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

Abstract

Disclosed herein are polypeptides, and methods of making and using such polypeptides, that comprise a bispecific antibody construct covalently linked to an extended recombinant polypeptide comprising a barcode fragment releasable from said 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.

1 . A polypeptide having an N-terminal amino acid and a C-terminal amino acid, the polypeptide comprising:
 (a) an extended recombinant polypeptide (XTEN), comprising:
 a barcode fragment (BAR) releasable from said polypeptide upon digestion by a protease; 
   (b) a bispecific antibody construct (BsAb), comprising:
 a first antigen binding fragment (AF1) that specifically binds to cluster of differentiation 3 T cell receptor (CD3), which 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), wherein said CDR-H3 comprises an 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) positioned between said XTEN and said bispecific antibody construct,   wherein said XTEN is characterized in that:
 (i) it comprises at least 100, or at least 150 amino acids; 
 (ii) at least 90% of its amino acid residues are identified herein by glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); 
 (iii) it comprises at least 4 different types of amino acids identified herein by G, A, S, T, E, or P; and 
 (iv) said XTEN is formed from a plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acids in length, 
 wherein said plurality of non-overlapping sequence motifs comprise:
 (1) a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of said set of non-overlapping sequence motifs is repeated at least two times in said XTEN; and 
 (2) a non-overlapping sequence motif that occurs only once within said XTEN; 
 
   
       wherein said barcode fragment (BAR) includes at least part of said non-overlapping sequence motif that occurs only once within said XTEN; 
       wherein said barcode fragment (BAR) differs in sequence and molecular weight from all other peptides fragments that are releasable from said polypeptide upon complete digestion of said polypeptide by said protease; and 
       wherein said barcode fragment (BAR) does not include said N-terminal amino acid or said C-terminal amino acid of said polypeptide. 
     
     
         2 . The polypeptide of  claim 1 , wherein:
 a. said set of non-overlapping sequence motifs are each independently identified herein by SEQ ID NOS: 179-200 and 1715-1722;   b. said set of non-overlapping sequence motifs are each independently identified herein by SEQ ID NOS: 186-189;   c. said set of non-overlapping sequence motifs comprise at least two, at least three, or all four of the sequence motifs SEQ ID NOS: 186-189.   
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The polypeptide of  claim 1 , wherein:
 a. said XTEN comprises a length of from 100 to 3,000, from 150 to 3,000, from 100 to 1,000, or from 150 to 1,000 amino acid residues;   b. said 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;   c. at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of said XTEN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); and/or   d. said XTEN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence set forth in Table 3a.   
     
     
         6 - 8 . (canceled) 
     
     
         9 . The polypeptide of  claim 1 , wherein said barbode fragment (BAR):
 a. does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in said XTEN;   b. has a glutamic acid at its C-terminus;   c. has an N-terminal amino acid that is immediately preceded by a glutamic acid residue;   d. is positioned a distance from either said N-terminus of said polypeptide or said C-terminus of said polypeptide, wherein said distance is from 10 to 150 amino acids, or from 10 to 125 amino acids in length;   e. is characterized in that:
 i. it does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in said XTEN; 
 ii. it has a glutamic acid at its C-terminus; 
 iii. it has an N-terminal amino acid that is immediately preceded by a glutamic acid residue; and 
 iv. it is positioned a distance from either said N-terminus of said polypeptide or said C-terminus of said polypeptide, wherein said distance is from 10 to 150 amino acids, or from 10 to 125 amino acids in length; optionally wherein said glutamic acid residue that precedes said N-terminal amino acid of said barcode fragment (BAR) is not immediately adjacent to another glutamic acid residue; and/or 
   f. does not include a second glutamic acid residue at a position other than the C-terminus of said barcode fragment unless said second glutamic acid is immediately followed by a proline.   
     
     
         10 - 15 . (canceled) 
     
     
         16 . The polypeptide of  claim 1 , wherein said XTEN is positioned:
 a. N-terminal of said bispecific antibody construct (BsAb), and wherein said barcode fragment (BAR) is positioned within 200, within 150, within 100, or within 50 amino acids of said N-terminus of said polypeptide;   b. N-terminal of said bispecific antibody construct (BsAb), and wherein said barcode fragment (BAR1) is positioned at a location that is between 10 and 200, between 30 and 200, between 40 and 150, or between 50 and 100 amino acids from said N-terminus of said protein;   c. C-terminal of said bispecific antibody construct (BsAb), and said barcode fragment (BAR) is positioned at a location that is between 10 and 200, between 30 and 200, between 40 and 150, or between 50 and 100 amino acids from said C-terminus of said protein; and/or   d. C-terminal of said bispecific antibody construct (BsAb), and said barcode fragment (BAR) is positioned at a location that is between 10 and 200, between 30 and 200, between 40 and 150, or between 50 and 100 amino acids from said C-terminus of said protein.   
     
