provide TCE cancer therapeutics with increased therapeutic indices.

Description

[Technical Field]

[0001] Sequence listing description: A computer-readable sequence listing will be submitted electronically along with this application, and the entire sequence listing will be incorporated into this application by reference. The sequence listing is contained in a file created on June 14, 2021, with the filename "789-601_20-1832-WO_ST25_FINAL.txt", and its size is 1502kb.

[0002] References This application relates to the following U.S. Provisional Patent Application No. 63 / 044,301, filed on June 25, 2020, with the title of the invention "BARCODED BISPECIFIC COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME"; the following U.S. Provisional Patent Application No. 63 / 077,503, filed on September 11, 2020, with the title of the invention "BARCODED BISPECIFIC COMPOSITIONS AND METHODS FOR MAKING AND USING THE"; the following U.S. Provisional Patent Application No. 63 / 108,783, filed on November 2, 2020, with the title of the invention "BARCODED BISPECIFIC COMPOSITIONS AND METHODS FOR MAKING AND USING THE": This invention claims priority to U.S. Provisional Patent Application No. 63 / 166,857, filed on 26 March 2021, titled "SAME," and to U.S. Provisional Patent Application No. 63 / 196,408, filed on 3 June 2021, titled "HER2 TARGETED BISPECIFIC COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME," all of which are incorporated herein by reference in their entirety. [Background technology]

[0003] background Bispecific T cell engagers (TCEs) represent a highly potent modality in cancer therapy, redirecting T cell cytotoxicity towards tumors expressing selected tumor-associated antigens, thus circumventing the requirement for T cell recognition of tumor antigens. TCE activity depends on their ability to activate T cells by effectively stimulating T cell receptors (TCRs). Their extremely potent action stems from the minimal requirement for initiating cytotoxicity: just three TCRs that are stimulated and coalesce to form immune synapses between T cells and target cells. In addition to their induction of cytotoxicity, their potency also involves downstream cytokine-driven actions of T cell activation that enhance and amplify the anti-tumor immune response. Therefore, TCEs are promising for immunotherapy in patients whose tumors harbor inadequate mutations or have otherwise evaded immune surveillance. However, this modality is not without its challenges; the use of TCEs in solid tumors is limited by their extremely potent action and on-target, off-tumor toxicity in healthy tissues.

[0004] While TCEs are considered highly effective in inducing remission in patients with hematological malignancies, their use in solid tumors is limited by their extremely potent effects on normal tissues that express the target, even at low levels, and their on-target toxicity. Rare but impressive clinical responses have been observed in tumors that do not typically respond to immunotherapy (e.g., microsatellite-stable colorectal and prostate cancers), but toxicities such as cytokine release syndrome at low doses have hindered dose escalation to reveal the clinical potential of this modality. Grade 4 cytokine release syndrome induced in patients treated with Ichnos ISB 1302 HER2 TCE at doses of <1 ug / kg highlights the challenges faced by TCEs, even when targeted to relatively tissue-limited targets.

[0005] Clinical trials of blinatumomab (an approved CD3xCD19 bispecific antibody) have revealed cytokine release syndrome (CRS) to be one of the major safety-related adverse events. CRS and on-target toxicity at low drug doses significantly impair the therapeutic index and potential of TCE modalities for solid tumors in clinical settings. For example, a clinical trial of catumaxomab (CD3xEpCAM) was terminated at a 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. Pasotuxizumab (PSMA-targeted TCE) showed a good response but was hampered by CRS at doses higher than 40 μg / day. The literature contains numerous other examples of the challenges of CRS and on-target toxicity presented by TCEs.

[0006] Attempts to circumvent CRS, involving complex molecular designs, have failed due to toxicity and / or enhanced immunogenicity. This presents a significant unmet need for novel strategies that can overcome the challenges of therapeutic index in solid tumors. If the potency of TCE could be utilized and the challenges of CRS and on-target toxicity could be controlled, it would be possible to create potent therapies that could potentially be used against a wide range of cancers.

[0007] Therapeutic drugs, such as active pharmaceutical ingredients (APIs), contain polypeptides, and these polypeptides may be produced in a manner that results in a mixture of polypeptides that can affect the activity of the API. This mixture of polypeptides may 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 API and therefore may affect the safety and / or efficacy of the polypeptide API. For example, protein-based prodrugs for cancer treatment may be manipulated using activation mechanisms that target tumors. More specifically, a therapeutic full-length protein may be in an inactivated (non-cytotoxic) prodrug form, but truncated variants of its full-length construct may 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 compared to the full-length protein, have lower selectivity and toxicity to tumor cells, or exhibit a less desirable pharmacokinetic profile (e.g., resulting in a narrower therapeutic range). As a result, the detection and quantification of protein structure variations may be important for evaluating the biological properties of biopharmaceuticals (e.g., clinical safety and pharmacological efficacy) and for developing new biopharmaceuticals (e.g., those with increased efficacy and reduced side effects). Existing techniques and methods for identifying and quantifying "contaminating" shortening products may have one or more drawbacks, such as limited sensitivity, ease of use, efficiency, or effectiveness. [Overview of the project] [Means for solving the problem]

[0008] overview This invention addresses a long-standing, unmet need for a TCE cancer treatment with an increased therapeutic index. In doing so, the invention leverages the therapeutic potential of TCEs by providing an XTEN-modified protease-activated bispecific T-cell engager (XPAT). XPAT represents a novel strategy that improves the toxicity profile while maintaining the potency of the T-cell engager against solid tumors, thereby enabling a significant increase in the therapeutic index and expanding the target landscape of this potent modality. In certain specific embodiments, the XPAT of this invention targets tumors with HER2. More specifically, AMX-818 is a HER2-targeting, conditionally activated prodrug TCE designed to leverage the abnormally regulated protease activity in tumors while preserving 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, (a) an elongated recombinant polypeptide (XTEN) comprising a barcode fragment (BAR) that can be released from the polypeptide upon digestion by a protease; (b) a bispecific antibody construct (BsAb) that specifically binds to the differentiated antigen group 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 ( A bispecific antibody construct comprising (c) CDR-H3, wherein CDR-H3 comprises a first antigen-binding fragment (AF1) comprising 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 (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 glycine (G), a (iii) being ranin (A), serine (S), threonine (T), glutamate (E), or proline (P), and (iv) comprising at least four different amino acids, G, A, S, T, E, or P, and (iv) the XTEN being formed from a plurality of non-overlapping sequence motifs, each having a length of 9 to 14 amino acids; the plurality of non-overlapping sequence motifs comprising (1) a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif in the set is repeated at least twice in the XTEN; and (2) a non-overlapping sequence motif appearing only once in the XTEN; the barcode fragment (BAR) comprising at least a portion of the non-overlapping sequence motif appearing only once in the XTEN; and the barcode fragment (BAR) being different in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide upon complete digestion of the polypeptide by the protease;The following polypeptides are provided herein, wherein the barcode fragment (BAR) does not contain either 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 comprises amino acid sequences identified herein by SEQ ID NOs. 179-200 and 1715-1722. In certain embodiments, the set of non-overlapping sequence motifs each independently comprises amino acid sequences 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 SEQ ID NOs. 186-189 of the sequence motifs. 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 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 with respect to the sequences listed in Table 3a. In certain embodiments, the barcode fragment (BAR) does not contain glutamic acid directly adjacent to another glutamic acid, if present, in the XTEN. In certain embodiments, the barcode fragment (BAR) has glutamic acid at its C-terminus. In certain embodiments, the barcode fragment (BAR) has an N-terminal amino acid immediately preceding a glutamic acid residue. In certain embodiments, the barcode fragment (BAR) is located at a certain distance from either the N-terminus or the C-terminus of the polypeptide, the distance being 10 to 150 amino acids or 10 to 125 amino acids in length. In certain embodiments, the barcode fragment (BAR) is characterized by (i) not containing a glutamic acid directly adjacent to another glutamic acid, if present in the XTEN; (ii) having a glutamic acid at its C-terminus; (iii) having an N-terminal amino acid immediately preceding a glutamic acid residue; and (iv) being located at a certain distance from either the N-terminus or the C-terminus of the polypeptide, such that the distance is 10 to 150 amino acids or 10 to 125 amino acids in length. In certain embodiments, the glutamic acid residue preceding the N-terminal amino acid of the barcode fragment (BAR) is not directly adjacent to another glutamic acid residue.

[0012] In certain embodiments, 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 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 from 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, 30 and 200 amino acids, 40 and 150 amino acids, or 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 from 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, 30 and 200 amino acids, 40 and 150 amino acids, or 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 long. In certain embodiments, the barcode fragment (BAR) is between 4 and 20 amino acids, 5 and 15 amino acids, 6 and 12 amino acids, or 7 and 10 amino acids long.

[0013] In certain embodiments, the barcode fragment (BAR) comprises the amino acid sequence described 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 that spans between 5% and 50%, 7% and 40%, or 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 terms of sequence and molecular weight from all other peptide fragments that can be released 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 with the sequences identified herein by SEQ ID NOs.7001-7626. In certain embodiments, the protease cleaves at the C-terminal side of a glutamic acid residue 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, which, in its uncleaved state, has a structural configuration of AF1-AF2-RS-XTEN, AF2-AF1-RS-XTEN, XTEN-RS-AF1-AF2, or XTEN-RS-AF2-AF1 from the N-terminus to the C-terminus. In certain embodiments, the release segment (RS) is fused to the bispecific antibody construct (BsAb) by 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 with respect to the sequences listed in Table C. In certain embodiments, 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.

[0015] In a particular embodiment, the CDR-L1 of AF1 includes the amino acid sequence of SEQ ID NO: 1 or 2, the CDR-L2 of AF1 includes the amino acid sequence of SEQ ID NO: 4 or 5, and the CDR-L3 of AF1 includes the amino acid sequence of SEQ ID NO: 6. In a particular embodiment, the CDR-L1 of AF1 includes the amino acid sequence of SEQ ID NO: 1, the CDR-L2 of AF1 includes the amino acid sequence of SEQ ID NO: 4 or 5, and the CDR-L3 of AF1 includes the amino acid sequence of SEQ ID NO: 6. In a particular embodiment, the CDR-L1 of AF1 includes the amino acid sequence of SEQ ID NO: 2, the CDR-L2 of AF1 includes the amino acid sequence of SEQ ID NO: 4 or 5, and the CDR-L3 of AF1 includes the amino acid sequence of SEQ ID NO: 6. In a particular embodiment, the CDR-L1 of AF1 includes the amino acid sequence of SEQ ID NO: 1, the CDR-L2 of AF1 includes the amino acid sequence of SEQ ID NO: 4, and the CDR-L3 of AF1 includes the amino acid sequence of SEQ ID NO: 6. In a particular embodiment, the CDR-L1 of AF1 includes the amino acid sequence of SEQ ID NO: 2, the CDR-L2 of AF1 includes the amino acid sequence of SEQ ID NO: 5, and the CDR-L3 of AF1 includes the amino acid sequence of SEQ ID NO: 6. In a particular embodiment, 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 or being identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequences of SEQ ID NOs. 60, 64, 65, and 67, respectively. In a particular embodiment, 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 or being identical to, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequences of SEQ ID NOs. 61, 64, 65, and 67, respectively.In a particular embodiment, 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 or being identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequences of SEQ ID NOs. 51, 52, 53, and 59, respectively. In a particular embodiment, 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 or being identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequences of SEQ ID NOs. 51, 52, 54, and 59, respectively.

[0016] In a particular embodiment, 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 or being identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequences of SEQ ID NOs. 51, 52, 55, and 59, respectively. In a particular embodiment, 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 or being identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequences of SEQ ID NOs. 51, 52, 56, and 59, respectively. In a particular embodiment, 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 FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, FR-L3 comprises the amino acid sequence of SEQ ID NO: 53, 54, 55, or 56, FR-L4 comprises the amino acid sequence of SEQ ID NO: 59, FR-H1 comprises the amino acid sequence of SEQ ID NO: 60 or 61, FR-H2 comprises the amino acid sequence of SEQ ID NO: 64, FR-H3 comprises the amino acid sequence of SEQ ID NO: 65, and FR-H4 comprises the amino acid sequence of SEQ ID NO: 67.

[0017] In a particular embodiment, 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 FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, FR-L3 comprises the amino acid sequence of SEQ ID NO: 53, FR-L4 comprises the amino acid sequence of SEQ ID NO: 59, FR-H1 comprises the amino acid sequence of SEQ ID NO: 60, FR-H2 comprises the amino acid sequence of SEQ ID NO: 64, FR-H3 comprises the amino acid sequence of SEQ ID NO: 65, and FR-H4 comprises the amino acid sequence of SEQ ID NO: 67.

[0018] In a particular embodiment, 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 FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, FR-L3 comprises the amino acid sequence of SEQ ID NO: 54, FR-L4 comprises the amino acid sequence of SEQ ID NO: 59, FR-H1 comprises the amino acid sequence of SEQ ID NO: 61, FR-H2 comprises the amino acid sequence of SEQ ID NO: 64, FR-H3 comprises the amino acid sequence of SEQ ID NO: 65, and FR-H4 comprises the amino acid sequence of SEQ ID NO: 67.

[0019] In a particular embodiment, 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 FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, FR-L3 comprises the amino acid sequence of SEQ ID NO: 55, FR-L4 comprises the amino acid sequence of SEQ ID NO: 59, FR-H1 comprises the amino acid sequence of SEQ ID NO: 61, FR-H2 comprises the amino acid sequence of SEQ ID NO: 64, FR-H3 comprises the amino acid sequence of SEQ ID NO: 65, and FR-H4 comprises the amino acid sequence of SEQ ID NO: 67.

[0020] In a particular embodiment, 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 FR-L1 comprises the amino acid sequence of SEQ ID NO: 51, FR-L2 comprises the amino acid sequence of SEQ ID NO: 52, FR-L3 comprises the amino acid sequence of SEQ ID NO: 56, FR-L4 comprises the amino acid sequence of SEQ ID NO: 59, FR-H1 comprises the amino acid sequence of SEQ ID NO: 61, FR-H2 comprises the amino acid sequence of SEQ ID NO: 64, FR-H3 comprises the amino acid sequence of SEQ ID NO: 65, and FR-H4 comprises the amino acid sequence of SEQ ID NO: 67.

[0021] In a particular embodiment, the first antigen-binding fragment (AF1) exhibits a higher melting temperature (T) in an in vitro assay compared to the melting temperature of the anti-CD3 binding fragment. m ); or when the first antigen-binding fragment is incorporated into the test bispecific antigen-binding construct, the T of the test bispecific antigen-binding construct is the same as that of the control bispecific antigen-binding construct. m Compared to T mAs is apparent from, it exhibits higher thermal stability than the anti-CD3 binding fragment having the sequence set forth in SEQ ID NO: 206, and 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 m of the first antigen-binding fragment 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 T m of the anti-CD3 binding fragment consisting of the sequence of SEQ ID NO: 206. In certain embodiments, the first antigen-binding fragment (AF1) comprises a heavy chain variable region (VH I ), and the VH I comprises an amino acid sequence having at least 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: 102 or 105. In certain embodiments, the first antigen-binding fragment (AF1) comprises a light chain variable region (VL I ), and the VL I comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or being identical to any one of the amino acid sequences of SEQ ID NO: 101, 103, 104, 106 or 107. In certain embodiments, the VH I and the VL IThe first antigen-binding fragment (AF1) is linked by a linker containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences listed in Table A. In certain embodiments, the first antigen-binding fragment (AF1) contains an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to any one of the amino acid sequences of SEQ ID NOs. 201-205, or is identical thereto. 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, which 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) less than or equal to 6.6. In some embodiments, the first antigen-binding fragment (AF1) exhibits an isoelectric point (pI) between 6.0 and 6.6, including both ends.

[0022] In certain embodiments, the first antigen-binding fragment (AF1) exhibits a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lower than the isoelectric point (pI) of a reference antigen-binding fragment having the sequence shown in SEQ ID NO: 206. In certain embodiments, the first antigen-binding fragment (AF1) is determined by an in vitro antigen-binding assay containing a human or cyno CD3 antigen to have a dissociation constant (K) between approximately 10 nM and approximately 400 nM. dIt specifically binds to human or cyno CD3 at a constant (K). In certain embodiments, the first antigen-binding fragment (AF1) is determined by an in vitro antigen-binding assay to have a dissociation constant (K) 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. d ) specifically binds to human or cyno CD3. In certain embodiments, the first antigen-binding fragment (AF1) has a dissociation constant (K) in the in vitro antigen-binding assay. d Determined by ), it exhibits a weak binding affinity to CD3, which is less than half, one-third, one-quarter, one-fifth, one-sixth, one-seventh, one-eighth, one-ninth, or one-tenth of the binding affinity of the 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 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 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) is a heavy-chain variable region (VH) containing an amino acid sequence identified herein by SEQ ID NOs. 778-783. II ); and the light chain variable region (VL) containing the amino acid sequences identified herein by sequence numbers 878-883. II ) includes. In a particular embodiment, the VH II and the VL IIThe first and second antigen-binding fragments are linked by a linker containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences listed 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 contains two or three amino acids, which are glycine, serine, or proline. In certain embodiments, the peptide linker contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences listed in Table B.

[0024] In a particular embodiment, the XTEN is a first elongated recombinant polypeptide (XTEN1) formed from a plurality of non-overlapping sequence motifs, 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 elongated recombinant polypeptide (XTEN2) comprising a second barcode fragment (BAR2) that can be released 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); and the XT EN2 is characterized by (i) containing at least 100 or at least 150 amino acids, (ii) at least 90% of its amino acid residues being glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P); and (iii) containing at least four different amino acids which 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 which can be released 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 a particular embodiment, XTEN1 is located at the C-terminus of the bispecific antibody construct, and XTEN2 is located at the N-terminus of the bispecific antibody construct.

[0025] In certain embodiments, the XTEN2 is formed from a second set of non-overlapping sequence motifs, each having a length of 9 to 14 amino acids, wherein the second set of non-overlapping sequence motifs includes (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) includes 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 is each independently identified herein by sequence numbers 179-200 and 1715-1722. In certain embodiments, the second set of non-overlapping sequence motifs is each independently identified herein by sequence numbers 186-189. In certain embodiments, the second set of non-overlapping sequence motifs includes at least two, at least three, or all four of sequence numbers 186-189 of the sequence motifs. In a particular embodiment, the XTEN2 includes lengths of 100-3,000, 150-3,000, 100-1,000, or 150-1,000 amino acid residues. In a particular embodiment, the XTEN2 includes lengths 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 XTEN2 are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). In certain embodiments, XTEN2 has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequences listed in Table 3a. In certain embodiments, the second barcode fragment (BAR2) does not contain glutamate directly adjacent to another glutamate present in XTEN2. In certain embodiments, the second barcode fragment (BAR2) has glutamate at its C-terminus. In a particular embodiment, the second barcode fragment (BAR2) has an N-terminal amino acid immediately preceding a glutamic acid residue. In a particular embodiment, the second barcode fragment (BAR2) is located at a certain distance from either the N-terminus 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 a particular embodiment, the second barcode fragment (BAR2) is characterized by (i) not containing a glutamic acid directly adjacent to another glutamic acid present in XTEN2; (ii) having a glutamic acid at its C-terminus; (iii) having an N-terminal amino acid immediately preceding a glutamic acid residue; and (iv) being located at a certain distance from either the N-terminus 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 BAR2 is not directly adjacent to another glutamic acid residue. In certain embodiments, the second barcode fragment (BAR2) does not contain 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 proline. In certain embodiments, 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 from the N-terminus of the polypeptide. In a particular embodiment, 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, 30 and 200 amino acids, 40 and 150 amino acids, or 50 and 100 amino acids from the N-terminus of the protein. In a particular embodiment, 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 from the C-terminus of the polypeptide. In a particular embodiment, 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, 30 and 200 amino acids, 40 and 150 amino acids, or 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) includes the amino acid sequence listed in Table 2. In certain embodiments, the XTEN2 has a length defined by its proximal and distal ends, where (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, 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 that can be released 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 with respect to the sequences identified herein by SEQ ID NOs. 7001-7626. In a particular embodiment, the first release segment (RS1) and the second release segment (RS2) are 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, which, in its uncleaved state, has a structural configuration from the N-terminus to the C-terminus as 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-diabody RS2-XTEN2, or XTEN2-RS2-diabody RS1-XTEN1, wherein the diabody is the light chain variable region (VL) of AF1. I ), the heavy chain variable region (VH) of AF1 I ), the light chain variable region (VL) of AF2 II ), and the heavy chain variable region (VH) of AF2. II ) includes. In a particular embodiment, 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) by a second spacer. In a particular embodiment, the second spacer comprises at least four amino acids, which are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). In a particular embodiment, the second spacer comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences listed in Table C.

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

[0031] In certain embodiments, the polypeptide has a terminal phase half-life at least twice as long as a bispecific antibody construct that is not linked to any XTEN. In certain embodiments, the polypeptide is less immunogenic than a bispecific antibody construct that is not linked to any XTEN, as confirmed by measuring the production of IgG antibodies that selectively bind to the bispecific antibody construct after administration of equivalent doses to a subject. In certain embodiments, the polypeptide exhibits an apparent molecular weight coefficient 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 with respect to sequences identified herein, such as the sequences in Table D.

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

[0033] In certain embodiments, the composition exists as a pharmaceutical combination with at least one additional therapeutic agent selected from the group consisting of antibodies, antibody fragments, antibody conjugates, cytotoxic agents, toxins, radionuclides, immunomodulators, phototherapeutic agents, radiosensitizers, hormones, anti-angiogenic agents, and combinations thereof.

