EGFR antigen-binding fragments and compositions comprising the same

Abstract

To provide a bispecific antibody effective for use in cancer therapy.SOLUTION: Provided is a polypeptide comprising an antibody-binding fragment (AF1), wherein said AF1 comprises a light chain complementary determining region (CDR-L), a heavy chain complementary determining region (CDR-H), a light chain framework region (FR-L), and a heavy chain framework region (FR-H), wherein said AF1 a. specifically binds to epidermal growth factor-receptor (EGFR), and b. comprises FR-LA H1, FR-LA H2, FR-LA H3, and FR-LA H4, each having a specific amino acid sequence.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] CROSS-REFERENCE This application claims the benefit of U.S. Provisional Patent Application No. 62 / 866,749, filed June 26, 2019, and U.S. Provisional Patent Application No. 63 / 043,486, filed June 24, 2020, both of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on June 25, 2020, is named 32808-779_601_SL.txt and is 2,131,102 bytes in size. [Background technology]

[0003] Many approved cancer treatment drugs are cytotoxic drugs that kill both normal cells and tumor cells.The therapeutic benefit of these cytotoxic drugs depends on the higher sensitivity of tumor cells than normal cells, which allows them to achieve clinical response using a dose that does not cause unacceptable side effects.However, essentially all of these non-specific drugs cause some damage to normal tissue, even if it is not serious, and in many cases, limit their therapeutic suitability.

[0004] Bispecific antibodies can offer a different approach to cytotoxic drugs by directing immune effector cells to kill cancer cells. Bispecific antibodies combine the advantages of different binding specificities from two monoclonal antibodies into a single composition, allowing for combinations of approaches or applications not possible with monospecific antibodies. In one embodiment, this approach relies on one arm of the bispecific antibody binding to a tumor-associated antigen or marker, while the other arm, upon binding to CD3 molecules on T cells, triggers their cytotoxic activity by releasing effector molecules such as TNF-α, IFN-γ, interleukins 2, 4, and 10, perforin, and granzymes. Advances in antibody engineering have led to the development of several bispecific antibody formats and compositions for redirecting effector cells to tumor targets, including bispecific antibodies that function by recruiting and activating polyclonal T cell populations at tumor sites and do so without the need for costimulation or traditional MHC recognition. However, in addition to the fact that some bispecific compositions have very short half-lives, require continuous infusion for 4-8 weeks to maintain blood levels within a therapeutic window long enough to achieve a therapeutic effect, or have variable efficacy, there remains the dual problem of certain patients experiencing a severe side effect termed "cytokine storm" or "cytokine release syndrome" (Lee DW et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014 124(2):188-195), which is mediated by the release of TNF-α and IFN-γ, among other cytokines. Thus, there remains an unmet need in the art for the development of effective bispecific antibodies for use in cancer therapy. Summary of the Invention

[0005] The present invention relates to an anti-epidermal growth factor receptor (EGFR) antigen-binding fragment incorporated into a chimeric fusion protein and methods of using or making the same. In one aspect, a polypeptide comprising an antibody-binding fragment (AF1) is disclosed herein, wherein AF1 comprises a light chain complementarity-determining region (CDR-L), a heavy chain complementarity-determining region (CDR-H), a light chain framework region (FR-L), and a heavy chain framework region (FR-H), and AF1 specifically binds to the epidermal growth factor receptor (EGFR), and comprises FR-H1, FR-H2, FR-H3, and FR-H4, wherein FR-H1 has the amino acid sequence of any one of SEQ ID NOs: 14 to 16, FR-H2 has the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19, FR-H3 has the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, and FR-H4 has the amino acid sequence of any one of SEQ ID NOs: 22 to 24. In some embodiments, AF1 comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with, or is identical to, the amino acid sequence of any one of SEQ ID NOs: 37-51. In certain embodiments, AF1 is a chimeric or humanized antigen-binding fragment. In one embodiment, AF1 is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, a linear antibody, and a single-chain variable fragment (scFv).

[0006] In another embodiment, AF1 comprises a variable heavy (VH) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of SEQ ID NOs: 28-32. In certain embodiments, AF1 comprises a variable light (VL) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of SEQ ID NOs: 25-27.