     
         17 - 19 . (canceled) 
     
     
         20 . The polypeptide of  claim 1 , wherein:
 a. said barcode fragment (BAR) is at least 4 amino acids in length;   b. said barcode fragment (BAR) is between 4 and 20, between 5 and 15, between 6 and 12, or between 7 and 10 amino acids in length;   c. said barcode fragment (BAR) comprises an amino acid sequence set forth in Table 2;   d. said XTEN has a length defined by a proximal end and a distal end, wherein (1) said proximal end is positioned, relative to said distal end, closer to said bispecific antibody construct (BsAb), and wherein (2) said barcode fragment (BAR) is positioned within a region of said XTEN that extends, as measured from said distal end, between 5% and 50%, between 7% and 40%, or between 10% and 30% of said length of said XTEN;   e. said XTEN further comprises additional one or more barcode fragments, wherein said additional one or more barcode fragments each differ in sequence and molecular weight from all other peptides fragments that are releasable from said polypeptide upon complete digestion of said polypeptide by said protease;   f. said release segment (RS) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence identified herein by SEQ ID NOS: 7001-7626;   g. said protease cleaves on the C-terminal side of glutamic acid residues that are not followed by proline, optionally wherein said protease is a Glu-C protease; and/or   h. the polypeptide is expressed as a fusion protein, wherein said fusion protein, in an uncleaved state, has a structural arrangement from N-terminus to C-terminus identified herein by AF1-AF2-RS-XTEN, AF2-AF1-RS-XTEN, XTEN-RS-AF1-AF2, or XTEN-RS-AF2-AF1.   
     
     
         21 - 28 . (canceled) 
     
     
         29 . The polypeptide of  claim 1 , wherein said release segment (RS) is fused to said bispecific antibody construct (BsAb) by a spacer, optionally wherein:
 a. said spacer comprises at least 4 types of amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); or   b. said spacer comprises an amino acid sequence having at least 80%, 90%, or 100% sequence identity to a sequence set forth in Table C.   
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . The polypeptide of  claim 1 , wherein said CDR-H1 and said CDR-H2 of said first antigen binding fragment (AF1) comprise amino acid sequences of SEQ ID NOS: 8 and 9, respectively; and/or
 a. said CDR-L1 of said AF1 comprises an amino acid sequence of SEQ ID NO:1 or 2;
 said CDR-L2 of said AF1 comprises an amino acid sequence of SEQ ID NO:4 or 5; and 
 said CDR-L3 of said AF1 comprises an amino acid sequence of SEQ ID NO:6; 
   b. said CDR-L1 of said AF1 comprises an amino acid sequence of SEQ ID NO:1;
 said CDR-L2 of said AF1 comprises an amino acid sequence of SEQ ID NO:4 or 5; and 
 said CDR-L3 of said AF1 comprises an amino acid sequence of SEQ ID NO:6; 
   c. said CDR-L1 of said AF1 comprises an amino acid sequence of SEQ ID NO:2;
 said CDR-L2 of said AF1 comprises an amino acid sequence of SEQ ID NO:4 or 5; and 
 said CDR-L3 of said AF1 comprises an amino acid sequence of SEQ ID NO:6; 
   d. said CDR-L1 of said AF1 comprises an amino acid sequence of SEQ ID NO:1;
 said CDR-L2 of said AF1 comprises an amino acid sequence of SEQ ID NO:4; and 
 said CDR-L3 of said AF1 comprises an amino acid sequence of SEQ ID NO:6; or 
   e. said CDR-L1 of said AF1 comprises an amino acid sequence of SEQ ID NO:2;
 said CDR-L2 of said AF1 comprises an amino acid sequence of SEQ ID NO:5; and 
 said CDR-L3 of said AF1 comprises an amino acid sequence of SEQ ID NO:6. 
   