[0034] Additional therapeutic agents include PD-1 / PD-L1(2) inhibitors, which are either anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-PD-L2 antibodies.

[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), pizilizumab (CT-011), PDR-001, JS001, STI-A1110, AMP-224, and AMP-514 (MEDI0680).

[0036] In one embodiment, the PD-1 / PD-L1(2) inhibitor may 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 that are separate from each other. In certain embodiments, the components are administered simultaneously or sequentially in separate dosage forms for use in the treatment of the same disease.

[0038] In certain embodiments, the polypeptides described herein are part of a drug combination for use as a pharmacopoeia to treat hyperproliferative disorders. Hyperproliferative disorders are selected from the group consisting of breast cancer, airway cancer, brain cancer, genital cancer, gastrointestinal cancer, urinary tract cancer, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, and their distant metastases.

[0039] In certain embodiments, the polypeptides described herein are used to prepare a pharmaceutical for treating a disease of interest. In certain embodiments, the disease is cancer.

[0040] In certain embodiments, there are methods for treating a disease of interest, the method comprising the step of administering one or more therapeutically effective doses of a pharmaceutical composition or drug combination to a subject in need thereof.

[0041] In one embodiment, the disease is cancer. In a particular embodiment, 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, appendiceal cancer, squamous cell carcinoma, salivary ductal carcinoma, adenoid cystic carcinoma, small intestine cancer, and gallbladder cancer.

[0042] A method is provided for treating a target disease, comprising the step of administering one or more therapeutically effective doses of the pharmaceutical composition described above to a subject in need thereof. In certain embodiments, the pharmaceutical composition is administered to the subject as one or more therapeutically effective doses in a therapeutically effective treatment process. In certain embodiments, the doses are administered to a human or animal by any clinically appropriate route and formulation. In certain embodiments, the subject is a mouse, rat, monkey, or human.

[0043] Nucleic acids comprising a polynucleotide sequence encoding a polypeptide described herein, or a reverse complement of said polynucleotide sequence, are also provided herein. An expression vector comprising said polynucleotide sequence and a recombinant regulatory sequence operably linked to said polynucleotide sequence is further provided herein. A host cell comprising said expression vector is provided herein. 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] Further aspects and advantages of the present disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes merely exemplary embodiments of the present disclosure. As will be apparent, other different embodiments of the present disclosure are possible, and some of their details can be modified in various obvious ways, all without departing from the present disclosure. Therefore, the drawings and description should be considered illustrative and not restrictive. Inclusion by reference

[0045] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. [Brief explanation of the drawing]

[0046] Various features of this disclosure are specifically described in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are utilized, and to the following appended drawings.

[0047] [Figure 1]Figure 1 shows a mixture of XTEN-modified protease-activated T cell engager ("XPAT") polypeptides containing XTENs of varying lengths. Full-length XPAT (top) contains a 288-amino acid-length XTEN at the N-terminus and an 864-amino acid-length XTEN at the C-terminus. In XPAT, various cleavages can occur in one or both of the N-terminal and C-terminal XTENs, for example, during fermentation, purification, or other steps in product preparation. Products with limited cleavage (cleavage near the distal end of the XTEN) may function similarly to the full-length construct, while severe cleavage (cleavage near the proximal end of the XTEN) may have significantly different pharmacological properties from their full-length counterparts. The presence of cleavage presents challenges in quantifying pharmacologically effective and ineffective variants in XPAT products. As illustrated in Figure 1 using full-length XPAT, each XTEN has a proximal and a distal end, with the proximal end located closer to the biologically active polypeptide (e.g., T cell engagers, cytokines, monoclonal antibodies (mAbs), antibody fragments, or other proteins to be XTENized) compared to the distal end.

[0048] [Figure 2]Figure 2 shows a mixture of XPAT polypeptides with barcoded XTENs of varying lengths. In full-length XPAT (top), the 288-amino acid-length N-terminal XTEN contains three cleavable fused barcode sequences "NA", "NB", and "NC" (from distal to proximal), and the 864-amino acid-length C-terminal XTEN contains three cleavable fused barcode sequences "CC", "CB", and "CA" (from proximal to distal). Each barcode is positioned to indicate the pharmacologically relevant length of the corresponding XTEN. For example, a minor N-terminal cleavage product of XPAT may lack the barcode "NA" (e.g., by cleavage) but have 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 cleavage product of XPAT may lack all three N-terminal barcodes (e.g., by cleavage), and may exhibit distinctly different pharmacological activity from the full-length construct. A specific proteolytic cleavage sequence is identified from the biologically active polypeptide of XPAT (wherein the tandem scFv constituting the active region of the T cell engager). Because it is present in full-length variants of XPAT (including full-length XPAT, minor cleavage, and major cleavage), this specific proteolytic cleavage sequence can be used as a reference to quantify the amounts of various cleavage products related to the total amount of biologically active protein.

[0049] [Figure 3]Figure 3 illustrates possible designs for barcoded XTENs by inserting a barcode-generating sequence into a generic (or standard) XTEN. The exemplary generic (or standard) XTEN (top) contains a non-repeating 12-mer motif of the sequence "BCDABDCDABDCBDCDABDCB", where sequence motifs "A", "B", "C", and "D" appear 3, 6, 5, and 7 times, respectively. The Glu-C protease digest of the exemplary generic XTEN (top panel) does not produce a specific peptide except at both ends ("NT" and "CT"). Insertion of the barcode-generating sequence "X" (e.g., a specific 12-mer) into the XTEN results in a specific proteolytic cleavage sequence (or barcode sequence) that is not present anywhere else in the XTEN. The barcode-generating sequence "X" may 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 the corresponding XTEN with a barcode. Those skilled in the art will understand that the barcode generation sequence ("X") can be the barcode sequence itself. Alternatively, the barcode generation sequence ("X") can be different from the resulting barcode sequence. For example, the barcode sequence may overlap with and thus contain a portion of a preceding or following 12mer motif.

[0050] [Figure 4-1]Figures 4A–4B illustrate the 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 generic XTEN (top panel) with a barcode-generating motif "X," in this example where the barcode-generating motif ("X") is itself a proteolytically cleavable barcode sequence. As shown in the bottom panel of Figure 4A, the barcode is positioned such that all severe cleavage forms of XTEN lack the barcode, and all limited cleavage 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 constructs containing biologically active proteins. In the other mixture, 13% of constructs lack the barcode (e.g., due to cleavage). Figures 4A-4B illustrate the use of barcoded XTEN to distinguish between two polypeptide mixtures that have substantially similar average molecular weights but clearly 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 (XTENized) and unmasked bispecific T cell engagers against target cells with varying levels of target antigen expression. Unmasked (de-XTENized) bispecificity demonstrates potent cytotoxicity against various tumor lineages with EC50 in the single-digit pM range, for example, when tested with SK-OV-3 and BT-474 cells. XTENization further demonstrates robust masking ability, for example, by protecting bispecific T cell engagers from immunological synapse formation, thereby resulting in reduced toxicity, as indicated by the rightward shift of the concentration-response curve.

[0052] [Figure 5-2]Figures 5A–5B illustrate the effective masking by XTEN to XTEN-modified protease-activated T cell engagers ("XPATs") in general, and particularly to HER2-XPATs. For example, the dose-dependent cytotoxicity of XTEN-modified (masked) HER2-XPATs (e.g., listed in Table D) and the corresponding de-XTEN-modified (unmasked, activated) HER2-PATs against two different HER2-expressing (e.g., cancer) cell lines was observed. Unmasked de-XTENized PAT (indicated by filled circles) yielded EC50 values ​​of 3.4 picomoles (pM) (SKOV3 cells) (Figure 5A) and 4.8 pM (BT474 cells) (Figure 5B), respectively, while the corresponding masked, XTENized PAT (indicated by filled squares) yielded EC50 values ​​of 44,474 pM (SKOV3 cells) and 49,370 pM (BT474 cells), demonstrating 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 in contact with non-cancerous tissue (cardiomyocytes). More specifically, Figure 6A shows the cytotoxicity of XTEN-modified (masked) HER2-XPAT (e.g., listed in Table D) and the corresponding de-XTEN-modified (unmasked, activated) HER2-PAT against cardiomyocytes. Cardiomyocyte death by T cell-induced cytolysis was observed in response to unmasked, de-XTEN-modified PAT (with an EC50 concentration of approximately 64 pM), whereas, in contrast to tumor cells, cardiomyocytes remained insoluble against death by masked XTEN-modified 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 it causes target cells to express target antigens at relatively moderate or low levels. For example, Figure 6B illustrates the cytotoxic effects of XTEN-modified and de-XTEN-modified protease-activated T cell engagers (PATs) on MCF-7, a cancer cell line with low HER2 expression, where masking with the XTEN polypeptide reduced the T cell-mediated cytotoxicity of the tested HER2-XPAT to approximately 1 / 104. As another example, the cytotoxicity of XTEN-modified (masked) HER2-XPAT and the corresponding de-XTEN-modified (unmasked, activated) HER2-PAT against another cancer cell line with moderate HER2 expression, MDA-MB-453, was measured (Figure 6C). [Figure 6-3] Same as above.

[0055] [Figure 7-1]Figures 7A–7D illustrate the activation of XTEN-bound bispecificity by proteolysis and the robust tumor regression induced thereby in the subjects. Figure 7A illustrates the equivalent efficacy induced by equimolar administration of a cleaved XTEN-bound HER2 T cell engager (indicated by a hexagon, "HER2-XPAT") and the corresponding unmasked HER2 T cell engager (indicated by a triangle, "HER2-PAT") in tumor-carrying mice (e.g., BT-474). (** indicates p<0.01). Notably, between the two tested XTEN-bound bispecificities, tumor regression was observed with the cleaved XTEN-bound construct (indicated by a hexagon) but not with the uncleaved XTEN-bound counterpart (indicated by a diamond), indicating that unmasked by proteolysis (de-XTENization) is an essential condition for efficacy. Figure 7B illustrates the efficacy of cleavage-type XTEN-modified bispecific (e.g., HER2-XPAT) to large tumors at a single dose (e.g., 2.1 milligrams per kilogram (mpk)). The non-cleavage construct used in this experiment is identical to the corresponding cleavage construct, but the release site is replaced with a similarly length non-cleavage sequence made from GASTEP amino acids (glycine, alanine, serine, threonine, glutamate, and / or proline). Figure 7C shows the efficacy of masked HER2-XPAT (filled triangles) at two different concentrations (15 nmol / Kg and 36 nmol / Kg) compared to unmasked HER2-PAT (filled squares) and non-cleavage-type XPAT (filled diamonds). Data for tumors treated with vehicle and vehicle + PBMC are also shown. Figure 7D shows the cleavage percentage of HER2-PAT in vivo in mice carrying BT-474 tumors. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above.

[0056] [Figure 8]Figure 8 illustrates the lymphocyte marginal trend induced in subjects by administration of XTEN-modified HER2-XPAT. For example, with a single intravenous infusion of XTEN-modified HER2-XPAT (e.g., 25 mg / kg in a 10 ml / kg administration volume) (e.g., 25 mg / kg in a 10 ml / kg administration volume), a decrease in lymphocyte count (hematology) was observed in both male and female monkey subjects (indicated by triangles and squares, respectively), starting 6 hours after administration and continuing for at least 24 to 72 hours after administration.

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

[0058] [Figure 10-1] Figures 10A-10C. Figure 10A shows a dose escalation of a single-dose, single-target HER2-XPAT, demonstrating tolerance at all doses up to 42 mg / kg. Figure 10B shows a tapering scheme for single-target HER2-PAT, where the maximum tolerated dose of unmasked HER2-PAT is 0.2 mg / kg. Figure 10C shows the plasma concentration of masked HER2-PAT (450-fold higher tolerance 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 absence of peripheral T cell activation in response to HER2-XPAT compared to HER2-PAT. Figures 11C-11E show the 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 NHP and humans showed minimal cleavage to unmasked TCE, even under inflammatory conditions. AMX-818 with a tandem scFv-bound fluorescent label (DyL650) was incubated in specified plasma samples at 37°C for 7 days. The samples were then run on a gel, and metabolites of similar size and unmasked active form of AMX-818 were quantified using a LI-COR detector. Human samples from inflammatory diseases were derived from patients with rheumatoid arthritis, lupus, inflammatory bowel disease, and multiple sclerosis. Human samples from cancer were derived from patients with tumors of the lung, breast, and colon. Sample size: healthy NHP N=4, healthy humans N=4, "inflammatory" NHP N=6, "inflammatory" humans N=27, humans with cancer N=11.

[0061] [Figure 13-1] Figures 13A and 13B provide additional data illustrating the preferred safety profile of the HER2-XPAT of the present invention. The data in Figure 13A show comparable PK between the HER2-XPAT form and the uncleaved HER2-PAT form, 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 evaluated on CD4+ and CD8+ T cells by flow cytometry after 48 hours of co-incubation of PBMC and SKOV3 cells at a specified concentration of test material in an effector:target ratio of 5:1. 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 with a 5:1 effector:target ratio for specified concentrations of the test substance. 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 was preferentially cleaved to unmasked TCE in human tumors transplanted into live mice, with minimal cleavage observed in healthy tissue. A single dose of 1.8 mg / kg (13 nM) of red fluorescently labeled (Alexa) XPAT was injected into mice transplanted with human tumors (Figure 16A). Two days later, tumors and healthy organs were harvested, and protease cleavage was measured in vivo (results shown in Figure 16B). Green fluorescently labeled (Dyl800) XPAT was added after tissue harvesting in the presence of a protease inhibitor to account for artificial cleavage that may result from the release of unrelated intracellular proteases during processing. By comparing the cleavage products produced by red and green fluorescently labeled XPAT, we can determine whether cleavage occurred in vivo or artificially during tissue processing in various tissues. Generally, 20% of XPAT in tumors is activated within two days of injection into tumor-transplanted mice (n=31 across 9 tumor types, Figure 16C). [Figure 16-2] Same as above. [Figure 16-3] Same as above.

[0065] A patent or application file must include at least one color drawing. A copy of this patent or patent application publication with the color drawing will be provided by the Patent Office upon request and payment of the fee. [Modes for carrying out the invention]

[0066] Detailed description of preferred embodiments There are significant unmet needs in cancer treatment. While TCEs have been shown to be effective in inducing remission in certain cancers, they have not led to the development of a broad range of therapeutics due to their extremely high potency in healthy tissue and on-target, off-tumor toxicity. As an explanation, TCEs may bridge T cells and tumor cells, activating tumor cells through T cell mediation, and further activating the initiation of a cytokine amplification cascade, thereby promoting further death and potentially resulting in long-term immunity. T cells activated by TCEs release cytolytic perforin / granzymes in an antigen-MHC recognition-independent manner. This results in a two-fold response: direct tumor cell death and amplification of tumor death by initiating a potent cytokine response from tumor cells. Direct tumor cell death leads to the release of tumor antigens. Among the many cytokine responses, notably, are increased interferon-γ, which stimulates CD8 T cell activity and antigen presentation by APCs; increased IL-2, which leads to increased proliferation of activated T cells; and increased CXCL9 and 10 responses, which increase T cell recruitment. In summary, the release of tumor antigens and the initiation of a cytokine response may lead to the activation of an endogenous T cell response, resulting in epitope expansion and potentially inducing long-term immunity.

[0067] The toxicity challenges associated with TCEs stem from the fact that most tumor targets are expressed to some extent in healthy tissues, and that normal cells can also produce cytokine responses that lead to cytokine release syndrome (CRS). These two potent responses of healthy tissue to TCE-induced T cell activation result in a general lack of therapeutic indices for these activators.

[0068] The present invention overcomes the shortcomings of existing TCEs by providing a conditionally activated TCE, XPAT, or XTEN-modified protease-activated bispecific T cell engager (referred to herein as HER2-XPAT, exemplified as AMX818) that targets HER2. More specifically, the XPAT of the present invention leverages the abnormally regulated protease activity present in tumors against healthy tissue, thereby enabling an expansion of the therapeutic index. The XPAT core consists of two single-chain antibody fragments (scFv) targeting CD3 and a tumor target (in exemplary embodiments, the tumor target is HER2). Two unstructured polypeptide masks (XTENs) are bound to the core, and these XTENs structurally reduce target engagement to either the tumor target and / or CD3, and extend the protein half-life. The properties of the XTEN polymers also minimize potential immunogenicity. This is because its lack of a stable tertiary structure is unfavorable for antibody binding, and the absence of hydrophobic, aromatic, and positively charged residues that would serve as anchor residues for peptide MHC II binding reduces its potential for binding to T cell epitopes. In humans, minimal immunogenicity of the XTEN polymer was observed in >200 patients treated with drugs containing XTEN, in relation to the morphology of human growth hormone and factor VIII with extended half-lives. The protease cleavage site at the base of the XTEN mask enables the proteolytic activity of XPAT in the tumor microenvironment, thereby detaching small, extremely potent TCEs that can redirect cytotoxic T cells to kill tumor cells expressing the target. In healthy tissues where protease activity is tightly regulated, XPAT should remain inactive, primarily as an intact prodrug, and therefore expand the 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, furthermore, lead to increased T-cell immune action for improved solid tumor eradication. The release site used for XPAT can be cleaved by proteases across a wide range of tumors, which are collectively involved in the prominent features of any cancer (growth; survival and death; angiogenesis; invasion and metastasis; inflammation; and immune evasion). Thus, the TCE activity of XPAT is localized to tumors by leveraging enhanced protease activity that is upregulated at all stages of cancer and tumor development but tightly regulated in healthy tissues.

[0070] AMX-818, an exemplary HER2-XPAT component, has been optimized to achieve a desirable balance: providing sufficient protection in healthy tissues while maintaining the required intensity of action in tumors across a wide range of cancers. To reduce the potential for T cell activation by the prodrug, a longer XTEN polymer mask (576 amino acids for the HER2 site compared to 256 amino acids) was selected, along with a lower binding affinity to the α-CD3 domain. To ensure sufficient activation of AMX-818 in tumors, the protease release site at the base of XTEN was engineered to be cleaved by at least eight different proteases from three different classes reported to be overexpressed or abnormally regulated in cancer. These include several matrix metalloproteinases (MMPs), matryptases, uPAs, and the cysteine ​​protease, regmine. 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 inflammatory tissue where HER2 expression is absent, or if AMX-818 encounters HER2 expressed in healthy tissue where protease is tightly controlled. This AND gate feature is expected to lead to preferential activation in tumors where both elevated protease activity and high HER2 expression are present.

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

[0072] Summary of data generated from AMX-818, an example of HER2-XPAT. The target binding and in vitro bioactivity of AMX-818 have been characterized in multiple studies. Equilibrium binding analysis utilizing surface plasmon resonance demonstrated remarkably similar affinities between human HER2 and CD3 and cynomolgus monkey HER2 and CD3 for AMX-818 and its metabolites, supporting the use of cynomolgus monkeys as a species for toxicity and PK studies. AMX-818 (PAT), activated by proteolysis, 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 to HER2 by a factor of ten and its affinity to CD3 by approximately a factor of six for both species. AMX-818 bound to human and cynomolgus monkey HER2 with affinities of 24.9 nM and 20.1 nM, respectively, while its CD3 affinities for humans and cynomolgus monkeys 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). The extremely potent action of TCEs stems from the minimal requirement for initiating cytotoxicity: just three TCRs that are stimulated, aggregate, and form immune synapses between T cells and target cells. While it is well known that T cell engagers induce cytotoxicity, their potency also involves downstream cytokine-driven actions that enhance and amplify the anti-tumor immune response. T cell activation by AMX-818, its prototype AMX-818-P1, and its proteolytic metabolites was characterized in vitro using Jurcutt NFAT-reporter cells and primary human PBMCs in the presence of HER2-highly expressing tumor cells, BT-474 (breast) and SKOV-3 (ovary). Human T cells were also evaluated 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, we evaluated the upregulation of PD-L1, a ligand for PD-1, on the surface of the SKOV3 tumor target. This is because it is induced in response to IFN-γ secreted by activated T cells.

[0074] AMX-818(PAT) stimulates Jarcut NFAT-luciferase reporter T cells in the presence of BT-474 cells to reduce EC levels in the 70 pM range. 50While activation occurred at a detectable level, responses to masked AMX-818 and AMX-818-P1 were significantly attenuated by four orders of magnitude, with the maximum response being 80–90% lower compared to that 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 insufficient for activation, and that co-engagement of both CD3 and tumor targets was required for effective T cell receptor (TCR) stimulation. AMX-818 metabolites masked individually, AMX-818(1x-C) and AMX-818(1x-N), showed intermediate activity. AMX-818-NoClvSite did not induce detectable T cell activation, suggesting that the minimal response observed with AMX-818 was likely driven by proteolytic cleavage.

[0075] AMX-818(PAT) and its prodrug AMX-818 induced similar levels of CD69 and PD-1 expression on the surface of both CD4+ and CD8+ T cell subsets, as well as PD-L1 expression on SKOV3 tumor cells. 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 inflammatory 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. Primary cardiomyocytes (low to moderate HER2 expression) were selected to show more physiological cytolytic targets for AMX-818 and its proteolytic metabolites, as cardiac tissue is known to express HER2 and, although rare, cardiotoxicity has been observed in patients treated with some HER2-targeted therapies. A luminescence-based cytotoxicity assay was performed in a 1:1 effector:target cell ratio, and the cell-free supernatant was collected for measurement of TCE-inducing cytokines.