     
     
         33 - 37 . (canceled) 
     
     
         38 . The polypeptide of  claim 1 , wherein said first antigen binding fragment (AF1):
 a. comprises four chain variable domain framework regions 1 (FR-H1), 2 (FR-H2), 3 (FR-H3), and 4 (FR-H4), each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or being identical to an amino acid sequence of:
 i. SEQ ID NOS: 60, 64, 65, and 67, respectively; or 
 ii. SEQ ID NOS: 61, 64, 65, and 67, respectively; 
   b. comprises four light chain variable domain framework regions (FR-L): FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or being identical to amino acid sequences of:
 i. SEQ ID NOS: 51, 52, 53, and 59, respectively; 
 ii. SEQ ID NOS: 51, 52, 54, and 59, respectively; 
 iii. SEQ ID NOS: 51, 52, 55, and 59, respectively; 
 iv. SEQ ID NOS: 51, 52, 56, and 59, respectively; and/or 
   c. further comprises:
 i. 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:
 said FR-L1 comprises an amino acid sequence of SEQ ID NO:51; 
 said FR-L2 comprises an amino acid sequence of SEQ ID NO:52; 
 said FR-L3 comprises an amino acid sequence of SEQ ID NO:53, 54, 55, or 56; 
 said FR-L4 comprises an amino acid sequence of SEQ ID NO:59; 
 said FR-H1 comprises an amino acid sequence of SEQ ID NO:60 or 61; 
 said FR-H2 comprises an amino acid sequence of SEQ ID NO:64; 
 said FR-H3 comprises an amino acid sequence of SEQ ID NO:65; and 
 said FR-H4 comprises an amino acid sequence of SEQ ID NO:67; 
 
 ii. 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:
 said FR-L1 comprises an amino acid sequence of SEQ ID NO:51; 
 said FR-L2 comprises an amino acid sequence of SEQ ID NO:52; 
 said FR-L3 comprises an amino acid sequence of SEQ ID NO:53; 
 said FR-L4 comprises an amino acid sequence of SEQ ID NO:59; 
 said FR-H1 comprises an amino acid sequence of SEQ ID NO:60; 
 said FR-H2 comprises an amino acid sequence of SEQ ID NO:64; 
 said FR-H3 comprises an amino acid sequence of SEQ ID NO:65; and 
 said FR-H4 comprises an amino acid sequence of SEQ ID NO:67; 
 
 iii. 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:
 said FR-L1 comprises an amino acid sequence of SEQ ID NO:51; 
 said FR-L2 comprises an amino acid sequence of SEQ ID NO:52; 
 said FR-L3 comprises an amino acid sequence of SEQ ID NO:54; 
 said FR-L4 comprises an amino acid sequence of SEQ ID NO:59; 
 said FR-H1 comprises an amino acid sequence of SEQ ID NO:61; 
 said FR-H2 comprises an amino acid sequence of SEQ ID NO:64; 
 said FR-H3 comprises an amino acid sequence of SEQ ID NO:65; and 
 said FR-H4 comprises an amino acid sequence of SEQ ID NO:67; 
 
 iv. 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:
 said FR-L1 comprises an amino acid sequence of SEQ ID NO:51; 
 said FR-L2 comprises an amino acid sequence of SEQ ID NO:52; 
 said FR-L3 comprises an amino acid sequence of SEQ ID NO:55; 
 said FR-L4 comprises an amino acid sequence of SEQ ID NO:59; 
 said FR-H1 comprises an amino acid sequence of SEQ ID NO:61; 
 said FR-H2 comprises an amino acid sequence of SEQ ID NO:64; 
 said FR-H3 comprises an amino acid sequence of SEQ ID NO:65; and 
 said FR-H4 comprises an amino acid sequence of SEQ ID NO:67; 
 
 v. 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:
 said FR-L1 comprises an amino acid sequence of SEQ ID NO:51; 
 said FR-L2 comprises an amino acid sequence of SEQ ID NO:52; 
 said FR-L3 comprises an amino acid sequence of SEQ ID NO:56; 
 said FR-L4 comprises an amino acid sequence of SEQ ID NO:59; 
 said FR-H1 comprises an amino acid sequence of SEQ ID NO:61; 
 said FR-H2 comprises an amino acid sequence of SEQ ID NO:64; 
 said FR-H3 comprises an amino acid sequence of SEQ ID NO:65; and 
 said FR-H4 comprises an amino acid sequence of SEQ ID NO:67. 
 
   
     
     
         39 - 48 . (canceled) 
     
     
         49 . The polypeptide of  claim 1 , wherein said first antigen binding fragment (AF1) exhibits a higher thermal stability than an anti-CD3 binding fragment consisting of a sequence set forth in SEQ ID NO: 206, as evidenced in an in vitro assay by:
 a higher melting temperature (T m ) of said first antigen binding fragment relative to that of said anti-CD3 binding fragment; or   upon incorporating said first antigen binding fragment into a test bispecific antigen binding construct, a higher T m  of said test bispecific antigen binding construct relative to that of a control bispecific antigen binding construct, wherein said test bispecific antigen binding construct comprises said first antigen binding fragment and a reference antigen binding fragment that binds to an antigen other than CD3; and wherein said control bispecific antigen binding construct consists of said anti-CD3 binding fragment consisting of said sequence of SEQ ID NO:206 and said reference antigen binding fragment, optionally wherein said Tm of said first antigen binding fragment is at least 2° C. greater, or at least 3° C. greater, or at least 4° C. greater, or at least 5° C. greater than said Tm of said anti-CD3 binding fragment consisting of said sequence of SEQ ID NO:206.   
     
     
         50 . (canceled) 
     
     
         51 . The polypeptide of  claim 1 , wherein:
 a. said first antigen binding fragment (AF1) comprises a heavy chain variable region (VH I ), wherein said VH I  comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical to an amino acid sequence of SEQ ID NO:102 or 105,   b. said first antigen binding fragment (AF1) comprises a light chain variable region (VL I ), wherein said VL I  comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS: 101, 103, 104, 106, or 107: optionally wherein:
 i. said VHI and said VLI is linked by a linker comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in Table A; 
   c. said first antigen binding fragment (AF1) comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity or being identical to an amino acid sequence of any one of SEQ ID NOS: 201-205;   d. said first antigen binding fragment (AF1) specifically binds human or cynomolgus monkey (cyno) CD3, optionally wherein said first antigen binding fragment (AF1) specifically binds human CD3, wherein said first antigen binding fragment (AF1) binds a CD3 complex subunit identified herein by CD3 epsilon, CD13 delta, CD3 gamma, or CD3 zeta unit of CD3;   e. said first antigen binding fragment (AF1) binds a CD3 epsilon fragment of CD3;   f. said first antigen binding fragment (AF1) exhibits an isoelectric point (pI) that is less than or equal to 6.6: optionally wherein said first antigen binding fragment (AF1) exhibits an isoelectric point (pI) that is between 6.0 and 6.6, inclusive;   g. said first antigen binding fragment (AF1) exhibits an isoelectric point (pI) that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the pI of a reference antigen binding fragment consisting of a sequence shown in SEQ ID NO: 206;   h. said first antigen binding fragment (AF1) specifically binds human or cyno CD3 with a dissociation constant (Kd) constant between about between about 10 nM and about 400 nM, as determined in an in vitro antigen-binding assay comprising a human or cyno CD3 antigen;   i. said first antigen binding fragment (AF1) specifically binds human or cyno CD3 with a dissociation constant (Kd) of less than about 10 nM, or less than about 50 nM, or less than about 100 nM, or less than about 150 nM, or less than about 200 nM, or less than about 250 nM, or less than about 300 nM, or less than about 350 nM, or less than about 400 nM, as determined in an in vitro antigen-binding assay; and/or   j. said first antigen binding fragment (AF1) exhibits a binding affinity to CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker relative to that of an antigen binding fragment consisting of an amino acid sequence of SEQ ID NO:206, as determined by the respective dissociation constants (Kd) in an in vitro antigen-binding assay.   
     
     
         52 - 64 . (canceled) 
     
     
         65 . The polypeptide of  claim 1 , wherein:
 a. said first antigen binding fragment (AF1) is:
 i. a chimeric or a humanized antigen binding fragment; and/or 
 ii. is an Fv, Fab, Fab′, Fab′-SH, linear antibody, or single-chain variable fragment (scFv), 
   b. said second antigen binding fragment (AF2) is an Fv, Fab, Fab′, Fab′-SH, linear antibody, a single domain antibody, or single-chain variable fragment (scFv);   c. said first and second antigen binding fragments are configured as an (Fab′)2 or a single chain diabody;   d. said second antigen binding fragment (AF2) comprises:
 a heavy chain variable region (VH II ) comprising an amino acid sequence identified herein by SEQ ID NOS: 778-783, and 
 a light chain variable region (VL II ) comprising an amino acid sequence identified herein by SEQ ID NOS: 878-883; and/or 
   e. said VH II  and said VL II  is linked by a linker comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in Table A.   
     