[0077] AMX-818(PAT) demonstrated very potent cytotoxicity against BT-474 tumor cells, reaching a semi-maximal inhibitory concentration (IC) in the range of 5-11 pM. 50 The cytotoxic response was nearly complete target cell death at the ) value. When human cardiomyocytes with lower HER2 expression—cells not expected to exhibit abnormally regulated protease activity—were used as targets, the cytotoxic response was reduced to approximately 1 / 13th, and maximum death was incomplete, reaching only 50-60%. The cytotoxic response with AMX-818 to both BT-474 and cardiomyocytes was strongly attenuated, and the mean IC value was reduced. 50The values ​​were shifted 2500 to 3000 times, demonstrating the effective functional masking of the prodrug by its XTEN polymer mask. The mask provides synergistic protection from cytotoxicity that far outweighs their combined effect on reducing target binding by interfering with the formation of functional immune synapses necessary to initiate target cell death. AMX-818 and its prototype AMX-818-P1 showed comparable cytotoxicity consistent with their nearly identical composition. The cytotoxicity of the individually 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 single masking. 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 potency of AMX-818 and its metabolites in inducing cytokine secretion in the supernatant from cytotoxicity assays was very similar to that observed for cytotoxicity against both BT-474 and cardiomyocyte target cells (AMX-818(PAT) was the most potent, >AMX-818 alone in masked form >AMX-818, and AMX-818 was the least potent). 50 The values ​​were, on average, higher than those for the cytotoxic response, demonstrating that the cytotoxic assay is more sensitive than the cytotoxic one. The response to AMX-818 was several orders of magnitude lower than that of AMX-818(PAT) in the presence of both BT-474 and cardiomyocytes, while the metabolites AMX-818(1x-N) and AMX-818(1x-C), masked individually, showed intermediate responses.

[0079] In the presence of cardiomyocytes, the maximum cytokine levels induced by AMX-818 at its highest concentration tested (300 nM) were significantly lower than those induced by AMX-818(PAT), with the exception of IL-6. Elevated IL-6 levels were detected at 300 nM concentrations of both AMX-818 and AMX-818-NoClvSite in the presence of both BT-474 and cardiomyocytes, and these levels were, in most cases, 2–6 times higher than the maximum levels produced by AMX-818(PAT). Notably, this contrasts with observations in cynomolgus monkeys, where peak systemic levels of IL-6 were >9 times higher with AMX-818(PAT) at a 0.2 mg / kg MTD than with AMX-818 at a 42 mg / kg MTD.

[0080] When evaluating cytokine induction in the absence of HER2-expressing target cells, 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 its highest concentration tested (500 nM), soluble AMX-818 induced low IL-10 levels from all PBMC donors. IL-6 was a noteworthy exception; at 500 nM, in soluble form, AMX-818 induced IL-6 levels from all donors by a mean 4.6-fold increase, exceeding levels induced by anti-CD3 antibody-positive controls. High IL-6 levels were also induced from two of the five donors in wet plate-coated form, and lower IL-6 levels were observed 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, nor were they observed in cynomolgus monkeys administered AMX-818 at doses of ≤50 mg / kg. In contrast, activated AMX-818 (PAT) induced high IL-6 levels in cynomolgus at doses ≤0.3 mg / kg, accompanied by increases in additional inflammatory cytokines.

[0081] In vivo pharmacological, 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 circulating AMX-818 was evaluated in cynomolgus monkeys administered high doses of AMX-818 or under pro-inflammatory conditions, and in vitro after long-term incubation in plasma from patients with cancer or systemic autoimmune disease. Preferential cleavage of AMX-818 in tumors clearly resulted in protease-dependent efficacy, but its peripheral stability in NHP provided a larger safety margin compared to AMX-818(PAT), thereby predicting an increased therapeutic index. Taken together, these data support the condition-dependent potent mask of AMX818, enabling tumor-localized activity through stability and effective masking occurring simultaneously in circulation and peripheral tissues.

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

[0083] In the BT474 model, administration of equimolar doses 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 the protease cleavage of its mask, as demonstrated by the lack of significant tumor growth inhibition in mice treated with AMX-818 (AMX-818-NoClvSite), a form lacking its protease release site. AMX-818-P1 and AMX-818(PAT) induced equivalent T cell activation in the tumor microenvironment, as assessed by flow cytometry by upregulation of the activation markers CD25 and CD69 on CD4 and CD8+ T cells. Importantly, consistent with the need for dual engagement of both HER2 and CD3 for T cell activation and their redirection to death, T cells were not activated peripherally, even by unmasked AMX-818 (PAT) which does not express human HER2.

[0084] A single dose of 2.1 mg / kg of AMX-818 is appropriate for large, established BT-474 tumors (478 mm). 3 In mice carrying the average tumor volume () of , the efficacy was sufficient to induce tumor regression within 4 days of administration. 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 is replaced by a non-HER2-binding scFv) or the lack of activity of AMX-818 itself when administered to tumor-bearing mice lacking hPBMCs. These findings further support the need for dual engagement for AMX-818's activity and provide a safety measure that AMX-818 should be cleaved in normal tissue where HER2 is absent.

[0085] In mice carrying HER2-low-expressing HT-55, 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-dependent and protease-dependent efficacy. The efficacy of AMX-818 in a model expressing 25,000 HER2 receptors suggests the potential to treat patients with multiple cancer types, including tumors with low HER2 expression levels. Ultimately, the preferential non-masking of AMX-818 (PAT) and fluorescently labeled AMX-818 in tumors was significant compared to <2% in combined cardiac, brain, and liver tissues after 2 days of incubation in BT-474 tumor-bearing mice. This supports the idea that localized abnormal regulation of proteases is the primary occupant factor in tumors, while protease inhibition is dominant in normal tissues.

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

[0087] As used herein and in the claims, the singular forms “a,” “an,” and “the” include multiple referents unless otherwise explicitly indicated by the context. For example, the term “a cell” includes multiple cells, including mixtures thereof.

[0088] The terms “polypeptide,” “peptide,” and “protein” are used herein synonymously to refer to polymers of amino acids of any length. Such polymers may be linear or branched, may contain modified amino acids, or may have non-amino acid intercalations. These terms also encompass amino acid polymers modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation, 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 its D or L optical isomers, as well as amino acid analogs and peptide mimetic compounds. Standard one- or three-letter codes are used to represent amino acids.

[0090] "Host cells" include individual cells or cell cultures that may or may have been recipients of the target vector. Host cells include offspring of a single host cell. Offspring may not necessarily be completely identical to the original parent cell (in terms of morphology or total DNA complement genomics) due to naturally occurring or genetically engineered mutations.

[0091] A "chimeric" protein contains at least one fusion polypeptide that includes a region located in a sequence different from its naturally occurring position. These regions may normally exist in separate proteins and be brought together into the fusion peptide, or they may normally exist in the same protein but be positioned in a new configuration within the fusion polypeptide. Chimeric proteins may be produced, for example, by chemical synthesis, or by translation of polynucleotides in which the peptide regions encode in a desired relationship.

[0092] The terms “conjugated,” “linked,” “fused,” and “fused” are used synonymously herein. These terms refer to the joining of two or more chemical elements or components by any means, including chemical conjugation or recombinant means.

[0093] The terms "polynucleotide," "nucleic acid," "nucleotide," and "nucleotide" are used synonymously. These refer to nucleotides, deoxyribonucleotides, or ribonucleotides, or analogs thereof, in polymeric form of any length. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be conjugated before or after the polymer is assembled. Non-nucleotide components may also be interspersed in the nucleotide sequence. Polynucleotides may be further modified after polymerization, such as by conjugation with labeling components.

[0094] As used herein, polynucleotides having “homologous” or being “homologous” are those that hybridize under stringent conditions as defined herein and have sequence identity of 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% with respect to their sequences.

[0095] The terms “identity percentage” and “identity %” refer, when applied to polynucleotide sequences, to the percentage of residue matching between at least two polynucleotide sequences aligned using a standard algorithm. Such algorithms can optimize the alignment between two sequences by inserting gaps in a standardized and reproducible manner into the sequences to be compared, thereby achieving a more meaningful comparison of the two sequences. The identity percentage may be measured over the length of the entire defined polynucleotide sequence, for example, as defined by a particular sequence number, or over a shorter length, for example, 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 residues. It is understood that such lengths are illustrative only, and any fragment length supported by the sequences shown in tables, figures, or sequence listings herein may be used to describe the length over which the identity percentage can be measured.

[0096] The “amino acid sequence identity percentage (%)” for polypeptide sequences identified herein is defined as the percentage of amino acid residues in the query sequence that are identical to amino acid residues in a second reference polypeptide sequence or a portion thereof, after the sequences have been aligned and gaps introduced as necessary to achieve the maximum sequence identity percentage, with no conservative substitutions being considered part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the skills in the art, for example, using commonly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. The identity percentage may be measured over the length of the entire defined polypeptide sequence, for example, as defined by a specific sequence number, or over a shorter length, for example, over the length of a fragment taken from a 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 consecutive residues. It is understood that such lengths are illustrative only, and any fragment length supported by the sequences shown in tables, figures, or sequence listings herein may be used to describe the length over which the identity percentage can be measured.

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

[0098] The term “substantially non-repeating XTEN” as used herein means (1) an XTEN sequence in which there are few or no instances of four consecutive amino acids of the same amino acid type, and (2) an XTEN having 12 or fewer subsequence scores (as defined in the preceding paragraph herein), or an XTEN in which there is no pattern in the order of sequence motifs constituting the polypeptide sequence from N-terminus to C-terminus.

[0099] A “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. This term includes vectors that primarily function for the insertion of DNA or RNA into cells, vector replications that primarily function for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. It also includes vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide that can be transcribed and translated into polypeptides when introduced into a suitable host cell. An “expression system” usually implies a suitable host cell comprising an expression vector capable of functioning to produce a desired expression product.

[0100] The term "t" 1 / 2 When used herein, " is ln(2) / K el This refers to the terminal phase half-life calculated as follows: K el This is the terminal phase elimination rate constant calculated by linear regression of the terminal linear portion of the logarithmic concentration-time curve. Half-life typically refers to the time required for half of the administered substance accumulated in a living organism to be metabolized or eliminated by normal biological processes. 1 / 2 In this specification, "terminal phase half-life," "elimination half-life," and "cyclic half-life" are used synonymously.

[0101] The terms “antigen,” “target antigen,” or “immunogen” are used herein synonymously to refer to a structure or binding determinant to which an antibody fragment or a therapeutic agent based on an antibody fragment binds or has specificity for such a structure.

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

[0103] As used herein, “treatment,” “to treat,” “to alleviate,” or “to relieve” are synonymous. These terms refer to an approach to obtain a beneficial or desired outcome, including but not limited to therapeutic and / or preventive benefits. “Therapeutic benefit” means the eradication or remission of the underlying disorder being treated. Therapeutic benefit is also achieved by the eradication or remission of one or more physiological symptoms associated with the underlying condition, such that the subject shows improvement despite the subject still being affected by the underlying disorder. For preventive benefits, a composition may be administered to a subject at risk of developing a particular condition, or to a subject reporting one or more physiological symptoms of a disease, even if the disease has not been diagnosed.

[0104] When used herein, “therapeutic effect” refers to a physiological effect resulting from the fusion polypeptides of this disclosure, other than the ability of a bioactive protein to induce the production of antibodies against an antigen epitope, including but not limited to the cure, mitigation, remission, or prevention of a disease in a human or other animal, or the enhancement of the physical or mental health of a human or animal. Determining a therapeutically effective dose is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure provided herein.

[0105] The terms “therapeutic dose” and “therapeutic size” as used herein refer to the amount of a bioactive protein, either alone or as part of a fusion protein composition, that, when administered to a subject in a single dose or repeated dose, can produce any detectable beneficial effect on any symptom, aspect, measurable parameter or characteristic of a disease or condition. Such effect does not necessarily have to be beneficial. A disease may also refer to a disorder or disease.

[0106] The term “therapeutic effective dose regimen,” as used herein, refers to a schedule of sequential doses of a bioactive protein, either alone or as part of a fusion protein, in which doses are administered in therapeutically effective amounts to produce a sustained beneficial effect on any symptom, aspect, measurement parameter or characteristic of a disease or condition.

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

[0108] In some embodiments, the polypeptide comprises a first XTEN (e.g., one described in the “Elongated Recombinant Polypeptide (XTEN)” section below, or one described elsewhere in this specification). In some embodiments, the polypeptide further comprises a second XTEN (e.g., one described in the “Elongated Recombinant Polypeptide (XTEN)” section below, or one described elsewhere in this specification). In some embodiments, the polypeptide comprises an XTEN ("N-terminal XTEN") at or near its N-terminus. In some embodiments, the polypeptide comprises an XTEN ("C-terminal XTEN") at or near its C-terminus. 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] Since bispecific antibodies (BsAbs) and bioactive polypeptides ("BPs") are linked to one or more XTENs within the polypeptide, the polypeptide may be called an XTEN-containing fusion polypeptide: "BPXTEN".

[0110] XTEN may comprise one or more barcode fragments (as further described below) that are releaseable (configured to be releaseable) from XTEN during protease-mediated digestion of the fusion polypeptide (or BPXTEN). 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 releaseable from the polypeptide during protease-mediated complete digestion of the polypeptide.

[0111] The (fusion) polypeptide may include, for example, one or more reference fragments (as further described below) that are releaseable (configured to be releaseable) from the polypeptide during protease digestion that releases a barcode fragment from the polypeptide. In some embodiments, each reference fragment may be a single reference fragment that differs in terms of sequence and molecular weight from all other peptide fragments that are releaseable from the polypeptide during protease digestion of the polypeptide.

[0112] In some embodiments, the polypeptide has an N-terminal amino acid and a C-terminal amino acid. The polypeptide is an elongated recombinant polypeptide (XTEN) comprising a barcode fragment (BAR) that can be released from the polypeptide upon protease digestion; and a bispecific antibody construct (BsAb) comprising a first antigen-binding fragment (AF1) that specifically binds to the differentiated antigen group 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). (c) a bispecific antibody construct comprising CDR-H3 comprising 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 (c) a release segment (RS) located between XTEN and the bispecific antibody construct, wherein XTEN (i) comprises at least 100 or at least 150 amino acids, and (ii) at least 90% of its amino acid residues are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) (iii) being identified herein by proline (P); (iii) comprising at least four different amino acids identified herein by G, A, S, T, E, or P; and (iv) XTEN being formed from a plurality of non-overlapping sequence motifs, each having a length of 9 to 14 amino acids; the plurality of non-overlapping sequence motifs comprising (1) a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif in the set is repeated at least twice in XTEN; and (2) a non-overlapping sequence motif that appears only once in XTEN; a barcode fragment (BAR) comprising at least a portion of the non-overlapping sequence motif that appears only once in XTEN; a barcode fragment (BAR) differing in sequence and molecular weight from all other peptide fragments that can be released from a polypeptide upon complete digestion of the polypeptide by a protease; and a barcode fragment (BAR) not comprising the N-terminal or C-terminal amino acids of the polypeptide.Polypeptides can be expressed as fusion proteins. In their uncleaved state, the fusion proteins may have a structural configuration identified herein by AF1-AF2-RS-XTEN, AF2-AF1-RS-XTEN, XTEN-RS-AF1-AF2, or XTEN-RS-AF2-AF1, from the N-terminus to the C-terminus.

[0113] In some embodiments of the polypeptides of this disclosure, XTEN is a first elongated recombinant polypeptide (XTEN1); a plurality of non-overlapping sequence motifs forming XTEN1 are the first plurality of non-overlapping sequence motifs; BAR is a first barcode fragment (BAR1); and RS is a first release segment (RS1). In some embodiments, the polypeptide further comprises (d) a second elongated recombinant polypeptide (XTEN2) comprising a second barcode fragment (BAR2) that can be released from the polypeptide upon protease digestion; and (e) a second release segment (RS2) located between the second XTEN (XTEN2) and a bispecific antibody construct (BsAb); XTEN2 (i) comprises at least 100 or at least 150 amino acids; and (ii) at least 90% of its amino acid residues are glycine (G), alanine (A), (iii) characterized by comprising at least four different amino acids identified herein by serine (S), threonine (T), glutamate (E), or proline (P); and (iii) comprising at least four different amino acids identified herein by 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 can be released from the polypeptide upon complete digestion of the polypeptide by a protease; and the second barcode fragment (BAR2) does not contain either the N-terminal 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 may be formed from a second plurality of non-overlapping sequence motifs, each having a length of 9 to 14 amino acids, the second plurality of non-overlapping sequence motifs comprising (1) a second set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif in 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 in the second XTEN; and a second barcode fragment (BAR2) comprising at least a portion of the non-overlapping sequence motif that appears only once in the second XTEN. The polypeptide is expressed as a fusion protein, which, in its uncleaved state, may have a structural configuration 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, from N-terminus to C-terminus. The diabody is the light chain variable region (VL) of AF1. I ), the heavy chain variable region of AF1 (VH I ), AF2 light chain variable region (VL II ), and the heavy chain variable region of AF2 (VH II ) may include.

[0114] In some embodiments of the polypeptides of this disclosure, (a) the first elongated 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 with respect to the sequences listed in Table 3a, and (b) the bispecific antibody construct (BsAb) comprises (I) light chain complementarity determining regions 1 (CDR-L1), 2 ( A first antigen-binding fragment (AF1) comprising CDR-L2) 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 each comprise the amino acid sequences of SEQ ID NOs. 8, 9, and 10, respectively, and (II) light chain variable regions (VL) identified herein by SEQ ID NOs. 778-783. II ) and the heavy chain variable region (VH) identified herein by sequence numbers 878-883. II (c) The first release segment (RS1) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences identified herein by SEQ ID NOs. 7001-7626; (d) The second elongated recombinant polypeptide (XTEN2) comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences listed in Table 3a; (e) The second release segment The ment (RS2) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences identified herein by sequence numbers 7001 to 7626, and the polypeptide may have a structural configuration identified herein 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 from the N-terminus to the C-terminus.

[0115] Elongated recombinant polypeptide (XTEN) Chain length and amino acid composition In some embodiments, XTEN contains at least 100 or at least 150 amino acids. In some embodiments, XTEN is 100 to 3,000 or 150 to 3,000 amino acids in length. In some embodiments, XTEN is 100 to 1,000 or 150 to 1,000 amino acids in length. In some embodiments, 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, and less At least 850, at least 900, at least 950, at least 1,000, at least 1,100, at least 1,200, at least 1,300, at least 1,400, at least 1,500, at least 1,600, at least 1,700, at least 1,800, at least 1,900, or at least 2,000 amino acids. In some embodiments, the XTEN has a length of up to (approximately) 100, up to (approximately) 150, up to (approximately) 200, up to (approximately) 250, up to (approximately) 300, up to (approximately) 350, up to (approximately) 400, up to (approximately) 450, up to (approximately) 500, up to (approximately) 550, up to (approximately) 600, up to (approximately) 650, up to (approximately) 700, up to (approximately) 750, up to (approximately) 800, and The maximum number of amino acids is approximately 850, the maximum is approximately 900, the maximum is approximately 950, the maximum is approximately 1,000, the maximum is approximately 1,100, the maximum is approximately 1,200, the maximum is approximately 1,300, the maximum is approximately 1,400, the maximum is approximately 1,500, the maximum is approximately 1,600, the maximum is approximately 1,700, the maximum is approximately 1,800, the maximum is approximately 1,900, or the maximum is approximately 2,000 amino acids.In some embodiments, XTEN has lengths of approximately 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, and 800. )950, (approx.)1,000, (approx.)1,100, (approx.)1,200, (approx.)1,300, (approx.)1,400, (approx.)1,500, (approx.)1,600, (approx.)1,700, (approx.)1,800, (approx.)1,900, or (approx.)2,000 amino acids, or having a length in the range between any two of the above. In some embodiments, at least 90% of the amino acid residues of 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 XTEN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). In some embodiments, XTEN comprises at least four different amino acids, which are G, A, S, T, E, or P, assigned substantially randomly to any other non-overlapping sequence motif constituting the XTEN polypeptide. In some embodiments, XTEN (e.g., XTEN1, XTEN2, etc.) is characterized by (i) comprising at least 100 or at least 150 amino acids; (ii) at least 90% of the amino acid residues of XTEN being glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P); and (iii) comprising at least four different amino acids from G, A, S, T, E, or P, which are substantially randomly assigned to any other non-overlapping sequence motif constituting the XTEN polypeptide. As used herein, it will be understood by those skilled in the art that the term “glutamate” is synonymous with “glutamic acid” and refers to a glutamic acid residue, 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. In some embodiments, the sum of the total number of amino acids in the first XTEN and the total number of amino acids in the second XTEN 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 arrangement motifs In some embodiments, XTEN comprises or is formed from a plurality of non-overlapping sequence motifs. In some embodiments, at least one of the non-overlapping sequence motifs is repeated (or repeated at least twice in XTEN), and at least one of the non-overlapping sequence motifs is not repeated (or is seen only once in XTEN). In some embodiments, the plurality of non-overlapping sequence motifs comprises (a) a set of (repeating) non-overlapping sequence motifs, where each non-overlapping sequence motif in the set is repeated at least twice in XTEN; and (b) a non-overlapping (not repeating) sequence motif that appears (or is not seen) only once in 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, a set of non-overlapping sequence motifs includes a set of non-overlapping (repeating) sequence motifs, each non-overlapping sequence motif in the set of non-overlapping sequence motifs that (1) is repeated at least twice in XTEN and (2) is between 9 and 14 amino acids in length. In some embodiments, the set of (repeating) non-overlapping sequence motifs includes 12-mer sequence motifs identified in Table 1 herein by SEQ ID NOs. 179-200 and 1715-1722. In some embodiments, the set of (repeating) non-overlapping sequence motifs includes 12-mer sequence motifs identified in Table 1 herein by SEQ ID NOs. 186-189. In some embodiments, the set of (repeating) non-overlapping sequence motifs includes 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 XTEN construction. [Table 1-1] [Table 1-2] *This shows individual motif sequences that form a "family sequence" when used together in various permutations.