     
         66 - 70 . (canceled) 
     
     
         71 . The polypeptide of  claim 1 , wherein said first and second antigen binding fragments are fused together by a peptide linker, optionally wherein
 a. said peptide linker comprises 2 or 3 types of amino acids are glycine, serine, or proline; and/or   b. said peptide linker comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in Table B.   
     
     
         72 . (canceled) 
     
     
         73 . (canceled) 
     
     
         74 . The polypeptide of  claim 1 , wherein:
 said XTEN is a first extended recombinant polypeptide (XTEN1);   said plurality of non-overlapping sequence motifs, from which said XTEN1 is formed, is a first plurality of non-overlapping sequence motifs;   said BAR is a first barcode fragment (BAR1); and   said RS is a first release segment (RS1); and   the polypeptide further comprising:   (d) a second extended recombinant polypeptide (XTEN2), comprising:
 a second barcode fragment (BAR2) releasable from said polypeptide upon digestion by said protease; and 
   (e) a second release segment (RS2) positioned between said second XTEN (XTEN2) and said bispecific antibody construct (BsAb),   wherein said XTEN2 is characterized in that:
 (i) it comprises at least 100, or at least 150 amino acids; 
 (ii) at least 90% of its amino acid residues are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); and 
 (iii) it comprises at least 4 different types of amino acids are G, A, S, T, E, or P; 
   
       wherein said second barcode fragment (BAR2) differs in sequence and molecular weight from all other peptides fragments that are releasable from said polypeptide upon complete digestion of said polypeptide by said protease; and 
       wherein said second barcode fragment (BAR2) does not include said N-terminal amino acid or said C-terminal amino acid of said polypeptide, optionally wherein
 a. said XTEN1 is positioned N-terminal of said bispecific antibody construct and said XTEN2 is positioned C-terminal of said bispecific antibody construct; 
 b. said XTEN1 is positioned C-terminal of said bispecific antibody construct and said XTEN2 is positioned N-terminal of said bispecific antibody construct. 
 
     
     
         75 . (canceled) 
     
     
         76 . (canceled) 
     
     
         77 . The polypeptide of  claim 74 , wherein:
 (iv) said XTEN2 is formed from a second plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acids in length, wherein said second plurality of non-overlapping sequence motifs comprise:
 (1) a second set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of said second set of non-overlapping sequence motifs is repeated at least two times in said second XTEN; and 
 (2) a non-overlapping sequence motif that occurs only once within said second XTEN; and 
   wherein said second barcode fragment (BAR2) includes at least part of said non-overlapping sequence motif that occurs only once within said second XTEN; optionally wherein
 i. said second set of non-overlapping sequence motifs are each independently identified herein by SEQ ID NOS: 179-200 and 1715-1722; or 
 ii. said second set of non-overlapping sequence motfis are each independently identified herein by SEQ ID Nos: 186-189. 
   
     
     
         78 . (canceled) 
     
     
         79 . (canceled) 
     
     
         80 . The polypeptide of  claim 77 , wherein:
 a. said second set of non-overlapping sequence motifs comprise at least two, at least three, or all four of the sequence motifs SEQ ID NOS: 186-189;   b. said XTEN2 comprises a length of from 100 to 3,000, from 150 to 3,000, from 100 to 1,000, or from 150 to 1,000 amino acid residues;   c. said XTEN2 comprises a length of at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acid residues; and/or   d. said XTEN2 has at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence set forth in Table 3a.   
     
     
         81 - 84 . (canceled) 
     
     
         85 . The polypeptide of  claim 74 , wherein:
 a. said second barcode fragment (BAR2) does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in said XTEN2;   b. said second barcode fragment (BAR2) has a glutamic acid at its C-terminus;   c. said second barcode fragment (BAR2) has an N-terminal amino acid that is immediately preceded by a glutamic acid residue;   d. second barcode fragment (BAR2) is positioned a distance from either said N-terminus of said polypeptide or said C-terminus of said polypeptide, wherein said distance is from 10 to 150 amino acids, or from 10 to 125 amino acids in length;   e. said second barcode fragment (BAR2) is characterized in that:
 i. it does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in said XTEN2 
 ii. it has a glutamic acid at its C-terminus; 
 iii. it has an N-terminal amino acid that is immediately preceded by a glutamic acid residue; and 
 iv. it is positioned a distance from either said N-terminus of said polypeptide or said C-terminus of said polypeptide, wherein said distance is from 10 to 150 amino acids, or from 10 to 125 amino acids in length; 
   f. said glutamic acid residue that precedes said N-terminal amino acid of said BAR2 is not immediately adjacent to another glutamic acid residue;   g. said second barcode fragment (BAR2) does not include a second glutamic acid residue at a position other than the C-terminus of said second barcode fragment (BAR2) unless said second glutamic acid is immediately followed by a proline;   h. said XTEN2 is positioned N-terminal of said bispecific antibody construct (BsAb), and wherein said second barcode fragment (BAR2) is positioned within 200, within 150, within 100, or within 50 amino acids of said N-terminus of said polypeptide;   i. said XTEN2 is positioned N-terminal of said bispecific antibody construct (BsAb), and wherein said second barcode fragment (BAR2) is positioned at a location that is between 10 and 200, between 30 and 200, between 40 and 150, or between 50 and 100 amino acids from said N-terminus of said protein;   j. said XTEN2 is positioned C-terminal of said bispecific antibody construct (BsAb), and wherein said second barcode fragment (BAR2) is positioned within 200, within 150, within 100, or within 50 amino acids of said C-terminus of said polypeptide;   k. said XTEN2 is positioned C-terminal of said bispecific antibody construct (BsAb), and said second barcode fragment (BAR2) is positioned at a location that is between 10 and 200, between 30 and 200, between 40 and 150, or between 50 and 100 amino acids from said C-terminus of said protein;   l. said second barcode fragment (BAR2) is at least 4 amino acids in length, optionally wherein said barcode fragment (BAR2) is between 4 and 20, between 5 and 15, between 6 and 12, or between 7 and 10 amino acids in length;   m. said second barcode fragment (BAR2) comprises an amino acid sequence set forth in Table 2;   n. said XTEN2 has a length defined by a proximal end and a distal end, wherein (1) said proximal end of said XTEN2 is positioned, relative to said distal end, closer to said bispecific antibody construct (BsAb), and wherein (2) said second barcode fragment (BAR2) is positioned within a region of said XTEN2 that extends, as measured from said distal end of said XTEN2, between 5% and 50%, between 7% and 40%, or between 10% and 30% of said length of said XTEN2; and/or;   o. said XTEN2 further comprises additional one or more barcode fragments, wherein said additional one or more barcode fragments of said XTEN2 each differ in sequence and molecular weight from all other peptides fragments that are releasable from said polypeptide upon complete digestion of said polypeptide by said protease.   
     
     
         86 - 100 . (canceled) 
     
     
         101 . The polypeptide of  claim 74 , wherein:
 a. said first release segment (RS1) and said second release segment (RS2) are identical in sequence;   b. said first release segment (RS1) and said second release segment (RS2) are not identical in sequence;   c. said second release segment (RS) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence identified herein by SEQ ID NOS: 7001-7626; and/or   d. said first releast segment (RS1) and said second release segment (RS2) are each a substrate for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence.   
     
     
         102 - 104 . (canceled) 
     
     
         105 . The polypeptide of  claim 74  expressed as a fusion protein, wherein:
 a. said fusion protein, in an uncleaved state, has a structural arrangement from N-terminus to C-terminus 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 
 
       said diabody comprises a light chain variable region (VL I ) of said AF1, a heavy chain variable region (VH I ) of said AF1, a light chain variable region (VL II ) of said AF2, and a heavy chain variable region (VH II ) of said AF2;
 b. said spacer of said first release segment (RS1) is a first spacer, and wherein said second release segment (RS2) is fused to said bispecific antibody construct (BsAb) by a second spacer, optionally wherein
 i. said second spacer comprises at least 4 types of amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); or 
 ii. said second spacer comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in Table C. 
 
 
     
     
         106 - 108 . (canceled) 
     
     
         109 . The polypeptide of  claim 74 , wherein:
 (a) said XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3a;   (b) said BsAb comprises:
 (I) said AF1 comprising light chain complementarity-determining regions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3) and heavy chain complementarity-determining regions 1 (CDR-H1), 2 (CDR-H2), and 3 (CDR-H3), wherein said CDR-H1, said CDR-H2, and said CDR-H3 comprise amino acid sequences of SEQ ID NOS: 8, 9, and 10, respectively; and 
 (II) said AF2 comprising a light chain variable region (VL II ) identified herein by SEQ ID NOS: 778-783 and a heavy chain variable region (VH II ) identified herein by SEQ ID NOS: 878-883; 
   (c) said RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOS: 7001-7626;   (d) said XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 3a; and   (e) said RS2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence identified herein by SEQ ID NOS: 7001-7626,   wherein said polypeptide has a structural arrangement from N-terminus to C-terminus 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.   
     