[0117] Barcode fragment In some embodiments, the polypeptide includes barcode fragments (e.g., first, second, or third barcode fragments of XTEN) that can be released from the polypeptide upon protease digestion. In some embodiments, the barcode fragments are (1) a portion of XTEN containing at least a portion of a sequence motif that appears (or is not seen) only once within XTEN (not recurring, non-overlapping); and (2) distinct in terms of sequence and molecular weight from all other peptide fragments that can be released from the polypeptide upon complete protease digestion of the polypeptide. It will be understood by those skilled in the art that the term “barcode fragment” (or “barcode” or “barcode sequence”) may refer to either a cleavably fused portion of XTEN within the polypeptide, or a peptide fragment resulting from its release from the polypeptide.

[0118] In some embodiments, the barcode fragment does not contain either the N-terminal or C-terminal amino acids of the polypeptide. As will be further elaborated below or elsewhere in this specification, in some embodiments, the barcode fragment is releaseable (configured to be releaseable) during Glu-C digestion of the fused polypeptide. In some embodiments, the barcode fragment does not contain glutamic acid in the XTEN that is directly adjacent to another glutamic acid, if present. In some embodiments, the barcode fragment has glutamic acid at its C-terminus. It will be understood by those skilled in the art that when cleavably fused in the XTEN, the C-terminus of the barcode fragment may point to the "last" (or most C-terminal) amino acid residue in the barcode fragment, even if other "non-barcode" amino acid residues are located on the C-terminal side of the barcode fragment in the same XTEN. In some embodiments, the 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 directly adjacent to another glutamic acid residue. In some embodiments, the barcode fragment does not contain a (second) glutamic acid residue at any position other than the C-terminus of the barcode fragment, unless proline immediately follows the glutamic acid. In some embodiments, the barcode fragment is located at a distance of 10 to 150 or 10 to 125 amino acids from either the N-terminus or the C-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 N-terminus of the polypeptide, or at that amino acid location, or within any of the aforementioned ranges. In some embodiments, the barcode fragment is located within 200 amino acids, 150 amino acids, 100 amino acids, or 50 amino acids from 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 at those amino acid locations, or within any of the aforementioned ranges. In some embodiments, the barcode fragment is located within 200 amino acids, 150 amino acids, 100 amino acids, 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 by (i) not containing glutamic acid directly adjacent to another glutamic acid if present in XTEN; (ii) having glutamic acid at its C-terminus; (iii) having an N-terminal amino acid immediately preceding a glutamic acid residue; and (iv) being located at a certain distance from either the N-terminus or the C-terminus of the polypeptide, such that the distance is 10 to 150 amino acids or 10 to 125 amino acids in length. In some embodiments, the barcode fragment (i) does not contain either the N-terminal or C-terminal amino acids of the polypeptide, (ii) does not contain a glutamic acid directly adjacent to another glutamic acid in the XTEN, (iii) has glutamic acid at its C-terminus, (iv) has an N-terminal amino acid immediately preceding a glutamic acid residue, and (v) is located at a certain distance from either the N-terminus or C-terminus of the polypeptide, the distance being 10 to 150 or 10 to 125 amino acids in length. In some embodiments, the glutamic acid residue preceding the N-terminal amino acid is not directly adjacent to another glutamic acid residue.In some embodiments, the barcode fragment does not contain glutamic acid residues at positions other than the C-terminus of the barcode fragment, unless glutamic acid is immediately followed by proline. It will be understood by those skilled in the art that, where the term “distance” is used herein, it may refer to the number of amino acid residues from the N-terminus of the polypeptide to the most N-terminal amino acid residue of the barcode fragment, or the number of amino acid residues from the C-terminus of the polypeptide to the most C-terminal amino acid residue of the barcode fragment. In some embodiments, for a barcoded XTEN fused to a bioactive polypeptide, at least one barcode fragment (or at least two or three barcode fragments) contained in the barcoded XTEN is located at at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids from the bioactive 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 of length 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids, or within a range of any of the aforementioned values. In some embodiments, the barcode fragment is of length 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 some embodiments, the barcode fragment includes an amino acid sequence identified in Table 2 of this specification by sequence numbers 68-77. Table 2. Exemplary barcode fragments that may be released during Glu-C digestion. [Table 2]

[0119] In some embodiments of the polypeptides of this 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) may be located within a region of the XTEN that spans between 5% and 50%, 7% and 40%, or 10% and 30% of the length of the XTEN, measured from the distal end.

[0120] In some embodiments of the polypeptides of this 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 that can be released 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 in this specification. In some embodiments, the set of barcode fragments comprises a second barcode fragment (or further barcode fragments), such as those described above or elsewhere in this specification. In some embodiments, the set of barcode fragments comprises a third barcode fragment, such as those described above or elsewhere in this specification. The set of fused barcode fragments within an N-terminal XTEN may be referred to as the N-terminal set of barcodes ("N-terminal set"). A set of fused barcode fragments within a C-terminal XTEN may be referred to as a C-terminal set of a barcode ("C-terminal set"). In some embodiments, an N-terminal set includes a first barcode fragment and a second barcode fragment. In some embodiments, an N-terminal set further includes a third barcode fragment. In some embodiments, a C-terminal set includes a first barcode fragment and a second barcode fragment. In some embodiments, a C-terminal set further includes a third barcode fragment. In some embodiments, the second barcode fragment is located on the N-terminal side of the first barcode fragment in the same set. In some embodiments, the second barcode fragment is located on the C-terminal side of the first barcode fragment in the same set. In some embodiments, the third barcode fragment is located on the N-terminal side of both the first and second barcode fragments. In some embodiments, the third barcode fragment is located on the C-terminal side of both the first and second barcode fragments. In some embodiments, the third barcode segment is located between the first barcode segment and the second barcode segment.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, each barcode fragment in this set of barcode fragments being (1) part of the second XTEN and (2) different in terms of sequence and molecular weight from all other peptide fragments that can be released from the polypeptide upon complete digestion of the polypeptide by a protease.

[0121] XTEN with exemplary barcode Table 3a shows the amino acid sequences of 13 exemplary barcoded XTENs containing one barcode (e.g., SEQ ID NOs. 8002-8003, 8005-8009, and 8013), two barcodes (e.g., SEQ ID NOs. 8001, 8004, and 8012), or three barcodes (e.g., SEQ ID NOs. 8011). Of these 13 exemplary barcoded XTENs, six (SEQ ID NOs. 8001-8003, 8008-8009, and 8011) will be fused to the C-terminus of the bioactive protein, and seven (SEQ ID NOs. 8004-8007, 8010, and 8012-8013) will be fused to the N-terminus of the bioactive protein. In some embodiments, XTEN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequences identified herein by sequence numbers 8001-8020 in Table 3a. Table 3a. Exemplary XTEN with barcode [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 one of those 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 with any one of sequence numbers 601-659 listed in Table 3b. In some embodiments, XTEN sequences 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 sequence numbers 601-659 listed in Table 3b are obtained by one or more mutations of the corresponding sequences from Table 3b (e.g., mutations less than 10, less than 8, less than 6, less than 5, less than 4, less than 3, or less than 2). In some embodiments, one or more mutations include deletion of a glutamate residue, insertion of a glutamate residue, substitution of a glutamate residue, or substitution of a glutamate residue, or any combination thereof. In some embodiments where the XTEN sequence differs from any one of sequence numbers 601-659 listed in Table 3b, but has at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity with respect to it, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the XTEN sequence and the corresponding sequence in Table 3b includes glutamic acid deletion, glutamic acid residue insertion, glutamic acid residue substitution, glutamic acid residue substitution, or any combination thereof.In some embodiments, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the XTEN sequence and the corresponding sequence in Table 3b includes substitutions at glutamic acid residues, or substitutions of glutamic acid residues, or both. The term “substitution at the first amino acid” as used herein refers to the substitution of a second amino acid residue at a first amino acid residue, resulting in the second amino acid residue occupying the substitutional position in the resulting sequence. For example, “substitution at glutamic acid” refers to the substitution of a non-glutamic acid residue (e.g., serine (S)) at a glutamic acid residue (E). The term “substitution at the first amino acid” as used herein refers to the substitution of a first amino acid residue at a second amino acid residue, resulting in the first amino acid residue occupying the substitutional position in the resulting sequence. For example, “substitution at glutamic acid” refers to the substitution of a glutamic acid residue at a non-glutamic acid residue (e.g., serine (S)). Table 3b. An exemplary general-purpose XTEN for operation to create a barcode-equipped XTEN. [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, amino acid mutations are performed for constructing barcoded XTEN sequences with respect to XTENs of medium length compared to those in Table 3b, and 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 a general-purpose XTEN in Table 3b.

[0124] Additional examples of generic XTEN sequences that may be used in accordance with this disclosure include U.S. Patent Application Publications 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1, or International Patent Application Publication No. This information is disclosed in documents WO2010091122A1, WO2010144502A2, WO2010144508A1, WO2011028228A1, WO2011028229A1, WO2011028344A2, WO2014 / 011819A2, or WO2015 / 023891.

[0125] In some embodiments, a fused barcoded XTEN within the polypeptide chain ("N-terminal XTEN"), adjacent to the N-terminus of the polypeptide chain, may be bound to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at its N-terminus to facilitate the purification of the fusion protein. In some embodiments, a fused barcoded XTEN within the polypeptide chain ("C-terminal XTEN"), located at the C-terminus of the polypeptide chain, may contain or be bound to the sequence EPEA at its C-terminus to facilitate the purification of the fusion protein. In some embodiments, the fusion polypeptide comprises both an N-terminal barcoded XTEN and a C-terminal barcoded XTEN, the N-terminal barcoded XTEN being bound at its N-terminus to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49), and the C-terminal barcoded XTEN being bound at its C-terminus to the sequence EPEA, thereby facilitating the 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 chromatography methods known in the Art, including but not limited to IMAC chromatography, C-tagXL affinity matrices, 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 in this specification may be cleavably fused within the XTEN and may be released from the XTEN during protease digestion of the polypeptide (and may be configured to be released). In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease cleaves at the C-terminal side of glutamic acid residues that are not followed by proline. It will be understood by those skilled in the art that barcoded XTENs (XTENs containing barcode fragments) are designed to achieve high efficiency, precision, and accuracy of protease digestion. For example, it will be understood by those skilled in the art that adjacent Glu-Glu(EE) residues in the XTEN sequence may result in various cleavage patterns during Glu-C digestion. Therefore, when a Glu-C protease is used for barcode release, the barcoded XTEN or barcode fragment may not contain any Glu-Glu(EE) sequences. Those skilled in the art will also understand that, if the dipeptide Glu-Pro(EP) sequence is present in the fusion polypeptide, it cannot be cleaved by the Glu-C protease during the barcode release process.

[0127] Three-dimensional arrangement of BPXTEN structure In some embodiments, the BPXTEN fusion protein comprises a single BP and a single XTEN. Such a BPXTEN may have at least the following conformational permutations, described from the N-terminus to the C-terminus, respectively: BP-XTEN, XTEN-BP, BP-S-XTEN, and XTEN-S-BP.

[0128] In some embodiments, the BPXTEN comprises a C-terminal XTEN and, optionally, a spacer array (S) between the XTEN and the BP (for example, as described herein, e.g., in Table C). Such a BPXTEN is represented by formula I (described from N-terminus to C-terminus): (BP)-(S) x -(XTEN) (I) It may be represented by the formula, where BP is the bioactive protein described below herein; S is a spacer sequence having between 1 and about 50 amino acid residues (for example, as described herein, e.g., in Table C); x is either 0 or 1; and XTEN may be any of the ones described above or elsewhere herein.

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

[0130] In some embodiments, BPXTEN includes both an N-terminal XTEN and a C-terminal XTEN. Such a BPXTEN (e.g., XPAT in Figures 1-2) is given by Equation III: (XTEN)-(S) y -(BP)-(S) z -(XTEN) (III) It may be represented by the formula, where BP is the bioactive protein described herein as follows; S is a spacer sequence having between 1 and about 50 amino acid residues (as described herein, for example, in Table C), which may optionally include a BP release segment (as described herein, for example, in more detail below); y is either 0 or 1; z is either 0 or 1; and XTEN may be any of those described herein above or anywhere else.

[0131] Bioactive polypeptides

[0132] Bioactive proteins (BPs) fused to XTEN (as described above or elsewhere in this specification), particularly those disclosed below in this specification, including sequences identified herein by Tables 6a–6f, are well known in the art, along with their corresponding nucleic acid and amino acid sequences. Descriptions and sequences of these BPs are available in public databases, e.g., Chemical Abstracts Services Databases (e.g., CAS Registry), GenBank, The Universal Protein Resource (UniProt), and subscription-based databases, e.g., GenSeq (e.g., Derwent). The polynucleotide sequence may be a wild-type polynucleotide sequence encoding a given BP (e.g., either full-length or mature), or in some cases, the sequence may be a variant of a wild-type polynucleotide sequence (e.g., a polynucleotide encoding a wild-type bioactive protein), where the polynucleotide DNA sequence is optimized, e.g., for expression in a particular species, or a polynucleotide encoding a wild-type protein variant, e.g., a site-directed mutant or allele variant. It is well within the capabilities of those skilled in the art to use the wild-type or consensus cDNA sequence or codon-optimized variant of BP to produce the BPXTEN constructs intended by the present invention, using methods known in the art and / or in conjunction with the guidance and methods provided herein.

[0133] The BPs to be included in BPXTEN (a fusion polypeptide comprising at least one BP and at least one XTEN) may include any protein of any biological, therapeutic, prophylactic, or diagnostic interest, or any protein that, when administered to a subject, is useful in mediating biological activity or in preventing or alleviating disease, disorder, or condition. BPs in which increased pharmacokinetic parameters, increased solubility, improved stability, masking of activity, or any other enhanced pharmaceutical properties are sought, or BPs in which the extension of the terminal phase half-life would result in improved efficacy, safety, reduced administration frequency, and / or improved patient compliance. Therefore, BPXTEN fusion protein compositions are prepared with a variety of objectives in mind, including improving the therapeutic efficacy of a bioactive compound by, for example, increasing in vivo exposure or extending the length in which BPXTEN remains within the therapeutic range when administered to a subject, compared to BPs not linked to XTEN.

[0134] BP may be a natural, full-length protein, or it may be a fragment or sequence variant of a bioactive protein that retains at least a portion of the bioactivity of the natural protein.

[0135] In one embodiment, the BP incorporated into the composition of interest may be a recombinant polypeptide having a sequence corresponding to a naturally occurring protein. In some embodiments, the BP may be a sequence variant, fragment, homolog, or mimic of a natural sequence that retains at least a portion of the biological activity of the natural BP. In non-limiting examples, the BP may be a sequence exhibiting at least about 80% sequence identity to the protein sequences identified herein, or alternatively, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In a further non-limiting example, BP is a bispecific sequence comprising a first binding domain and a second binding domain, wherein the first binding domain, having a specific binding affinity for a tumor-specific marker or antigen on target cells, exhibits at least approximately 80% sequence identity with respect to the VL and VH sequences of a pair of anti-CD3 antibodies identified herein by Table 6f, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 A second binding domain exhibiting 8%, 99%, or 100% sequence identity and having a specific binding affinity to 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 the pair of anti-target cell antibodies identified herein by Table 6a. In one embodiment, the BPXTEN fusion protein may comprise a single BP molecule linked to XTEN. In some embodiments, BPXTEN may comprise 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, when used in vivo or in in vitro assays, BPs will exhibit binding specificity to a given target (or a given number of targets), or other desired biological characteristics. For example, BPs can be agonists, receptors, ligands, antagonists, enzymes, antibodies (e.g., monospecific or bispecific), or hormones. BPs known to be used for or useful for a disease or disorder, where the natural BP has a relatively short terminal phase half-life and improvements in pharmacokinetic parameters (which can be released as needed from the fusion protein by cleaving the spacer sequence) allow for lower-frequency administration or enhanced pharmacological effects, are of particular interest. BPs with a narrow therapeutic range between the minimum effective dose or blood concentration (Cmin) and the maximum tolerable dose or blood concentration (Cmax) are also of interest. In such cases, ligation of BPs to fusion proteins containing selected XTEN sequences can result in improvements to these properties, and thus make them more useful as therapeutic or prophylactic agents compared to BPs not ligated to XTEN.

[0137] The BPs included in the compositions of the present invention may be useful in treating a variety of therapeutic or disease categories, including but not limited to glucose and insulin disorders, metabolic disorders, cardiovascular diseases, coagulation and hemorrhagic disorders, growth disorders or conditions, endocrine disorders, eye diseases, kidney diseases, liver diseases, tumorigenic conditions, inflammatory conditions, and autoimmune conditions.

[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 the 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 present on all differentiated T cells. The anti-CD3-containing fusion protein of the present invention may be used in particular to delay the onset of type 1 diabetes, including the use of anti-CD3 as a therapeutic effector, as well as the use of anti-CD3 as a targeted portion for a second therapeutic BP in a BPXTEN composition. The sequence of the variable region of anti-CD3 and the creation of anti-CD3 are described in U.S. Patents No. 5,885,573 and No. 6,491,916.

[0139] The BPs of the composition in question are not limited to natural, full-length polypeptides, but also include their recombinant versions and bioactive and / or pharmacologically active variants or fragments. For example, it will be understood that various amino acid substitutions can be added to GP to create variants without departing from the spirit of the invention with respect to the bioactivity or pharmacological properties of BP. Examples of conserved amino acid substitutions in polypeptide sequences are shown in Table 5. However, in embodiments of BPXTEN where the sequence identity of BP is less than 100% compared to a particular sequence disclosed herein, the invention intends to substitute a given amino acid residue of a given BP at any position in the sequence of BP, including an adjacent amino acid residue, with any of the other 19 natural L-amino acids. If any one substitution results in an undesirable change in bioactivity, one of the alternative amino acids can be used, and the construct can be evaluated by the methods described herein, or by using any of the teachings and guidelines on conservative and non-conservative mutations described herein, for example, U.S. Patent No. 5,364,934 (the contents of which are incorporated herein by reference in their entirety), or by methods generally known to those skilled in the art. In addition, variants may also include polypeptides in which one or more amino acid residues are added or deleted at the N or C terminus of the full-length natural amino acid sequence of BP, and which retain at least a portion of the biological activity of the natural 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 given sequence, or alternatively, at least about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or 100% sequence identity. 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, which has a specific binding affinity for tumor-specific markers or antigens on target cells, has at least about 80% sequence identity with respect to the VL and VH sequences of the pair of anti-CD3 antibodies identified herein by Table 6f, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. A second binding domain exhibiting 97%, 98%, 99%, or 100% sequence identity and having a specific binding affinity to effector cells may be a bispecific sequence exhibiting at least approximately 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 the pair of anti-target cell antibodies identified herein as shown in Table 6a. The BPs of the embodiments described above 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 higher, activity compared to the corresponding natural BP sequence will be considered suitable for incorporation into the target BPXTEN.BP found to retain a suitable activity level can be ligated to one or more XTEN polypeptides as described above or elsewhere in this specification. In one embodiment, BP found to retain a suitable activity level can be ligated 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 form a chimeric fusion protein.

[0142] T cell engager The additional structural configurations of BPXTEN relate to XTEN-modified protease-activated T cell engagers ("XPAT" (singular or plural)) (e.g., bispecific T cell engagers) in which BP is a bispecific antibody. In some embodiments, the XPAT composition comprises (1) a first portion including a first binding domain and a second binding domain; and (2) a second portion including a release segment; and (3) a third portion including a bulking portion. In some embodiments, the XPAT composition has the configuration of formula Ia (described from N-terminus to C-terminus): (Part 1) - (Part 2) - (Part 3) (Ia) The formula comprises, wherein the first part is bispecific, comprising two scFvs, the first binding domain having a specific binding affinity to a tumor-specific marker or antigen of the target cell and the second binding domain having a specific binding affinity to effector cells; the second part comprises a release segment (RS) that can be cleaved by a mammalian protease (the protease may be activated by being tumor-specific or antigen-specific, as will be further fully described below herein); and the third part is a bulking portion. In the embodiments described above, the binding domain of the first portion 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 pair of binding domains are linked by a polypeptide linker (as will be further fully described below herein)). In one embodiment, the first portions VL and VH are listed in Tables 6a-6f; RS is identified herein by the group of sequences listed in Tables 8a-8b (as will be further fully described below); and the bulking portion 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 portion is an XTEN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences listed in Tables 3a-3b. In the embodiments described above, the composition is a recombinant fusion protein. In some embodiments, the portions are linked by chemical conjugation.