     
         110 . The polypeptide of  claim 1 , wherein the polypeptide:
 a. has a terminal half-life that is at least two-fold longer compared to the bispecific antibody construct not linked to any XTEN;   b. is less immunogenic compared to the bispecific antibody construct not linked to any XTEN, as ascertained by measuring production of IgG antibodies that selectively bind to said bispecific antibody construct after administration of comparable doses to a subject;   c. exhibits an apparent molecular weight factor under physiological conditions that is greater than about 3, greater than about 4, greater than about 5, or greater than about 6; and/or   d. comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in Table D.   
     
     
         111 - 113 . (canceled) 
     
     
         114 . A pharmaceutical composition comprising a polypeptide having an N-terminal amino acid and a C-terminal amino acid, the polypeptide comprising:
 a. an extended recombinant polypeptide (XTEN), comprising:
 a barcode fragment (BAR) releasable from said polypeptide upon digestion by a protease; 
   b. a bispecific antibody construct (BsAb), comprising:
 a first antigen binding fragment (AF1) that specifically binds to cluster of differentiation 3 T cell receptor (CD3), which 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), wherein said CDR-H3 comprises an 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) positioned between said XTEN and said bispecific antibody construct,
 wherein said XTEN is characterized in that: 
 (i) it comprises at least 100, or at least 150 amino acids; 
 (ii) at least 90% of its amino acid residues are identified herein by glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); 
 (iii) it comprises at least 4 different types of amino acids identified herein by G, A, S, T, E, or P; and 
 (iv) said XTEN is formed from a plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acids in length, 
 wherein said plurality of non-overlapping sequence motifs comprise:
 (1) a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of said set of non-overlapping sequence motifs is repeated at least two times in said XTEN; and 
 (2) a non-overlapping sequence motif that occurs only once within said XTEN; 
 
   
       wherein said barcode fragment (BAR) includes at least part of said non-overlapping sequence motif that occurs only once within said XTEN; 
       wherein said barcode fragment (BAR) differs in sequence and molecular weight from all other peptides fragments that are releasable from said polypeptide upon complete digestion of said polypeptide by said protease; and
 wherein said barcode fragment (BAR) does not include said N-terminal amino acid or said C-terminal amino acid of said polypeptide; 
 
       and one or more pharmaceutically suitable excipients. 
     
     
         115 . The pharmaceutical composition of  claim 114 , wherein said pharmaceutical composition:
 a. is formulated for intradermal, subcutaneous, oral, intravenous, intra-arterial, intraabdominal, intraperitoneal, intrathecal, or intramuscular administration;   b. is in a liquid form or frozen;   c. is in a pre-filled syringe for a single injection, optionally wherein said pharmaceutical composition is formulated as a lyophilized powder to be reconstituted prior to administration; and/or   d. is in a pharmaceutical combination comprising at least one additional therapeutic agent selected form the group consisting of an antibody, an antibody fragment, an antibody conjugate, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof; optionally wherein:
 i. said additional therapeutic agent is a PD-1/PD-L1(2) inhibitor; 
 ii. the PD-1/PD-L1(2) inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody; 
 iii. the PD-1/PD-L1(2) is an anti-PD-1 antibody selected from the group comprising nivolumab (Opdivo, BMS-936558, MDX1106), pembrolizumab (Keytruda, MK-3475, lambrolizumab), pidilizumab (CT-011), PDR-001, JS001, STI-A1110, AMP-224 and AMP-514 (MEDI0680); and/or 
 iv. the PD-1/PD-L1(2) inhibitor is an anti-PD-L1 antibody selected from the group comprising atezolizumab (Tecentriq, MPDL3280A), durvalumab (MEDI4736), avelumab (MSB0010718C), BMS-936559 (MDX11105) and LY3300054. 
   
     
     
         116 - 123 . (canceled) 
     
     
         124 . The pharmaceutical composition of  claim 114 , wherein:
 a. the PD-1/PD-L1(2) inhibitor is an anti-PD-L2 antibody;   b. the components are administered in separate dosage forms simultaneously or sequentially for use in the treatment of the same disease;   c. are for use as medicament for treating hyper-proliferative disorder, optionally wherein the hyper-proliferative disorders are selected from the group consisting of cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases.   
     
     
         125 - 130 . (canceled) 
     
     
         131 . A method of treating a disease in a subject, comprising administering to said subject in need thereof one or more therapeutically effective doses of a pharmaceutical composition comprising a polypeptide comprising having an N-terminal amino acid and a C-terminal amino acid, the polypeptide comprising:
 a. an extended recombinant polypeptide (XTEN), comprising:
 a barcode fragment (BAR) releasable from said polypeptide upon digestion by a protease; 
   b. a bispecific antibody construct (BsAb), comprising:
 a first antigen binding fragment (AF1) that specifically binds to cluster of differentiation 3 T cell receptor (CD3), which 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), wherein said CDR-H3 comprises an 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) positioned between said XTEN and said bispecific antibody construct,
 wherein said XTEN is characterized in that: 
 (i) it comprises at least 100, or at least 150 amino acids; 
 (ii) at least 90% of its amino acid residues are identified herein by glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); 
 (iii) it comprises at least 4 different types of amino acids identified herein by G, A, S, T, E, or P; and 
 (iv) said XTEN is formed from a plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acids in length, 
 wherein said plurality of non-overlapping sequence motifs comprise:
 (1) a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of said set of non-overlapping sequence motifs is repeated at least two times in said XTEN; and 
 (2) a non-overlapping sequence motif that occurs only once within said XTEN; 
 
   
       wherein said barcode fragment (BAR) includes at least part of said non-overlapping sequence motif that occurs only once within said XTEN;
 wherein said barcode fragment (BAR) differs in sequence and molecular weight from all other peptides fragments that are releasable from said polypeptide upon complete digestion of said polypeptide by said protease; and 
 wherein said barcode fragment (BAR) does not include said N-terminal amino acid or said C-terminal amino acid of said polypeptide. 
 
     
     
         132 . The method of  claim 131 , wherein:
 a. said disease is cancer;   b. said cancer is selected from the group consisting of glioblastoma, melanoma, cholangio carcinoma, small cell lung cancer, colorectal cancer, prostate cancer, vaginal cancer, angiosarcoma, non-small cell lung cancer, appendiceal cancer, squamous cell cancer, salivary duct carcinoma, adenoid cystic carcinoma, small intestine cancer, and gallbladder cancer;   c. the pharmaceutical composition is administered to the subject as one or more therapeutically effective doses;   d. the pharmaceutical composition is administered to the subject as one or more therapeutically effective doses over an effective dosing period; and/or   e. the subject is a mouse, rate, monkey, or human.   
     
     
         133 - 136 . (canceled) 
     
     
         137 . A nucleic acid comprising (a) a polynucleotide sequence encoding a polypeptide having an N-terminal amino acid and a C-terminal amino acid, the polypeptide comprising:
 a. an extended recombinant polypeptide (XTEN), comprising:
 a barcode fragment (BAR) releasable from said polypeptide upon digestion by a protease; 
   b. a bispecific antibody construct (BsAb), comprising:
 a first antigen binding fragment (AF1) that specifically binds to cluster of differentiation 3 T cell receptor (CD3), which 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), wherein said CDR-H3 comprises an 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) positioned between said XTEN and said bispecific antibody construct,
 wherein said XTEN is characterized in that: 
 (i) it comprises at least 100, or at least 150 amino acids; 
 (ii) at least 90% of its amino acid residues are identified herein by glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); 
 (iii) it comprises at least 4 different types of amino acids identified herein by G, A, S, T, E, or P; and 
 (iv) said XTEN is formed from a plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acids in length, 
 wherein said plurality of non-overlapping sequence motifs comprise:
 (1) a set of non-overlapping sequence motifs, wherein each non-overlapping sequence motif of said set of non-overlapping sequence motifs is repeated at least two times in said XTEN; and 
 (2) a non-overlapping sequence motif that occurs only once within said XTEN; 
 
   
       wherein said barcode fragment (BAR) includes at least part of said non-overlapping sequence motif that occurs only once within said XTEN;
 wherein said barcode fragment (BAR) differs in sequence and molecular weight from all other peptides fragments that are releasable from said polypeptide upon complete digestion of said polypeptide by said protease; and 
 wherein said barcode fragment (BAR) does not include said N-terminal amino acid or said C-terminal amino acid of said polypeptide; or (b) a reverse complement of said polynucleotide sequence of (a). 
 
     
     
         138 . An expression vector comprising said polynucleotide sequence of  claim 137  and a recombinant regulatory sequence operably linked to said polynucleotide sequence. 
     
     
         139 . A host cell, comprising said expression vector of  claim 138 , optionally wherein the host cell is a prokaryote,  E. coli , or a mammalian cell. 
     
     
         140 - 142 . (canceled)