[0143] In some embodiments, the XPAT composition has the stereochemical configuration of formula IIa (described from the N-terminus to the C-terminus): (Part 3) - (Part 2) - (Part 1) (IIa) The formula comprises a bispecific scFv having two scFvs, the first of which the first binding domain has a specific binding affinity to a tumor-specific marker or antigen on target cells, and the second binding domain has a specific binding affinity to effector cells; the second of which comprises a release segment (RS) that can be cleaved by a mammalian protease; and the third of which is a bulking portion. In the embodiments described above, the binding domain of the first portion may be in the order (VL-VH)1-(VL-VH)2 (wherein "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 (wherein the pair of 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 the group of sequences listed in Tables 8a-8b; the bulking portion 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 portion is XTEN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences listed in Tables 3a-3b. In the embodiments described above, the composition is a recombinant fusion protein. In some embodiments, the portions are linked by chemical conjugation.

[0144] In some embodiments, the XPAT composition has the stereochemical configuration of formula IIIa (described from N-terminus to C-terminus): (Part 5) - (Part 4) - (Part 1) - (Part 2) - (Part 3) (IIIa) The formula comprises, wherein the first part is bispecific, comprising two scFvs, the first binding domain having a specific binding affinity for a tumor-specific marker or antigen of a target cell, and the second binding domain having a specific binding affinity for effector cells; the second part comprises a release segment (RS) that can be cleaved by a mammalian protease; the third part is a bulking portion; the fourth part comprises a release segment (RS) that can be cleaved by a mammalian protease, which may be identical to or different from the second part; and the fifth part is a bulking portion, which may be identical to or different from the third part. In the embodiments described above, the binding domain of the first portion 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 pair of binding domains are linked by a polypeptide linker as described herein). In the embodiments described above, RS is identical to the sequence described in Tables 8a-8b. In the embodiments described above, the bulking portion 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 portion is an XTEN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences identified herein by the sequences listed in Tables 3a-3b. In the embodiments described above, the composition is a recombinant fusion protein. In some embodiments, the portions are linked by chemical conjugation.

[0145] Based on their design and specific components, the compositions in question are advantageously bispecific therapeutic agents that, when cleaved by proteases found in the target tissue or tissue ill-healthed by the disease, exhibit higher selectivity, longer half-lives, lower toxicity, and fewer side effects. Therefore, the compositions in question have an improved therapeutic index compared to bispecific antibody compositions known in the art. Such compositions are useful for treating certain diseases, including but not limited to cancer. Those skilled in the art will understand that the composition of the present invention achieves this reduction of nonspecific interactions through a combination of mechanisms, including steric hindrance by placing the binding domain on a bulky XTEN molecule and steric hindrance resulting from the tethering of the long, flexible XTEN polypeptide's flexible, unstructured characteristics to the composition, which, by being anchored to the composition, can vibrate and orbit the binding domain, thereby creating a barrier between the composition and the tissue or cell, as well as a reduction in the ability of the intact composition to penetrate cells or tissues, due to its large molecular weight compared to the size of the individual binding domains (both resulting from the actual molecular weight of XTEN and the large hydrodynamic radius of unstructured XTEN). However, the composition is designed such that, when it is in proximity to a target tissue or cell that has or secretes a protease capable of cleaving RS, or when the binding domain is internalized in the target cell or tissue upon ligand binding, the bispecific binding domain is released from the bulk of XTEN by the action of a protease, thereby removing the steric hindrance barrier and allowing its pharmacological effects to be exerted more freely. The target composition is used to treat various conditions in which selective delivery of a therapeutic bispecific antibody composition to cells, tissues, or organs is desired. In one embodiment, the target tissue may be cancer, such as leukemia, lymphoma, or a tumor of an organ or system.

[0146] Joint domain This disclosure envisions the use of single-stranded binding domains, e.g., Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single-domain antibodies, single-domain camelid antibodies, single-domain antibody molecules (scFv), and diabodies, which can bind to ligands or receptors associated with effector cells and to antigens of diseased tissues or cells, such as cancer, tumors, or other malignant tissues. In some embodiments, the antigen-binding fragment (AF) (e.g., a first antigen-binding fragment (AF1), or a second antigen-binding fragment (AF2)) may be (each independently) a chimeric or humanized antigen-binding fragment. The antigen-binding fragment (AF) (e.g., a first antigen-binding fragment (AF1), or a second antigen-binding fragment (AF2)) may be (each independently) Fv, Fab, Fab', Fab'-SH, a linear antibody, or a single-stranded variable fragment (scFv). Two antigen-binding fragments (e.g., first and second antigen-binding fragments) may be configured as (Fab')2 or single-stranded diabodies. In some embodiments, bispecific ones include a first binding domain having binding specificity to a target cell marker and a second binding domain having binding specificity to an effector cell antigen. In some embodiments, the first and second binding domains may be non-antibody scaffolds, e.g., antikalin, adnectin, finomer, affilin, affibody, centinlin, or DARPin. In other embodiments, the tumor cell target 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 area by considering the location of the target tissue protease and the presence of that protease in healthy tissue intended not to be targeted, and the presence of the target ligand in healthy tissue, but a greater presence of that ligand in unhealthy target tissue. The "therapeutic range" refers to the maximum difference between the minimum effective dose and the maximum tolerable dose of a given therapeutic composition.To facilitate the achievement of a broad therapeutic area, the binding domain of the first portion of the composition is shielded by the proximity of the bulking portion (e.g., XTEN), and as a result, the binding affinity of the intact composition to one or both ligands is reduced compared to the composition cleaved by a mammalian protease, thereby freeing the first portion from the shielding effect of the bulking portion.

[0147] Regarding single-chain binding domains, as is well established, Fv is the smallest antibody fragment containing a complete antigen recognition and binding site, consisting of a dimer of one heavy-chain variable domain (VH) and one light-chain variable domain (VL) associated non-covalently. Within each of the VH and VL chains are three complementarity-determining regions (CDRs) that interact to define the 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, scFv is created, each having three, four, or five CHRs within each binding domain. Framework sequences adjacent to the CDRs have a tertiary structure that is essentially conserved in native immunoglobulins across species, and framework residues (FRs) play a role in holding the CDRs in their proper orientation. Constant domains are not required for binding function but may help stabilize the VH-VL interaction. In some embodiments, the binding domain of the polypeptide may be a pair of VH-VL, VH-VH, or VL-VL domains from either the same or different immunoglobulins, but generally, it is preferable to use the respective VH and VL chains from the parent antibody to construct the single-strand binding domain. The order of the VH and VL domains in the polypeptide chain is not limited with respect to this invention, and the given order of domains can usually be reversed without any loss of function, but of course, the VH and VL domains are arranged so that the antigen-binding site can fold correctly. Therefore, the single-strand binding domains of the bispecific scFv embodiment of the composition in question 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 pair of binding domains are linked by the polypeptide linker described below in this specification).

[0148] Therefore, the arrangement of the binding domains in the exemplary bispecific single-chain antibodies disclosed herein may be such that the first binding domain is located on the C-terminal side of the second binding domain. The arrangement of the V chain may 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 configurations where the second binding domain is located on the N-terminal side of 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 side of” or “C-terminal side of” and their grammatical variants mean relative positions within the primary amino acid sequence, not being located at the absolute N or C-terminus of a bispecific single-chain antibody. Therefore, as a non-limiting example, the first binding domain "located on the C-terminal side of the second binding domain" means that the first binding is located on the carboxyl side of the second binding domain in the bispecific single-chain antibody, and does not rule out the possibility that additional sequences, such as a His tag, or another compound, such as 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, and each scFv comprises 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, and each domain comprises one VL domain and one VH.

[0150] In the scFv embodiment of the XPAT composition of the present invention, a first binding domain and a second binding domain are included, where the VL and VH domains are thought to be derived from monoclonal antibodies having binding specificity to tumor-specific markers or antigens of target cells and effector cell antigens, respectively. In other embodiments, the first and second binding domains each contain six CDRs derived from monoclonal antibodies having binding specificity to target cell markers, e.g., tumor-specific markers and effector cell antigens, respectively. In other embodiments, the first and second binding domains of the first portion of the composition in question may have three, four, or five CHRs within each binding domain. In other embodiments, embodiments of the present invention include a first binding domain and a second binding domain, each of which contains 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 tumor-specific markers or antigens of target cells and effector cell antigens, respectively. In one embodiment, the present invention provides a chimeric polypeptide assembly composition in which a 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 scFv 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 with the VL and VH sequences of a pair of anti-CD3 antibodies identified in Table 6a, or is identical to the sequences of that pair. In some embodiments, the second domain embodiment of the present invention comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-H3 regions, each of which is derived from a monoclonal antibody identified herein as an antibody listed in Table 6a. In the embodiments described above, the VH and / or VL domains may be configured as scFv, diabody, single-domain antibody, or single-domain camelid antibody.

[0151] In other embodiments, the second domain of the composition in question is derived from an anti-CD3 antibody identified herein as the antibody listed in Table 6a. In one embodiment described above, the second binding domain of the composition in question includes the VL and VH region sequences of a pair of anti-CD3 antibodies identified herein as the group of antibodies listed in Table 6a. In some embodiments, the present invention provides a chimeric polypeptide assembly composition in which the second binding domain includes 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 with the VL and VH sequences of a pair of huUCHT1 anti-CD3 antibodies in Table 6a, or is identical to the sequences of that pair. In the embodiments described above, the VH and / or VL domains may consist of 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 antitumor cell antibody identified as an antibody listed in Table 6f. In some embodiments, the present invention provides a chimeric polypeptide assembly composition in which the first 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 with, or identical to, the VL and VH sequences of a pair of antitumor cell antibodies identified in Table 6f. In the aforementioned embodiments, the first domain of the composition described comprises the VL and VH region sequences of a pair of antitumor cell antibodies disclosed herein. In the aforementioned embodiments, the VH and / or VL domains may constitute an scFv, a portion of a diabody, a single-domain antibody, or a single-domain camelid antibody.

[0153] In some embodiments, the chimeric polypeptide assembly composition comprises a first portion comprising a first binding domain and a second binding domain, where these binding domains are in a diabody configuration, and each binding domain comprises one VL domain and one VH domain. In one embodiment, the diabody embodiment of the present invention comprises a first binding domain and a second binding domain, where the VL and VH domains are derived from monoclonal antibodies having binding specificity to tumor-specific markers or antigens of target cells and effector cell antigens, respectively. In some embodiments, the diabody embodiment of the present invention comprises 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 tumor-specific markers or antigens of target cells and effector cell antigens, respectively. The diabody embodiments of the present invention include a first binding domain and a second binding domain, the VL and VH domains of which are thought to be derived from a monoclonal antibody having binding specificity to a tumor-specific marker or target cell antigen and effector cell antigen, respectively. In some embodiments, the diabody embodiments of the present invention include a first binding domain and a second binding domain, each of which includes 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 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 includes a pair of 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 the diabody 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 with the VL and VH sequences of a pair of anti-CD3 antibodies identified in Table 6a, or is identical to the sequences of that pair. In some embodiments, the present invention provides a chimeric polypeptide assembly composition in which the second binding domain of the diabody 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 with the VL and VH sequences of the huUCHT1 antibody in Table 6a, or is identical to the sequences of that pair. 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 a chimeric polypeptide assembly composition in which the first binding domain of the diabody 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 with the VL and VH sequences of an antitumor cell antibody identified in Table 6f, or is identical to the pair of sequences. In other embodiments, the first domain of the diabody of the composition is derived from an antitumor cell antibody described herein.

[0154] Methods for measuring the binding affinity and / or other biological activity of the target composition of the present invention may 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 shown, 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 assay (ELISA), binding equilibrium exclusion assay (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 with the bulking moiety cleaved and the bulking moiety removed compared to a chimeric polypeptide assembly with the bulking moiety bound, 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 target 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 target and periodically collecting biological samples (e.g., body fluids, e.g., blood or plasma or ascites) to determine the concentration and / or amount of the substance in the samples over time. The concentration of the substance in the biological samples can be determined using a variety of suitable methods, including enzyme-linked immunosorbent assay (ELISA), immunoblotting, and chromatographic techniques, including high-pressure liquid chromatography and high-performance protein-lipid chromatography. In some cases, the substance can be labeled with a detectable tag, e.g., a radioactive tag or a fluorescent tag, which can be used to determine the concentration of the substance in a sample (e.g., a blood or plasma sample). Various pharmacokinetic parameters can then be determined from the results, which can be done using a software package, e.g., SoftMax Pro software, or by manual calculations known in the art.

[0156] In addition, the physicochemical properties of the chimeric polypeptide assembly composition can be measured to confirm the retention of solubility, structure, and stability. The target composition is assayed, thereby determining the binding-dissociation constant (K). d , K on and K off ), the half-life of ligand-receptor complex dissociation, and the activity of the binding domain that inhibits the biological activity of the captured ligand compared to the free ligand (IC2). 50 This makes it possible to determine the binding characteristics of the ligand-binding domain, including the value. 50 "EC" refers to the concentration required to inhibit half of the maximum biological response of a ligand agonist, and is generally determined by competitive binding assays. 50 " is the concentration required to achieve half of the maximum biological response of the active substance, and is generally determined by ELISA or a cell-based assay (including the method of the examples described herein).

[0157] Anti-CD3 binding domain In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a first portion of a binding domain having binding affinity to T cells. In one embodiment, the second portion of the binding domain comprises VL and VH derived from a monoclonal antibody that binds to CD3. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody against CD3 epsilon and / or CD3 delta. Exemplary, non-limiting examples of VL and VH sequences of monoclonal antibodies against CD3 are shown in Table 6a. In one embodiment, the present invention provides a chimeric polypeptide assembly comprising a binding domain having binding affinity to CD3, comprising the anti-CD3 VL and VH sequences described in Table 6a. In some embodiments, the present invention provides a chimeric polypeptide assembly comprising a first portion of a binding domain having binding affinity to CD3 epsilon, comprising the anti-CD3 epsilon VL and VH sequences described in Table 6a. In some embodiments, the present invention provides a chimeric polypeptide assembly composition in which the second binding domain of the first portion of scFv comprises VH and VL regions, and each VH and VL region exhibits at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VL and VH sequences of the pair of huUCHT1 anti-CD3 antibodies in Table 6a, or is identical to the sequences of that pair. In some embodiments, the present invention provides a chimeric polypeptide assembly composition in which the binding domain has binding affinity to CD3, comprising the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the respective anti-CD3 VL and VH sequences described in Table 6a.In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a binding domain having binding affinity to CD3, including 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 sequence is 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 function in the cell surface expression of the TCR and in the signaling cascade that occurs when peptide:MHC ligands bind to the TCR. Typically, when an antigen binds 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, causing the T cell to rapidly divide and produce new T cells sensitized to attack the specific antigen that has been exposed to the TCR. The CD3 complex consists of four other membrane-bound polypeptides (CD3-gamma, -delta, and / or -zeta) along with the CD3 epsilon molecule. In humans, CD3-epsilon is encoded by the CD3E gene on chromosome 11. The intracellular domain for each CD3 chain contains an immunoreceptor-activated tyrosine motif (ITAM), which functions as a nucleation site in the intracellular signaling mechanism during T cell receptor engagement.

[0159] Several therapeutic strategies modulate T-cell immunity, particularly anti-human CD3 monoclonal antibodies (mAbs), which are widely used clinically in immunosuppressive regimens, by targeting TCR signaling. The CD3-specific mouse mAb OKT3 was the first mAb approved for use in humans (Sgro, C. Side-effects of a monoclonal antibody, muromonab CD3 / orthoclone OKT3: bibliographic review. Toxicology 105:23-29, 1995), and is 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)), in 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 documented 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)). In particular, Wong's research demonstrated that target cell death can be achieved by cross-linking CD3 T cells with target cells, and that neither FcR-mediated ADCC nor complement binding is required for bivalent anti-CD3 MABs to lyse target cells.

[0160] OKT3 exhibits both mitogen and T-cell killing activity in a time-dependent manner. Following initial T-cell activation leading to cytokine release, further administration of OKT3 subsequently blocks all known T-cell functions. This subsequent blockade of T-cell function is the reason why OKT3 has found broad applications as an immunosuppressant in therapeutic regimens for reducing or even eliminating allograft tissue rejection. Other antibodies specific to the CD3 molecule are disclosed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50; anti-human monoclonal CD3 epsilon antibodies are described in WO2005 / 118635 and WO2007 / 033230; the VL and VH sequences of the mouse anti-CD3 monoclonal Ab UCHT1 (muxCD3, Shalaby et al., J. Exp. Med. 175, 217-225 (1992)) and its humanized variant (hu UCHT1) are described in U.S. Patent Application Publication 20120034228, which discloses a binding domain capable of binding to epitopes of human and non-chimpanzee primate CD3 epsilon chains. 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, this disclosure relates to antigen-binding fragments (AF1) having a specific binding affinity to effector cell antigens, 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 effector cells, which are plasma cells, T cells, B cells, cytokine-induced killer cells (CIK cells), mast cells, dendritic cells, regulatory T cells (RegT cells), helper T cells, myeloid cells, or NK cells.

[0162] Various AF1s that bind to effector cell antigens are particularly useful for pairing with antigen-binding fragments that have binding affinity to HER2 antigens associated with diseased cells or tissues in the form of a composition to induce cell death of diseased cells or tissues. Binding specificity can be determined by complementarity-determining regions, i.e., CDRs, e.g., light chain CDRs or heavy chain CDRs. Often, binding specificity is determined by the light chain CDR and heavy chain CDR. A given combination of heavy chain CDR and light chain CDR provides a given binding pocket that confers greater affinity and / or specificity to the effector cell antigen compared to other reference antigens. A resulting bispecific composition having a first antigen-binding fragment (AF1) for HER2 linked by a short, flexible peptide linker to a second antigen-binding fragment (AF2) that has binding affinity to the effector cell antigen is bispecific because each antigen-binding fragment has a specific binding affinity to its respective ligand. In such compositions, it will be understood that AF2 directed at HER2 in diseased tissue and AF1 directed at an effector cell marker are used in combination to bring the effector cells into very close proximity to the cells of the diseased tissue and achieve cytolysis of the diseased tissue cells. Furthermore, AF1 and AF2 are incorporated into a specially designed polypeptide comprising a cleavable release segment and XTEN, which is a polypeptide specifically designed to confer a prodrug characteristic to the composition, activated when the release segment is cleaved 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 composition has a binding affinity to effector cell antigens expressed on the surface of T cells. In some embodiments, AF1 of the composition has a binding affinity to CD3. In some embodiments, AF1 of the composition has a binding affinity to members of the CD3 complex, which include all known CD3 subunits of the CD3 complex, either individually or in unrelated combined forms, such as CD3 epsilon, CD3 delta, CD3 gamma, and CD3 zeta. In some embodiments, AF1 has a binding affinity to CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta.

[0164] The antigen-binding fragments intended by this disclosure may originate 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 producing 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 by the recombinant DNA method (U.S. Patent No. 4,816,567). The structures of antibodies and their fragments, the variable regions (VH and VL) of the heavy and light chains of antibodies, the single-stranded variable region (scFv), the complementarity-determining region (CDR), and domain antibodies (dAb) are well understood. Methods for producing polypeptides having a desired antigen-binding fragment with binding affinity to a given antigen are well 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 a portion or fragment of an antibody that retains the ability to bind to an antigen that is a ligand for the corresponding intact antibody. In such embodiments, the antigen-binding fragment may be, but is not limited to, a CDR and intervening framework region, a variable or hypervariable region of the antibody's light chain and / or heavy chain (VL, VH), a variable fragment (Fv), a Fab' fragment, an F(ab')2 fragment, a Fab fragment, a single-chain antibody (scAb), a VHH camelid antibody, a single-chain variable fragment (scFv), a linear antibody, a single-domain antibody, a complementation-determining region (CDR), a domain antibody (dAb), a BHH or BNAR type single-domain heavy-chain immunoglobulin, a single-domain light-chain immunoglobulin, or other polypeptides known in the art that contain an antibody fragment capable of binding to an antigen. Antigen-binding fragments having CDR-H and CDR-L can be configured in an orientation from the N-terminus to the C-terminus, either (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-stranded diabody configuration; that is, the VL and VH of AF1 and AF2 can be configured to enable diabody configuration using linkers of appropriate length.

[0166] The various CD3-binding AF1s of this disclosure have been specifically modified to enhance their stability in the polypeptide embodiments described herein. Antibody protein aggregation remains a significant challenge in their development and remains a major area of ​​focus in antibody production. Antibody aggregation can be induced by partial unfolding of its domains, leading to monomer association, subsequent nucleation, and aggregate growth. The aggregation tendency of antibodies and antibody-based proteins can be influenced by external experimental conditions, but it is strongly dependent on the intrinsic antibody properties determined by their sequence and structure. While it is well known that proteins are only slightly stable in their folded state, it is often not well understood that most proteins are inherently prone to aggregation in their unfolded or partially unfolded states, and that the resulting aggregates can be extremely stable and long-lived. While a reduced aggregation tendency has also been shown to be accompanied by increased expression titers, reducing protein aggregation is beneficial throughout the development process and may provide a more efficient pathway to clinical research. For therapeutic proteins, aggregates are a significant risk factor for adverse immune responses in patients and can be formed by a variety of mechanisms. By controlling aggregation, the stability, manufacturability, attrition rate, safety, formulation, titer, immunogenicity, and solubility of proteins can be improved. Essential protein properties such as size, hydrophobicity, electrostatics, and charge distribution play a crucial role in protein solubility. Low solubility of therapeutic proteins due to surface hydrophobicity has been shown to make formulation development more difficult, potentially leading to inadequate in vivo distribution, undesirable pharmacokinetic behavior, and immunogenicity in vivo. Reducing the overall surface hydrophobicity of candidate monoclonal antibodies may also result in benefits and cost reductions related to purification and administration regimens. Individual amino acids can be identified through structural analysis as contributing to antibody aggregation and may be located in the CDR and framework regions. In particular, residues may be predicted to be at high risk of causing hydrophobic problems in a given antibody.In one embodiment, the disclosure provides an AF1 having the ability to specifically bind to CD3, wherein, with respect to a parent antibody or antibody fragment, it has at least one amino acid substitution of a hydrophobic amino acid in a framework region, the hydrophobic amino acid being isoleucine, leucine, or methionine. In some embodiments, the CD3 AF1 has at least two amino acid substitutions of hydrophobic amino acids in one or more framework regions, the hydrophobic amino acids being 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 the antibody or antibody fragment, it will have a net positive charge. A larger 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 the antibody or antibody fragment, it will have a negative charge. A negative charge generally results in decreased tissue uptake and a longer half-life. This charge can be manipulated by mutations in framework residues. These considerations were reflected in the design of the AF1 sequences of the embodiments described herein, in which individual amino acid substitutions were made to the parental 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 charge of a specific amino acid in the protein sequence. The charge estimate is called the acid dissociation constant or pKa value and is used to calculate the pI. 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 molecule 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 Disclosure provides an antigen-binding fragment (e.g., AF1 or AF2) for use in any of the polypeptide embodiments described herein, comprising CDR-L and CDR-H (see Table 6b), and comprising (a) 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 Disclosure provides an antigen-binding fragment (e.g., AF1 or AF2) for use in any of the polypeptide embodiments described herein, comprising CDR-L and CDR-H, wherein (a) it specifically binds to the differentiated antigen group 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 also comprise CDR-L, which comprises CDR-L1 having the amino acid sequence of SEQ ID NOs. 1 or 2, CDR-L2 having the amino acid sequence of SEQ ID NOs. 4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NOs. 6 In some embodiments, the peptide comprises an antigen-binding fragment (AF) (e.g., AF1 or AF2) containing CDR-L1, CDR-L2, and CDR-L3. In some embodiments, CDR-L1 of AF may contain the amino acid sequence of SEQ ID NO: 1 or 2, CDR-L2 of AF may contain the amino acid sequence of SEQ ID NO: 4 or 5, and CDR-L3 of AF may contain 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) containing CDR-L1, CDR-L2, and CDR-L3, CDR-L1 of AF may contain the amino acid sequence of SEQ ID NO: 2, CDR-L2 of AF may contain the amino acid sequence of SEQ ID NO: 4 or 5, and CDR-L3 of AF may contain 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) containing CDR-L1, CDR-L2, and CDR-L3, CDR-L1 of AF may contain the amino acid sequence of SEQ ID NO: 2, CDR-L2 of AF may contain the amino acid sequence of SEQ ID NO: 5, and CDR-L3 of AF may contain the amino acid sequence of SEQ ID NO: 6.

[0169] In some embodiments, the embodiments of the antigen-binding fragment (AF) described in the preceding paragraph (e.g., AF1 or AF2) further include a light chain framework region (FR-L) and a heavy chain framework region (FR-H) (see Table 6c), wherein the antigen-binding fragment (AF) exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 51, and at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, and 93% sequence identity to the amino acid sequence of SEQ ID NO: 52. It may also include FR-L2 which exhibits or is identical to 94%, 95%, 96%, 97%, 98%, and 99% sequence identity; FR-L3 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to or is identical to any one amino acid sequence of sequence number 53 to 56; and FR-L4 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to or is identical to the amino acid sequence of sequence number 59. The antigen-binding fragment (AF) is FR-L1 which shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 51, or is identical to it, and FR-L1 which shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 52, or is identical to it. It may also include FR-L3 which exhibits or is identical to L2, showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with or is identical to the amino acid sequence of SEQ ID NO: 59, and FR-L4 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with or is identical to the amino acid sequence of SEQ ID NO: 59.The antigen-binding fragment (AF) is FR-L1 which shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 51, or is identical to it, and FR-L1 which shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 52, or is identical to it. It may also include FR-L3 which shows or is identical to L2, showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 54, and FR-L4 which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 59. The antigen-binding fragment (AF) is FR-L1 which shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 51, or is identical to it, and FR-L1 which shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 52, or is identical to it. It may also include FR-L3 which shows or is identical to L2, showing at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 55, and FR-L4 which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 59.The antigen-binding fragment (AF) is FR-L1 which shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 51, or is identical to it, and FR-L1 which shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 52, or is identical to it. It may also include FR-L3 which shows or is identical to L2, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 56, and FR-L4 which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 59. The antigen-binding fragment (AF) is FR-H1 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one amino acid sequence of SEQ ID NO: 60-63, and FR which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 64. It may also include FR-H3 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of -H2, SEQ ID NO: 65 or 66, and FR-H4 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67.The antigen-binding fragment (AF) is either FR-H1, which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 60, or FR-H2, which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 64. It may also include FR-H3 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 65, and FR-H4 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67. The antigen-binding fragment (AF) is FR-H1 which shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 61, or is identical to it, and FR-H1 which shows at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 64, or is identical to it. It may also include FR-H3 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 65, and FR-H4 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect 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 include a light chain framework region (FR-L) and a heavy chain framework region (FR-H), where AF exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to the amino acid sequence of SEQ ID NO: 51, and at least 86% to the amino acid sequence of SEQ ID NO: 52. FR-L2 exhibits or is identical to FR-L2 exhibiting sequence identity of 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% for any one amino acid sequence of SEQ ID NOs. 53-56, FR-L3 exhibits or is identical to FR-L3 exhibiting sequence identity of at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% for the amino acid sequence of SEQ ID NO. 59, FR-L4 exhibits or is identical to the amino acid sequence of SEQ ID NO: 60 or 61 with 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity, while FR-H1 exhibits or is identical to the amino acid sequence of SEQ ID NO: 64 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity. It may also include FR-H2 which exhibits or is identical to sequence identity, FR-H3 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 65, and FR-H4 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 67.AF is FR-L1 which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 51, FR-L2 which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequence of SEQ ID NO: 52, and at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, FR-L3 exhibits or is identical to the amino acid sequence of SEQ ID NO: 94%, 95%, 96%, 97%, 98%, 99% sequence identity; FR-L4 exhibits or is identical to the amino acid sequence of SEQ ID NO: 59 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity; FR-H1 exhibits or is identical to the amino acid sequence of SEQ ID NO: 60 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity; and SEQ ID NO: 64. It may also include FR-H2 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence; FR-H3 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 65; and FR-H4 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67.AF is FR-L1, which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 51, and FR-L2, which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 52. FR-L3 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 54, FR-L4 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 59, sequence number FR-H1 exhibits or is identical to the amino acid sequence of sequence number 61 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity; FR-H2 exhibits or is identical to the amino acid sequence of sequence number 64 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity; sequence number 65 It may also include FR-H3 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67, and FR-H4 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67.AF is FR-L1, which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 51, and FR-L2, which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 52. FR-L3 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 55, FR-L4 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 59, sequence number FR-H1 exhibits or is identical to the amino acid sequence of sequence number 61 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity; FR-H2 exhibits or is identical to the amino acid sequence of sequence number 64 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity; sequence number 65 It may also include FR-H3 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67, and FR-H4 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67.AF is FR-L1, which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 51, and FR-L2, which shows or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 52. FR-L3 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 56, FR-L4 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 59, sequence number FR-H1 exhibits or is identical to the amino acid sequence of sequence number 61 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity; FR-H2 exhibits or is identical to the amino acid sequence of sequence number 64 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity; sequence number 65 It may also include FR-H3 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67, and FR-H4 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67.

[0170] In some embodiments, the 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 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or being identical to the amino acid sequence of SEQ ID NO: 102 or SEQ ID NO: 105 in Table 6d. In some embodiments, the Disclosure provides an AF for use in any of the polypeptide embodiments described herein, comprising a variable light chain (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or being 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 Disclosure provides antigen-binding fragments (e.g., AF1 or AF2) for use in any of the polypeptide embodiments described herein, which may include an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to any one of the amino acid sequences of SEQ ID NOs. 201-205 in Table 6e.

[0171] In some embodiments, the Disclosure provides antigen-binding fragments (e.g., AF1 or AF2) that bind to CD3 protein complexes with enhanced stability compared to CD3-binding antibodies or antigen-binding fragments known in the Art. Furthermore, the CD3 antigen-binding fragments of the Disclosure are designed to confer a greater degree of stability to the chimeric bispecific antigen-binding fragment composition into which they are integrated, 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 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 used as components of the chimeric bispecific antigen-binding fragment composition into which they are integrated exhibit desirable pharmaceutical properties, including high thermal stability and a low tendency to aggregate, resulting in improved expression and recovery during manufacturing and storage, and further promoting an extension of the serum half-life. Biophysical properties such as thermal stability are often limited by antibody variable domains whose intrinsic properties are quite different. High thermal stability is often associated with other desirable properties, including high expression levels and resistance to 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 defined as the "melting temperature" (T), which is the temperature at which half of the molecule denatures. m This is determined by measuring the ) temperature. The melting temperature of each heterodimer indicates its thermal stability. mIn vitro assays for determining the melting point of heterodimers are known in the art, including the methods described in the following examples. The melting point of heterodimers 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 heterodimers can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9), or as described in the following examples.

[0172] In some embodiments of the polypeptides of this disclosure, the antigen-binding fragment (e.g., AF1 or AF2) is used in an in vitro assay, and the melting temperature (T) of the anti-CD3 binding fragment of the first antigen-binding fragment is used. m A higher melting temperature compared to the control bispecific antigen-binding construct, or when the first antigen-binding fragment is incorporated into the test bispecific antigen-binding construct, the T of the test bispecific antigen-binding construct compared to the control bispecific antigen-binding construct. m Compared to T m As is evident, the test bispecific antigen-binding construct may exhibit higher thermal stability than the anti-CD3 binding fragment consisting of the sequence of Sequence ID No. 206 (see Table 6e). The test bispecific antigen-binding construct comprises a first antigen-binding fragment and a reference antigen-binding fragment that binds to antigens other than CD3, while the control bispecific antigen-binding construct consists of an anti-CD3 binding fragment consisting of the sequence of Sequence ID No. 206 (see Table 6e) and a reference antigen-binding fragment. The melting temperature of the first antigen-binding fragment (T m ) is the T of the anti-CD3 binding fragment consisting of the sequence of sequence number 206 (see Table 6e). m It could be at least 2°C higher, at least 3°C ​​higher, at least 4°C higher, or at least 5°C higher.

[0173] The thermal denaturation curves of the anti-CD3 bispecific antibody and reference binding, including the CD3 binding fragment and anti-CD3 binding fragment of the present disclosure, show that the constructs of the present disclosure are more resistant to thermal denaturation than the antigen binding fragment or control bispecific antibody consisting of the sequence shown in SEQ ID NO: 781 (see Table 6f), the control bispecific antigen binding fragment includes SEQ ID NO: 781 (see Table 6f) and a reference antigen binding fragment that binds to the HER2 embodiment described herein. In one embodiment, any polypeptide from the embodiments of the subject composition described herein includes the anti-CD3 AF of the embodiment described herein, and the T of AF m In an in vitro assay, the T of the antigen-binding fragment consisting of the sequence of Sequence ID No. 781 (see Table 6f) was determined by increasing the melting temperature. 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, any polypeptide among the embodiments of the subject composition 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 CD3 complex subunits 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) can be determined by an in vitro antigen-binding assay containing human or cyno CD3 antigen to have a dissociation constant (K) between approximately 10 nM and approximately 400 nM, or between approximately 50 nM and approximately 350 nM, or between approximately 100 nM and approximately 300 nM. dIt can specifically bind to human or cyno CD3 at a constant (K). In some embodiments, any polypeptide among the embodiments of the subject compositions described herein can be determined by an in vitro antigen binding assay to have a dissociation constant (K) weaker than about 10 nM, or about 50 nM, or about 100 nM, or about 150 nM, or about 200 nM, or about 250 nM, or about 300 nM, or about 350 nM, or weaker than about 400 nM. d It contains an antigen-binding fragment (AF) that specifically binds to human or cyno-CD3. For clarity, 400 K d The antigen-binding fragment (AF) containing 10 nM K d It binds to its ligand more weakly than those having a certain dissociation constant (K) in an in vitro antigen-binding assay. In some embodiments, any polypeptide among the embodiments of the subject compositions described herein has a dissociation constant (K) in an in vitro antigen-binding assay. d The present invention includes an antigen-binding fragment (AF) that specifically binds to human or cyno CD3 with a weak binding affinity of 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, or 1 / 10 of the amino acid sequence of the antigen-binding fragment of Sequence ID No. 781 (see Table 6f), as determined by ). In some embodiments, the present disclosure includes each dissociation constant (K) in an in vitro antigen-binding assay. dThe present invention provides a bispecific polypeptide comprising an antigen-binding fragment (AF) (anti-CD3 AF) that exhibits a weak binding affinity to CD3 of less than or equal to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 20, 1 / 50, 1 / 100, or 1 / 1000 compared to the anti-HER2 AF embodiments described herein, as determined by the above method. The binding affinity of the target composition to the target ligand can be assayed using a binding assay or competitive assay such as a Biacore assay or ELISA assay using a chip-bound receptor or binding protein as described in U.S. Patent No. 5,534,617, 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 a standard method such as the Scatchard analysis described in van Zoelen, et al., Trends Pharmacol Sciences (1998) 19)12):487, or other methods known in the art.

[0175] In relevant embodiments, the 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 imparts enhanced stability to the compositions of the Disclosure compared to corresponding compositions comprising CD3-binding antibodies or antigen-binding fragments known in the Art. In one embodiment, any polypeptide among the embodiments of the Subject Compositions described herein comprises a CD3-binding AF (anti-CD3 AF), where the anti-CD3 AF exhibits a pI between 6.0 and 6.6, including both ends. In some embodiments, any polypeptide among the embodiments of the Subject Compositions described herein comprises a CD3-binding AF (anti-CD3 AF), where the anti-CD3 AF exhibits a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lower than the pI of a reference antigen-binding fragment (e.g., consisting of the sequence shown in SEQ ID NO: 206 (see Table 6e)). In some embodiments, any polypeptide among the embodiments of the subject composition described herein comprises a CD3-binding AF (anti-CD3 AF) fused to another AF (anti-HER2 AF) that binds to the HER2 antigen, wherein the anti-CD3 AF exhibits a pI of 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 to the HER2 antigen or its epitope. In some embodiments, any polypeptide among the embodiments of the subject composition described herein comprises a CD3-binding AF (anti-CD3 AF) fused to an AF (anti-HER2 AF) that binds to the HER2 antigen, wherein the AF exhibits a pI of 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.Such a design, in which the pIs of the two antigen-binding fragments are within such a range, is particularly intended to result in the resulting fused antigen-binding fragments conferring a high degree of stability to the chimeric bispecific antigen-binding fragment composition into which they are incorporated, leading to improved expression and enhanced recovery of the soluble, non-aggregated 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 the two AFs (anti-CD3 AF and anti-HER2 AF) within a relatively narrow pI range may allow for the selection of buffers or other solutions in which both AFs (anti-CD3 AF and anti-HER2 AF) are stable, thereby promoting the overall stability of the composition. Antigen-binding fragments (AFs) may exhibit an isoelectric point (pI) less than or equal to 6.6. Antigen-binding fragments (AFs) may exhibit an isoelectric point (pI) between 6.0 and 6.6, including both ends. The antigen-binding fragment (AF) may exhibit a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lower than the isoelectric point (pI) of the reference antigen-binding fragment consisting of the sequence shown in Sequence ID No. 206 (see Table 6e). The antigen-binding fragment (AF) has a dissociation constant (K) between approximately 10 nM and approximately 400 nM. d The antigen-binding fragment (AF) has a dissociation constant (K) of less than approximately 10 nM, or less than approximately 50 nM, or less than approximately 100 nM, or less than approximately 150 nM, or less than approximately 200 nM, or less than approximately 250 nM, or less than approximately 300 nM, or less than approximately 350 nM, or less than approximately 400 nM. d) can specifically bind to human or cyno CD3 (e.g., determined in an in vitro antigen-binding assay). The antigen-binding fragment (AF) can exhibit a binding affinity for CD3 that is 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9 or less, or 1 / 10 or less as weak as that of the antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 206 (see Table 6e) (e.g., determined by each dissociation constant (K d )).

[0176] In certain embodiments, the VL and VH of the antigen-binding fragment are fused by a relatively long linker consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids that have a flexible characteristic 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 joined 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, which 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, wherein the first domain and the second domain, and the third domain and the last domain are fused together by one of the short linkers described above, and the second variable domain and the third variable domain are fused by one of the relatively long linkers described above. As will be recognized by those skilled in the art, the selection of the short linker and the relatively long linker is to prevent the adjacent variable domains from mispairing, thereby facilitating the formation of the single-chain diabody conformation comprising the VL and VH of the first antigen-binding fragment and the second antigen-binding fragment. Table 6b. Exemplary CD3 CDR Sequences [Table 6b] Table 6c. Exemplary CD3 FR sequence [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 a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity to the 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 VL and VH derived from a monoclonal antibody against HER-2. Monoclonal antibodies against HER-2 are known in the art. Exemplary, non-limiting examples of VL and VH sequences are shown in Table 6f. In one embodiment, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity to the tumor-specific marker HER-2, comprising the anti-HER-2 VL and VH sequences described in Table 6f. In some embodiments, the present invention provides a chimeric polypeptide assembly composition comprising a first partial binding domain having binding affinity to a tumor-specific marker comprising the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each of which is derived from the respective VL and VH sequences described in Table 6f. Preferably, in the embodiment, binding is determined by an in vitro binding assay, 10 -10 Larger 10 -7 K up to M d It has a value. In some embodiments, the polypeptide contains an antigen-binding fragment (anti-HER2 AF) that specifically binds to HER2, the anti-HER2 AF (e.g., AF1 or AF2) has a heavy chain variable region (VH) containing the amino acid sequence shown as SEQ ID NOs. 778-783 in Table 6f. II (2) Light chain variable region (VL) containing the amino acid sequences shown as sequence numbers 878-883 in Table 6f II) may also be included. It is particularly intended that the chimeric polypeptide assembly composition may contain any one of the aforementioned binding domains or sequence variants thereof (inso that the variant exhibits binding specificity to the described antigen). In one embodiment, the sequence variant is created by substituting different amino acids for amino acids in the VL or VH sequence. In the deletion variant, one or more amino acid residues in the VL or VH sequence described herein are removed. Thus, the deletion variant contains all fragments of the binding domain polypeptide sequence. In the substitution variant, one or more amino acid residues in the VL or VH (or CDR) polypeptide are removed and replaced with alternative residues. In one embodiment, the substitution is essentially conservative, and this type of conservative substitution is well known in the art. Furthermore, it is particularly intended that the composition comprising the first binding domain and the second binding domain disclosed herein can be used 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: Long linkers within molecules [Table A] Table B: Short linkers within a molecule [Table B]

[0178] In some embodiments of the polypeptides of this disclosure, the light chain variable region (VL) and heavy chain variable region (VH) pairs of antigen-binding fragments may be linked by a linker or a long linker (e.g., consisting of hydrophilic amino acids). Such linkers linking the light chain variable region (VL) and heavy chain variable region (VH) of antigen-binding fragments (e.g., a first antigen-binding fragment (AF1), a second antigen-binding fragment (AF2)) (each independently) contain an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences listed in Table A. Such linkers linking the light chain variable region (VL) and heavy chain variable region (VH) of antigen-binding fragments (e.g., a first antigen-binding fragment (AF1), a second antigen-binding fragment (AF2)) (each independently) may contain an amino acid sequence identical to the sequences listed in Table A. In some embodiments of the polypeptides of this 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 contain an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences listed in Table B. Such a peptide linker linking two antigen-binding fragments (e.g., a first and a second antigen-binding fragment) may contain an amino acid sequence identical to the sequences listed in Table B. In some cases, the first antigen-binding fragment is a single-stranded variable fragment (scFv). In some cases, the second antigen-binding fragment is a single-stranded variable fragment (scFv). The two single-stranded 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 this 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 in Table A. In such embodiments, the peptide linker used to link the two antigen-binding fragments may be S-1 or S-2 in Table B.In some embodiments, the present disclosure provides a polypeptide 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 domain and the second domain, and the third domain and the last domain are fused together by a short linker of hydrophilic amino acids identified herein by the sequences set forth in Table B, and the second variable domain and the third variable domain are fused by a long linker identified in Table A. As will be recognized by those skilled in the art, the selection of the short linker and the long linker is to prevent the adjacent variable domains from mispairing, thereby facilitating the formation of the single-chain diabody conformation comprising the VL and VH of the first binding portion and the second binding portion. Table C: Exemplary Spacers between Release Segment and Bispecific Antibody Constructs [Table C-1] [Table C-2]

[0179] In some embodiments of the polypeptides of this disclosure, release segments (RSs) (e.g., a first release segment (RS1), a second release segment (RS2), etc.) may be fused to a bispecific antibody construct (BsAb) by spacers. Such spacers (each independently) contain at least four amino acids, which are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P). The peptides of this disclosure may include a first release segment fused to the bispecific antibody construct by a first spacer and a second release segment fused to the bispecific antibody construct by a second spacer. The spacers (e.g., a first spacer, a second spacer, etc.) (each independently) contain an amino acid sequence having at least (about) 80%, at least (about) 90%, or 100% sequence identity with respect to the sequences listed in Table C. Each spacer (e.g., the first spacer, the second spacer, etc.) contains (independently) the same amino acid sequence as the sequence listed in Table C.

[0180] unstructured 3D structure Typically, the XTEN components of a fusion protein are designed to behave similarly to denatured peptide sequences under physiological conditions, despite the extended polymer length. Denaturation describes the state of a peptide in solution, characterized by a large degree of structural freedom in the peptide backbone. Most peptides and proteins adopt denatured structures in the presence of high concentrations of denaturants or at high temperatures. Peptides with denatured structures are characterized, for example, by the absence of long-range interactions, when determined by NMR, by a characteristic circular dichroism (CD) spectrum. The terms "denatured structure" and "unstructured structure" are used synonymously herein. In some cases, the present invention provides XTEN sequences that, under physiological conditions, may resemble denatured sequences that largely lack secondary structure. In other cases, XTEN sequences may substantially lack secondary structure under physiological conditions. "Largely lacking," as used in this context, means that less than 50% of the XTEN amino acid residues of the XTEN sequence contribute to the secondary structure, as measured or determined by the means described herein. When used in this context, "substantially absent" means that, as measured or determined by the means described herein, 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 the secondary structure.

[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 structures can be measured spectrophotometrically, for example, by circular dichroism spectroscopy in the far-UV spectrum region (190-250 nm). Secondary structural elements such as alpha helices and beta sheets each produce CD spectra characterized by their shape and size. Secondary structures 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 in, for example, U.S. Patent Application Publication No. 20030228309A1. For a given sequence, the algorithm can predict whether some secondary structures are present or none at all, expressed, for example, as the total and / or percentage of residues in the sequence that form an alpha-helix or beta-sheet, or as the percentage of residues in the sequence that are predicted to result in random coil formation (lack of secondary structure).

[0182] In some cases, the XTEN sequence used in the fusion protein composition of the present invention, when determined by the Chou-Fasman algorithm, may have a percentage of alpha helices in the range of 0% to less than about 5%. In other cases, the XTEN sequence of the fusion protein composition, when determined by the Chou-Fasman algorithm, may have a percentage of beta sheets in the range of 0% to less than about 5%. In some cases, the XTEN sequence of the fusion protein composition, when determined by the Chou-Fasman algorithm, may have a percentage of alpha helices in the range of 0% to less than about 5% and a percentage of beta sheets in the range of 0% to less than about 5%. In preferred embodiments, the XTEN sequence of the fusion protein composition will have a percentage of alpha helices in the range of less than about 2% and a percentage of beta sheets in the range of less than about 2%. In other cases, the XTEN sequence of the fusion protein composition, when determined by the GOR algorithm, may have a high percentage of random coils. In some embodiments, the XTEN array 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%, when determined by the GOR algorithm.

[0183] Net charge In other cases, the XTEN polypeptide may have unstructured features conferred by incorporating net-charged amino acid residues in the XTEN sequence and / or reducing the proportion of hydrophobic amino acids. 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 XTEN in a composition may be above +0.1 charge / residue or below -0.1 charge / residue. In other cases, the net charge of XTEN may 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] Since most human or animal tissues and surfaces have a net negative charge, XTEN sequences are designed to have a net negative charge, minimizing nonspecific interactions between XTEN-containing compositions and various surfaces such as blood vessels, healthy tissues, or various receptors. While not bound by any particular theory, XTEN can adopt an open conformation due to electrostatic repulsion between individual amino acids of the XTEN polypeptide, which have a high net negative charge individually and are distributed across the sequence of the XTEN polypeptide. Such a distribution of net negative charge in XTEN with elongated sequence length leads to an unstructured conformation, which in turn can effectively increase the hydrodynamic radius. Therefore, in one embodiment, the present invention provides XTEN in which the XTEN sequence contains about 8, 10, 15, 20, 25, or even about 30% glutamic acid. The XTEN in the compositions of the present invention generally have no positively charged amino acids or only low amounts of them. In some cases, XTEN may have less than 10% positively charged amino acid residues, or less than 7%, less than 5%, or less than 2% positively charged amino acid residues. However, the present invention envisions constructs that incorporate a limited number of positively charged amino acids, such as lysine, into XTEN, enabling conjugation between the epsilonamine of lysine and a reactive group on a peptide, a linker crosslink, or a drug or small molecule conjugated to the XTEN backbone. As described above, a fusion protein can be constructed comprising 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 components is determined by the number of lysine or other amino acids with reactive side chains (e.g., cysteine) incorporated into 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 in the composition of interest may have isoelectric points (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 isoelectric points between 1.5 and 4.5. In these embodiments, the XTEN incorporated into the BPXTEN fusion protein composition of the present invention will have a net negative charge under physiological conditions, which may contribute to an unstructured three-dimensional structure and reduced binding of the XTEN component to mammalian proteins and tissues.

[0186] Since hydrophobic amino acids can confer structure to polypeptides, the present invention provides hydrophobic amino acid content in XTENs that is typically less than 5%, less than 2%, or less than 1%. In one embodiment, the amino acid content of methionine and tryptophan in the XTEN components of a BPXTEN fusion protein is typically less than 5%, less than 2%, most preferably less than 1%. In some embodiments, the XTEN has a sequence having less than 10% positively charged amino acid residues, or less than 7%, less than 5%, or less than 2% positively charged amino acid residues, with the sum of methionine and tryptophan residues being less than 2%, and the sum of asparagine and glutamine residues being less than 10% of the total XTEN sequence.

[0187] Increase in hydrodynamic radius In some embodiments, XTEN can have a high hydrodynamic radius, conferring a correspondingly increased apparent molecular weight to the BPXTEN fusion protein into which XTEN is incorporated. By linking XTEN to a BP sequence, a BPXTEN composition may be obtained that has an increased hydrodynamic radius, an increased apparent molecular weight, and an increased apparent molecular weight coefficient compared to BP not linked to XTEN. For example, in therapeutic applications where an extended half-life is desired, a composition in which XTEN with a large hydrodynamic radius is incorporated into a fusion protein 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 may result in reduced renal clearance of circulating protein. The hydrodynamic radius of a protein is determined not only by its molecular weight but also by its structure, including its shape and density. While not bound by any particular theory, XTENs can adopt an open conformation due to electrostatic repulsion between individual charges of the peptide, or due to intrinsic flexibility, which is conferred by certain amino acids in a sequence that lack the potential to confer secondary structure. The open, elongated, unstructured conformation of an XTEN polypeptide may have a more proportionally large hydrodynamic radius compared to polypeptides with comparable sequence length and / or molecular weight that have secondary and / or tertiary structures, such as typical globular proteins. Methods for determining the hydrodynamic radius are well known in the art, including, for example, by using size exclusion chromatography (SEC), as described in U.S. Patents 6,406,632 and 7,294,513.By adding XTEN with increased length, the hydrodynamic radius parameter, apparent molecular weight, and apparent molecular weight coefficient are proportionally increased, thereby allowing BPXTEN to be tuned to a desired characteristic cutoff apparent molecular weight or hydrodynamic radius. Thus, in certain embodiments, a BPXTEN fusion protein can be constructed with XTEN such that the fusion protein has 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 embodiments described above, the large hydrodynamic radius conferred by XTEN in the BPXTEN fusion protein may result in a decrease in the renal clearance of the resulting fusion protein, which in turn leads to a corresponding increase in terminal phase half-life, an increase in mean retention time, and / or a decrease in renal clearance rate.

[0188] In some embodiments, XTEN of selected length and sequence may be selectively incorporated into BPXTEN to create a fusion protein having an apparent molecular weight 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 under physiological conditions. In some embodiments, XTEN of selected length and sequence may be selectively ligated to a BP that yields a BPXTEN fusion protein having an apparent molecular weight coefficient of at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 15, or an apparent molecular weight coefficient of at least 20 or greater, under physiological conditions. In some embodiments, the BPXTEN fusion protein has an apparent molecular weight coefficient of about 4 to about 20, or about 6 to about 15, or about 8 to about 12, or about 9 to about 10, relative to the actual molecular weight of the fusion protein, under physiological conditions. In some embodiments, the (fusion) polypeptide exhibits an apparent molecular weight coefficient greater than about 6 under physiological conditions.

[0189] Increased terminal phase half-life In some embodiments, the (fusion) polypeptide has a terminal phase half-life that is at least twice as long, at least three times longer, at least four times longer, or at least five times 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 protein described herein to a subject in need may result in at least a twofold, at least threefold, at least fourfold, at least fivefold, or longer increase in the therapeutic range for the fusion protein compared to the corresponding BP, which is not linked to XTEN and administered to the subject at an equivalent dose.

[0191] Low immunogenicity In some embodiments, the present invention provides compositions in which the XTEN sequence has low immunogenicity or is substantially non-immunogenic. The low immunogenicity of XTEN may be due to several factors, such as substantially non-repeating sequences, unstructured three-dimensional structure, high solubility, low or absent degree of self-aggregation, low or absent degree of proteolytic sites within the sequence, and low or absent degree of epitopes within the XTEN sequence.

[0192] Those skilled in the art will understand that polypeptides having short amino acid sequences with a high degree of repetition (e.g., a 200-amino acid sequence containing an average of 20 or more repeats in a limited set of 3 or 4-mers) and / or consecutive repeating amino acid residues (e.g., a 5 or 6-mer sequence having identical amino acid residues) tend to aggregate, form higher-order structures, or form contact points, resulting in crystalline or pseudocrystalline structures.

[0193] In some embodiments, the XTEN sequence is substantially non-repeating, and (1) the XTEN sequence does not have three consecutive amino acids of the same amino acid species unless the amino acid is serine, in which case three or fewer consecutive amino acids may be serine residues; and (2) the XTEN does not contain three-amino acid sequences (3mers) that appear more than 16, 14, 12, or 10 times within the XTEN sequence of 200 amino acid length. Those skilled in the art will understand that such substantially non-repeating sequences allow for the design of long XTEN sequences with a relatively low frequency of charged amino acids, which are less prone to aggregation and therefore more likely to aggregate if the sequence or amino acid residues are more repetitive in other ways.

[0194] Structural epitopes are formed by regions on the protein surface composed of numerous discontinuous amino acid sequences of a protein antigen. Precise protein folding allows these sequences to become clearly defined, stable spatial arrangements, or epitopes, that can be recognized as "foreign" by the host humoral immune system, resulting in the production of antibodies against the protein or the induction of a cell-mediated immune response. In the latter case, the immune response to the protein in an individual is heavily influenced by T cell epitope recognition, a function of the peptide bond specificity of the individual's HLA-DR allotype. Association of MHC class II peptide complexes with congeneral T cell receptors on the surface of T cells, along with cross-linking to certain other co-receptors such as the CD4 molecule, 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 antibody production 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, most notably 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 by selecting a sequence that does not bind sufficiently to the MHC receptor. The present invention provides a BPXTEN fusion protein having a substantially non-repeating XTEN polypeptide designed to reduce binding to the MHC II receptor and avoid the formation of an epitope to which a T cell receptor or antibody binds, resulting in a low degree of immunogenicity. Avoidance of immunogenicity is partly a direct result of the structural mobility of the XTEN sequence, i.e., the lack of secondary structure due to the selection and order of amino acid residues. Sequences that tend to adopt a tightly folded structure, for example, in aqueous solution or under physiological conditions, can yield a structural epitope, are of particular interest. With conventional therapeutic practices and administration methods, the administration of fusion proteins containing XTEN generally does not result in the formation of neutralizing antibodies against the XTEN sequence, and the immunogenicity of the BP fusion partner in the BPXTEN composition may also be reduced.

[0196] In one embodiment, the XTEN sequence used in the target fusion protein may not substantially contain epitopes recognized by human T cells. Excluding such epitopes to produce 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 are described (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Peptide sequences that can form oligomers without generating T cell epitopes or non-human sequences are of particular interest. This can be achieved by testing serial repeat sequences of these sequences for the presence of T cell epitopes and the appearance of non-human 6-15mer, particularly 9mer sequences, and then modifying the design of the XTEN sequence to exclude or disrupt epitope sequences. In some cases, XTEN sequences become substantially non-immunogenic due to the limited number of XTEN epitopes predicted to bind to MHC receptors. The reduction in the number of epitopes capable of binding to MHC receptors simultaneously leads to decreased potential for T cell activation and T cell helper function, reduced B cell activation or upregulation, and decreased antibody production. A low degree of 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 WO2010 / 144502A2, which is incorporated entirely herein by reference. The TEPITOPE score of a given peptide frame within a protein is determined by the K of its binding to a number of the most common human MHC alleles, as disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555. d This is the logarithm of (dissociation constant, affinity, and dissociation rate (off-rate)). The score is at least 20 log(10e) between approximately 10 and approximately -10.10 K d ~10e -10 K d This can be reduced by avoiding hydrophobic amino acids, such as M, I, L, V, or F, which can function as anchor residues during peptide presentation on MHC, over a range corresponding to the binding constraints. In some embodiments, the XTEN components incorporated into BPXTEN do not have predictive T cell epitopes with TEPITOPE scores of approximately -5 or greater, or -6 or greater, or -7 or greater, or -8 or greater, or -9 or greater. As used herein, the score "-9 or greater" includes TEPITOPE scores from 10 to -9, including both ends, but does not include the score of -10, as -10 is less than -9.

[0197] In some embodiments, the XTEN sequence of the present invention, including that incorporated into a target BPXTEN fusion protein, can be substantially non-immunogenic by limiting known proteolytic sites derived from the XTEN sequence, thereby reducing the processing of XTEN into small peptides capable of binding to MHC II receptors. In some embodiments, the XTEN sequence can be made substantially non-immunogenic by using a sequence substantially lacking secondary structure, thereby conferring resistance to many proteases due to the high entropy of the structure. Thus, by lowering the TEPITOPE score and eliminating known proteolytic sites from XTEN, an XTEN composition containing XTEN in a BPXTEN fusion protein composition may be substantially unable to bind to mammalian receptors, including those of the immune system. In one embodiment, the XTEN in the BPXTEN fusion protein has >100 nM K for mammalian receptors. d Binding affinity, or K500 nM to mammalian cell surface or circulating polypeptide receptors. d , or K greater than 1 μM d It may have.

[0198] Furthermore, the substantially non-repeating sequence and the lack of corresponding epitopes in such embodiments of XTEN may limit the ability of B cells to bind to or be activated by XTEN. While XTEN can come into contact with many different B cells across its extended sequence, each individual B cell can only come into contact with one or a few individual XTENs. 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 unfused corresponding BP. In one embodiment, up to three parenteral doses of BPXTEN in a mammal may result in anti-BPXTEN IgG that is detectable at a 1:100 serum dilution but not at a 1:1000 dilution. In some embodiments, up to three parenteral doses of BPXTEN in a mammal may result in anti-BP IgG that is detectable at a 1:100 serum dilution but not at a 1:1000 dilution. In some embodiments, 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 undetectable at a dilution of 1:1000. In the embodiments described above, the mammal may be a mouse, rat, rabbit, or cynomolgus monkey.

[0199] An additional feature of certain embodiments of XTEN having substantially non-repeating sequences compared to sequences with a low degree of non-repeating (such as those having three consecutive identical amino acids) may be that non-repeating XTENs form weaker contact (e.g., monovalent interactions) with antibodies, thereby reducing the likelihood of immunoclearance and potentially allowing the BPXTEN composition to remain in circulation for a longer period.

[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 equivalent doses to the 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 become biologically active or their biological activity increases when they are released from XTEN by cleaving selective cleavage sequences incorporated within spacer sequences in BPXTEN, as is more fully described below herein.

[0202] Any set of spacer sequences is selective in the fusion proteins encompassed by the present invention. Spacers may be provided to enhance the expression of host cell-derived fusion proteins or to reduce steric hindrance so that BP components can exhibit their desired tertiary structure and / or interact appropriately with their target molecules. For spacers and methods for identifying desired spacers, see, for example, George, et al. (2003) Protein Engineering 15:871-879, which is specifically incorporated herein by reference. In one embodiment, the spacer comprises one or more peptide sequences having a length between 1 and 50 amino acid residues, or a length of about 1 to 25 residues, or a length of about 1 to 10 residues. The spacer sequence may contain any of the 20 native L-amino acids, excluding the cleavage site, and preferably contains sterically hindrance-free hydrophilic amino acids, including but 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 more preferably a mixture 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 the BPXTEN fusion protein composition may further contain identical or different cleavage sequences that can be acted upon by a protease to release BP from the fusion protein.

[0203] In some cases, the incorporation of the cleavage sequence into BPXTEN is designed to enable the release of 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 bound to BP after cleavage do not significantly interfere with BP activity (e.g., receptor binding) and provide sufficient access to proteases to further cleave the cleavage sequence. In some embodiments, the cleavage site is a sequence that can be cleaved by endogenous proteases in the mammalian subject, so that BPXTEN can be cleaved after administration to the subject. In such cases, BPXTEN can function as a prodrug or a circulating depot for BP. Examples of cleavage sites intended by the present invention include, but are not limited to, polypeptide sequences that can be cleaved 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 saltase A. Sequences known to be cleaved by the aforementioned proteases are known in the art. Exemplary cleavage sequences and cut sites within the sequences are shown in Table 7a along with sequence variants. For example, thrombin (activated coagulation factor II) acts on the sequence LTPRSLLV (SEQ ID NO: 222), which is cleaved after the arginine at position 4 of the sequence [Rawlings ND, et al. (2008) Nucleic Acids Res., 36: D320]. Activated FIIa is produced by the cleavage of FII by FXa in the presence of phospholipids and calcium, and lies downstream of factor IX in the coagulation pathway. Once activated, its innate role in coagulation is to cleave fibrinogen, which then initiates clot formation.FIIa activity is strictly controlled and occurs only when coagulation is necessary for proper hemostasis. However, since coagulation is an ongoing process in mammals, involving the incorporation of the LTPRSLLV (SEQ ID NO: 222) sequence of BPXTEN between BP and XTEN, the XTEN domain is removed from the adjacent BP simultaneously with the activation of either an exogenous or essential coagulation pathway when coagulation is physiologically required, thereby releasing BP over time. Similarly, incorporation of other sequences into BPXTEN under the action of endogenous proteases provides sustained release of BP, which in certain cases may provide a higher degree of activity for BP from a “prodrug” form of BPXTEN.

[0204] In some cases, only two or three amino acids adjacent to the cut site (a total of four to six amino acids) are incorporated into the cleavage sequence. In other cases, known cleavage sequences may have one or more deletions or insertions, or one or two or three amino acid substitutions for any one or two or three amino acids in the known sequence, and these deletions, insertions, or substitutions result in a decrease or increase in sensitivity to the protease, rather than the absence of sensitivity, thus enabling adaptation of the rate of BP release from XTEN. Exemplary substitutions are shown in Table 7a. Table 7a: Protease cleavage sequences for BP release [Table 7a] The arrow below indicates the cutting site; NA: Not applicable; * The list of multiple amino acids before, between, or after the slash indicates alternative amino acids that may be substituted at that location; The hyphen "-" indicates that any of the amino acids may be substituted with the corresponding amino acid shown in the middle column.

[0205] In some embodiments, the BPXTEN fusion protein may further include a spacer sequence which may include one or more cleavage sequences configured to release BP from the fusion protein when acted upon by a protease. In some embodiments, one or more cleavage sequences may be sequences having 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 with the sequences from Table 7a.

[0206] In some embodiments, the disclosure provides BP-releasing segment peptides (or releasing segments (RS)) that are substrates for one or more mammalian proteases associated with or resulting from cells found in or near diseased tissue. Such proteases include, but are not limited to, metalloproteinases, cysteine ​​proteases, aspartate proteases, and serine proteases, as listed in Table 7b. The RS is particularly useful for incorporation into a target recombinant polypeptide and confers a prodrug form that can be activated by cleavage of the RS by a mammalian protease. As described herein, the RS is incorporated into a target recombinant polypeptide composition, ligating the incorporated binding site to XTEN (this configuration is further described below), and as a result, upon cleavage of the RS by the action of one or more proteases for which the RS is a substrate, the binding site and XTEN are released from the composition, the binding site is no longer protected by XTEN, and regains sufficient ability to bind to their ligands. In these recombinant polypeptide compositions comprising first and second antibody fragments, the composition is referred to herein as an activatable antibody composition (AAC). Table 7b: Proteases of target tissues [Table 7b-1] [Table 7b-2]

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

[0208] This disclosure envisions a release segment that is a substrate for one, two, or three different classifications of proteases, including metalloproteinases, cysteine ​​proteases, aspartate proteases, or serine proteases, including the proteases in Table 7b. In certain features, the RS functions as a substrate for proteases found in close association with or coexisting with disease tissues or cells, such as tumors, cancer cells, and inflammatory tissues, and upon cleavage of the RS, the binding portion, otherwise protected by the XTEN of the recombinant polypeptide composition (and thus having low binding affinity to each of their ligands), is released from the composition, regaining their full ability to bind to ligands on target and / or effector cells. In some embodiments, the RS of the recombinant polypeptide composition contains an amino acid sequence that is a substrate for cellular proteases located within targeted cells, including but not limited to the proteases in Table 7b. Another specific feature of the recombinant polypeptide composition in question is that the RS, which is a substrate for two or three classifications of proteases, is designed with a sequence that can be cleaved at different positions in the RS sequence by different proteases. Thus, the RS, which is a substrate for two, three, or more classifications of proteases, has two, three, or more different cleavage sites in the RS sequence, but nevertheless, cleavage by a single protease results in the release of the binding moiety and XTEN from the recombinant polypeptide composition containing the RS.

[0209] In one embodiment, the RS of this disclosure for incorporation into the target recombinant polypeptide composition is meprin, neprilysin (CD10), PSMA, BMP-1, A disintegrin and metalloproteinase (ADAM), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM having a thrombospongin motif (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloprotein 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), Regmine, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin X, Cathepsin D, Cathepsin E, Secretase, Urokinase (uPA), Tissue Plasminogen Activator (tPA), Plasmin, Thrombin, Prostate-Specific Antigen (PSA, KLK3), Human Neutrophil Elastase (HNE), Elastase, Tryptase,RS is a substrate for one or more proteases, including but not limited to type II transmembrane serine proteases (TTSP), DESC1, hepsin (HPN), matryptase, matryptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast-activating protein (FAP), kallikrein-related peptidases (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14. In one embodiment, RS is a substrate for ADAM17. In one embodiment, RS is a substrate for BMP-1. In one embodiment, RS is a substrate for cathepsin. In one embodiment, RS is a substrate for HtrA1. In one embodiment, RS is a substrate for regmine. In one embodiment, 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 matryptase. 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 the recombinant polypeptide composition in question is a substrate for at least two proteases, including but not limited to regmine, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase. In some embodiments, the RS of the target recombinant polypeptide composition is a substrate for regmain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase. Table 8a: BP-releasing segment sequence [Table 8a-1] [Table 8a-2] [Table 8a-3] Table 8b: Release 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, the site-specific activation (RS) for incorporation into a target recombinant polypeptide can be designed to be selectively sensitive to various proteases that are substrates, resulting in varying cleavage rates and efficiencies. Since a given protease may be present at different concentrations in disease tissues, including but not limited to tumors, hematological malignancies, or inflammatory tissues or sites, compared to healthy tissue or circulation, the disclosure provides RS having individual amino acid sequences engineered to have higher or lower cleavage efficiencies for a given protease, to ensure that when the recombinant polypeptide is near target cells or tissues and coexisting proteases, it is preferentially converted from prodrug form to active form (i.e., by separation and release of the binding moiety and XTEN from the recombinant polypeptide after cleavage of the RS) compared to the cleavage rate of the RS in healthy tissue or circulation, and as a result, the released antibody fragment binding moiety has a higher ability to bind to ligands in disease tissue compared to the prodrug form remaining in circulation. Such selective design can improve the therapeutic index of the resulting composition and reduce side effects compared to conventional therapeutics that do not incorporate such site-specific activation.

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

number

[0212] In some embodiments, the Disclosure provides an AAC comprising a plurality of RSs, each RS sequence being identified herein by the sequence group listed in Table 8a, and the RSs being linked to each other by 1 to 6 amino acids which are glycine, serine, alanine, and threonine. In one embodiment, the AAC comprises a first RS and a second RS distinct from the first RS, each RS sequence being identified herein by the sequence listed in Table 8a, and the RSs being linked to each other by 1 to 6 amino acids which are glycine, serine, alanine, and threonine. In some embodiments, the AAC comprises a first RS, a second RS distinct from the first RS, and a third RS distinct from the first and second RS, each sequence being identified herein by the sequence listed in Table 8a, and the first, second, and third RSs being linked to each other by 1 to 6 amino acids which are glycine, serine, alanine, and threonine. It is particularly intended that multiple RSs of an AAC can be linked to form a sequence that can be cleaved by multiple proteases at different cleavage rates or efficiencies. In some embodiments, the disclosure provides an AAC comprising RS1 and RS2 identified herein by the sequences listed in Tables 8a-8b, as well as XTEN1 and XTEN2, such as those described above or elsewhere herein, wherein RS1 is fused with XTEN1 at the binding site and RS2 is fused with XTEN2 at the binding site. Such a composition is intended to be more readily cleaved by a diseased target tissue expressing multiple proteases compared to healthy tissue or in normal circulation, resulting in the resulting fragment having binding sites that penetrate more readily into target tissue, e.g., tumors, and have enhanced ability to bind and link to target cells and effector cells (or only target cells in the case of an AAC designed with respect to a single binding site).

[0213] The RS of this disclosure is useful to be included in recombinant polypeptides as a therapeutic agent for the treatment of cancer, autoimmune diseases, inflammatory diseases, and other conditions where localization of recombinant polypeptide activity is desirable. The composition in question addresses an unmet need and is superior in one or more embodiments to conventional antibody therapeutics or bispecific antibody therapeutics that are active at injection, including an improved therapeutic ratio with enhanced terminal phase half-life, targeted delivery, and reduced toxicity to healthy tissue.

[0214] In some embodiments, the (fusion) polypeptide comprises a first release segment (RS1) located between the (first) XTEN and the biologically active polypeptide. In some embodiments, the polypeptide further comprises a second release segment (RS2) located between the biologically active polypeptide and the second XTEN. In some embodiments, RS1 and RS2 are sequence-identical. In some embodiments, RS1 and RS2 are sequence-distinguishable. In some embodiments, RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequences identified herein by the sequences or subsets thereof in Tables 8a-8b. 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 with the sequences identified herein by the sequences or subsets thereof in Tables 8a-8b. 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.

[0215] reference fragment

[0216] In some embodiments, the (fusion) polypeptide further comprises one or more reference fragments that can be released from the polypeptide upon protease digestion. In some embodiments, each of the one or more reference fragments comprises a portion of a biologically active 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 that can be released from the polypeptide upon protease digestion of the polypeptide.

[0217] Exemplary polypeptide

[0218] In some embodiments of the compositions of this disclosure, the polypeptide is a recombinant polypeptide comprising an amino acid sequence having at least (about) 80% sequence identity with respect to the sequences listed in Table D (consisting of SEQ ID NOs. 12 to 47) or a subset thereof. The polypeptide may also 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 at least (about) 100% sequence identity with respect to the sequences listed in Table D (SEQ ID NOs. 12 to 47) or a subset thereof. Polypeptides may contain amino acid sequences having at least (approximately) 90%, at least (approximately) 91%, at least (approximately) 92%, at least (approximately) 93%, at least (approximately) 94%, at least (approximately) 95%, at least (approximately) 96%, at least (approximately) 97%, at least (approximately) 98%, at least (approximately) 99%, or approximately 100% sequence identity with respect to the sequences (sequence numbers 12-47) or subsets of those listed in Table D. Polypeptides may contain amino acid sequences identical to the sequences (sequence numbers 12-47) or subsets of those listed in Table D. It is particularly intended that the compositions of this disclosure may contain sequence variants of the amino acid sequences listed in Table D, such as including an inserted linker sequence or a purified tag sequence bound thereto, insofar as the variants exhibit substantially similar or identical biological activity(s) 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 mixtures comprising a first set of polypeptides and a second set of polypeptides, comprising a plurality of polypeptides of varying lengths. In some embodiments, each polypeptide in the first set of polypeptides comprises a barcode fragment having a sequence and molecular weight different from the sequence and molecular weight of all other fragments that can be released from the first set of polypeptides (a) by protease digestion. In some embodiments, the second set of polypeptides lacks the barcode fragment of the first set of polypeptides (e.g., by cleavage). In some embodiments, both the first set of polypeptides and the second set of polypeptides each comprise a reference fragment that is common to both the first set of polypeptides and the second set of polypeptides and is (b) can be released by protease digestion. In some embodiments, the ratio of the first set of polypeptides to polypeptides containing the reference fragment is greater than 0.70. In some embodiments, the ratio of the first set of polypeptides to polypeptides containing 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 of the first set of polypeptides and the second set of polypeptides. In some embodiments, the protease is a protease that cleaves glutamic acid residues at the C-terminus. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, polypeptides of varying lengths include polypeptides comprising at least one elongated recombinant polypeptide (XTEN), such as those described above or elsewhere in this specification. In some embodiments, a first set of polypeptides comprises a full-length polypeptide, and the barcode fragment is part of the full-length polypeptide. In some embodiments, the full-length polypeptide is a (fusion) polypeptide, such as those described above or elsewhere in this specification. In some embodiments, the barcode fragment lacks (does not contain) both the N-terminal and C-terminal amino acids of the full-length polypeptide.In some embodiments, a mixture of polypeptides of varying lengths differs from one another by N-terminal cleavage, C-terminal cleavage, or cleavage of both the N-terminal and C-terminal of the full-length polypeptide. In some embodiments, a first set of polypeptides and a second set of polypeptides may differ in one or more pharmacological properties. Non-limiting exemplary properties include:

[0220] Polypeptide characterization methods The disclosure herein includes a method for evaluating the relative amount of a first set of polypeptides in a mixture to a second set of polypeptides in a mixture, wherein (1) each polypeptide in the first set of polypeptides shares a barcode fragment that occurs only once in the polypeptide, 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 set of polypeptides and the second set of polypeptides contains a reference fragment, respectively. The method may include the step of contacting the mixture with a protease to produce a plurality of proteolytic fragments obtained by cleaving the first set of polypeptides and the second set of polypeptides, wherein the plurality of proteolytic fragments include a plurality of reference fragments and a plurality of barcode fragments. The method may further include the step of determining the ratio of the amount of barcode fragments to the amount of reference fragments, thereby evaluating the relative amount of the first set of polypeptides 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 both the first set of polypeptides and the second set of polypeptides. In some embodiments, the proteolytic fragments include multiple reference fragments and multiple barcode fragments. In some embodiments, the protease cleaves first and second sets of polypeptides (or polypeptides of varying lengths) at the C-terminal side of glutamic acid residues 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, the step of determining the ratio of the amount of barcode fragments to the amount of reference fragments includes the step of identifying the barcode fragments and reference fragments derived from the mixture after contact with the protease. In some embodiments, the barcode fragments and reference fragments are identified based on their respective masses. In some embodiments, the barcode fragments and reference fragments are identified by mass spectrometry.In some embodiments, the barcode fragment and the reference fragment are identified by liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the step of determining the ratio of the barcode fragment to the reference fragment includes isobaric labeling. In some embodiments, the step of determining the ratio of the barcode fragment to the reference fragment includes spiking the mixture with one or both of the isotopically labeled reference fragment and the isotopically labeled barcode fragment. In some embodiments, polypeptides of varying lengths include polypeptides comprising at least one elongated recombinant polypeptide (XTEN) described above or anywhere else in this specification. In some embodiments, the XTEN is characterized in that (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, which are G, A, S, T, E, or P. In some embodiments, the barcode fragment, if present, is part of XTEN. In some embodiments, the mixture of polypeptides of varying lengths includes polypeptides described above or elsewhere in this specification. In some embodiments, the polypeptides of varying lengths include full-length polypeptides and fragments thereof. In some embodiments, the polypeptides of varying lengths essentially consist of full-length polypeptides and fragments thereof. In some embodiments, the mixture of polypeptides of varying lengths differs from one another by N-terminal cleavage, C-terminal cleavage, or cleavage of both the N-terminus and C-terminus of the full-length polypeptide. In some embodiments, the full-length polypeptide is a polypeptide described above or elsewhere in this specification. In some embodiments, the ratio of the amount of barcode fragment to reference fragment is greater than 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, or 0.99.

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

[0222] Enhanced capabilities for detecting and quantifying cleavage products (e.g., by isobaric labeling) can generate knowledge that can be useful in designing manufacturing processes that include purification steps to minimize the presence of unwanted variants in purified drug substance / product.

[0223] Recombinant generation The disclosures herein include nucleic acids. The nucleic acids may include polynucleotides (or polynucleotide sequences) encoding (fusion) polypeptides such as those described above or elsewhere in this specification, or the nucleic acids may include reverse complements of such polynucleotides (or polynucleotide sequences).

[0224] The disclosures herein include an expression vector comprising a polynucleotide sequence, such as any of those described in the preceding paragraphs, and a regulatory sequence operably ligated to the polynucleotide sequence.

[0225] The disclosures herein include host cells containing expression vectors such as any of those described in the preceding paragraphs. 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 Disclosure provides a method for producing the composition of interest. In one embodiment, the method comprises culturing host cells containing a nucleic acid construct encoding one of the polypeptides or XTEN-containing compositions described herein under conditions that promote the expression of the polypeptide or BPXTEN-fusion polypeptide, and subsequently recovering the polypeptide or BPXTEN-fusion polypeptide using a standard purification method (e.g., column chromatography, HPLC), wherein the recovered composition is such that 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 of production, 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 monomer-soluble form.

[0227] In some embodiments, the disclosure provides an expression vector encoding constructs useful for producing polypeptides and BPXTEN fusion polypeptides at high fermentation expression levels of functional proteins using E. coli or mammalian host cells, and for generating polypeptide construct compositions with high cytotoxic activity at high expression levels. In one embodiment, the method comprises: 1) preparing a polynucleotide encoding a polypeptide from 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 transcriptional and translational sequences suitable for high levels of 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. Preferably, the host cell is E. coli. By this method, polypeptide expression 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 the 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 properly folded. As used herein, the term “properly folded” means that the components of the antigen-binding fragment of the composition have the ability to specifically bind to their target ligand.In some embodiments, the Disclosure provides a method for producing a polypeptide or a BPXTEN fusion polypeptide, comprising the step of culturing a host cell containing a vector encoding a polypeptide including the polypeptide or a BPXTEN fusion polypeptide, under conditions effective for expressing a polypeptide product at a concentration greater than about 10 milligrams (mg / g) per gram of dry weight of the host cell in a fermentation reaction, 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 properly folded. In some embodiments, the Disclosure provides a method for producing a polypeptide or BPXTEN fusion polypeptide, comprising the step of culturing host cells containing a vector encoding a composition under conditions effective for expressing a polypeptide product at a concentration greater than about 10 milligrams (mg / g) per gram of dry weight of host cells in a fermentation reaction, 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 disclosures herein include pharmaceutical compositions comprising a (fusion) polypeptide, such as any of those described above or elsewhere herein, and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is formulated for intradermal, subcutaneous, oral, intravenous, intra-arterial, intra-abdominal, intraperitoneal, intravitreous, 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 contained 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 intraocularly, if otherwise), 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 therapeutically by any preferred route. Furthermore, the pharmaceutical composition may contain other pharmaceutically active compounds or any of the 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 in a therapeutically effective dose. In some of the aforementioned cases, the therapeutically effective dose results in an increase in the time spent in the therapeutic region for the fusion protein compared to the corresponding BP of a fusion protein that is not linked to XTEN and administered to the subject in an equivalent dose. The increase in time spent in the therapeutic region may be at least 3 times greater than that of the corresponding BP that is not linked to XTEN, or at least 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, at least 10 times, or at least 20 times greater than that of the corresponding BP that is not linked to XTEN.

[0232] In some embodiments, the present invention provides a method for treating a disease, disorder, or condition, comprising the step of administering a pharmaceutical composition to a subject using a plurality of sequential doses of the pharmaceutical composition administered using a therapeutically effective dose regimen. In one embodiment described above, the therapeutically effective dose regimen is not linked to XTEN and has at least two sequential C levels in relation to the blood levels of the fusion protein compared to the corresponding BP of the fusion protein administered to the subject with an equivalent dose regimen. max Peak and / or C mm Between troughs, this can result in a time increase of at least 3-fold, or at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold, or at least 20-fold. In some of the embodiments described above, administration of the fusion protein produces a comparable improvement in at least one measurement parameter, using a lower dosing frequency or a lower total dose in molar units of the fusion protein of the pharmaceutical composition, compared to the corresponding bioactive protein component that is not linked to XTEN(or XTEN) and administered to the subject using a therapeutically effective regimen for the subject.

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

[0234] A useful extended-release formulation in the present invention may be an oral formulation comprising a matrix and a coating composition. Suitable matrix materials include waxes (e.g., carnauba wax, 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, hydroxypropyl methylcellulose, and polyethylene glycol). Other suitable matrix tableting materials include microcrystalline cellulose, powdered cellulose, hydroxypropyl cellulose, and ethyl cellulose, along with other carriers and fillers. Tablets may also contain granules, coated powders, or pellets. Tablets may also be multilayered. Multilayer tablets are particularly preferred when the active ingredients have significantly different pharmacokinetic profiles. Optionally, the finished tablets 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, or polyvinyl alcohol, or a monomer material such as sugars (e.g., lactose, sucrose, fructose, mannitol, etc.), salts (e.g., sodium chloride, potassium chloride, etc.), organic acids (e.g., fumaric acid, succinic acid, lactic acid, and tartaric acid), or mixtures thereof. If necessary, an enteric polymer may be incorporated into the coating composition. Suitable enteric polymers include hydroxypropyl methylcellulose, succinic acid acetate, hydroxypropyl methylcellulose, phthalic acid, polyvinyl phthalic acid acetate, cellulose phthalate acetate, cellulose acetate trimellitate, shellac, zein, and polymethacrylates containing carboxyl groups. The coating composition may be plasticized by adding suitable plasticizers such as diethyl phthalate, citrate ester, polyethylene glycol, glycerol, acetylated glyceride, acetylated citrate ester, dibutyl sebacate, and castor oil. The coating composition may also contain fillers, which may be insoluble materials such as silicon dioxide, titanium dioxide, talc, kaolin, alumina, starch, powdered cellulose, MCC, or potassium polariphosphate. The coating composition can be applied as a solution or latex in an organic solvent or aqueous solvent or a mixture thereof. Solvents such as water, lower alcohols, lower chlorinated hydrocarbons, ketones, or mixtures thereof may be used.

[0236] The BPXTEN polypeptide of the present invention can be formulated according to known methods for preparing pharmaceutically useful compositions, thereby combining the polypeptide 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 propyl ethylene glycol, polyethylene glycol, and vegetable oils. The therapeutic formulations are available in the form of lyophilized formulations or aqueous solutions, as described in Remington's Pharmaceutical Sciences 16. th As described in edition, Osol, A. Ed. (1980), the active ingredient having the desired purity is prepared for storage by mixing it with a physiologically acceptable carrier, excipient, or stabilizer 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 PH1Ol), polymethacrylate, poly(ethyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride) (e.g., Eudragit RS-30D), hydroxypropyl methylcellulose (Methocel KlOOM, Premium CR Methocel KlOOM, Methocel E5, Opadry®), magnesium stearate, talc, triethyl citrate, ethylcellulose dispersion (Surelease®), and protamine sulfate. The slow-release agent may also include a carrier, which may include, for example, a solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption retarders. These slow-release agents may also include pharmaceutically acceptable salts, such as inorganic salts like hydrochloride, hydrobromide, phosphate, or sulfate, as well as salts of organic acids such as acetate, propionate, malonate, or benzoate. The composition may also contain liquids such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifiers, or pH buffers. Liposomes may also be used as carriers.

[0237] In some embodiments, the compositions of the present invention are encapsulated within liposomes, which have been shown to be useful for delivering beneficial active agents in a controlled manner over extended periods. Liposomes are closed bilayer membranes containing a captured aqueous volume. Liposomes may be monolayer vesicles having a single membrane bilayer or multilayer vesicles having multiple membrane bilayers, each separated from the next membrane bilayer by an aqueous layer. The resulting membrane bilayer structure is such that the hydrophobic (nonpolar) tail of the lipid is oriented toward the center of the bilayer, while the hydrophilic (polar) head is oriented toward the aqueous phase. In one embodiment, liposomes can be coated with a flexible, water-soluble polymer to avoid uptake by mononuclear phagocytic organs, primarily the liver and spleen. Suitable hydrophilic polymers for surrounding liposomes include, but are not limited to, PEG, polyvinylpyrrolidone, polyvinyl methyl ether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyethylene glycol, polyaspartamide, and hydrophilic peptide sequences, as described in U.S. Patents No. 6,316,024; No. 6,126,966; No. 6,056,973; and No. 6,043,094, the contents of which are incorporated herein by reference in their entirety.

[0238] Liposomes may be composed of any lipid or combination of lipids known in the art. For example, the vesicle-forming lipids may be naturally occurring lipids or synthetic lipids, including phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and sphingomyelin, as disclosed in U.S. Patent Nos. 6,056,973 and 5,874,104. The vesicle-forming lipid may also be a glycolipid, cerebroside, or cationic lipid, as disclosed in U.S. Patent No. 6,056,973, for example, 1,2-dioleyloxy-3-(trimethylamino)propane (DOTAP); N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE); N-[1[(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 3[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Choi); or dimethyldioctadecylammonium (DDAB). As disclosed in U.S. Patent Nos. 5,916,588 and 5,874,1...

Claims

1. A polypeptide containing the amino acid sequence of SEQ ID NO:

34.

2. The polypeptide according to claim 1, comprising the amino acid sequence of SEQ ID NO:

34.

3. A pharmaceutical composition comprising the polypeptide described in claim 1 or 2 and one or more pharmaceutically suitable excipients.

4. A pharmaceutical composition for treating a cancer expressing HER2 in a subject, wherein the cancer is a solid tumor, and the pharmaceutical composition comprises the polypeptide described in claim 1 or 2.

5. (a) a polynucleotide sequence encoding the polypeptide described in claim 1 or 2; or (b) a nucleic acid comprising the reverse complement of the polynucleotide sequence of (a).

6. An expression vector comprising a polynucleotide sequence according to claim 5 and a transcription or translation regulatory sequence operably linked to the polynucleotide sequence.

7. A host cell comprising the expression vector described in claim 6.

8. The pharmaceutical composition according to claim 4, wherein the cancer expressing HER2 is selected from breast cancer, colorectal cancer, non-small cell lung cancer, and ovarian cancer.

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