[0007] In some embodiments, AF1 further comprises CDR-H3, wherein CDRH3 has the amino acid sequence of SEQ ID NO: 6. In certain embodiments, AF1 further comprises CDR-H1, CDR-H2, and CDR-H3, which have the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. In certain embodiments, AF1 comprises CDR-L1, CDR-L2, and CDR-L3, which comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively.

[0008] In other embodiments, AF1 further comprises FR-L, including FR-L1, FR-L2, FR-L3, and FR-L4, wherein a. FR-L1 exhibits at least 90%, or at least 95%, sequence identity to, or is identical to, the amino acid sequence of SEQ ID NO: 7; b. FR-L2 exhibits at least 90%, or at least 95% sequence identity to, or is identical to, the amino acid sequence of SEQ ID NO: 8; c. FR-L3 exhibits at least 90%, or at least 95% sequence identity to, or is identical to, the amino acid sequence of SEQ ID NO: 9-11; and d. FR-L4 exhibits at least 90%, or at least 95% sequence identity to, or is identical to, the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the FR-L comprises a. FR-L1 having the amino acid sequence of SEQ ID NO: 7, b. FR-L2 having the amino acid sequence of SEQ ID NO: 8, c. FR-L3 having the amino acid sequence of SEQ ID NO: 9, and d. FR-L4 having the amino acid sequence of SEQ ID NO: 13. In another embodiment, the FR-L comprises a. FR-L1 having the amino acid sequence of SEQ ID NO: 7, b. FR-L2 having the amino acid sequence of SEQ ID NO: 8, c. FR-L3 having the amino acid sequence of SEQ ID NO: 10, and d. FR-L4 having the amino acid sequence of SEQ ID NO: 13. In yet another embodiment, the FR-L comprises a. FR-L1 having the amino acid sequence of SEQ ID NO: 7, b. FR-L2 having the amino acid sequence of SEQ ID NO: 8, c. FR-L3 having the amino acid sequence of SEQ ID NO: 11, and d. FR-L4 having the amino acid sequence of SEQ ID NO: 13.

[0009] In one embodiment, the FR-H comprises a. FR-H1 having the amino acid sequence of SEQ ID NO: 14, b. FR-H2 having the amino acid sequence of SEQ ID NO: 18, b. FR-H3 having the amino acid sequence of SEQ ID NO: 20, and c. FR-H4 having the amino acid sequence of SEQ ID NO: 22 or 23. In other embodiments, the FR-H comprises a. FR-H1 having the amino acid sequence of SEQ ID NO: 15, b. FR-H2 having the amino acid sequence of SEQ ID NO: 19, c. FR-H3 having the amino acid sequence of SEQ ID NO: 21, and d. FR-H4 having the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the FR-H comprises a. FR-H1 having the amino acid sequence of SEQ ID NO: 16, b. FR-H2 having the amino acid sequence of SEQ ID NO: 19, c. FR-H3 having the amino acid sequence of SEQ ID NO: 20, and d. FR-H4 having the amino acid sequence of SEQ ID NO: 22 or 23. In some embodiments, AF1 has at least one or at least two amino acid substitutions of hydrophobic amino acids in the framework regions relative to the amino acid sequence of SEQ ID NO: 52, wherein the hydrophobic amino acids are selected from isoleucine, leucine, or methionine, and the replacement amino acids are selected from arginine, threonine, or glutamine.

[0010] In one embodiment, the polypeptide further comprises a first release segment peptide (RS1) and / or a first extended recombinant polypeptide (XTEN1), wherein RS1 is a substrate for cleavage by a mammalian protease. In some embodiments, the fusion protein has, from N-terminus to C-terminus, the structural arrangement of AF1-RS1-XTEN1 or XTEN1-RS1-AF1 in the uncleaved state.

[0011] In some embodiments, RS1 is a substrate for a protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other embodiments, RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 53-671. In certain embodiments, RS1 comprises an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which is set forth in Table 5.

[0012] In some embodiments, the polypeptides disclosed herein further comprise a first extended recombinant polypeptide (XTEN1), wherein XTEN1 is characterized in that: a. it has at least about 36 amino acids; b. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues in the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P); and c. it has at least 4-6 different amino acids selected from G, A, S, T, E, and P. In certain embodiments, XTEN1 comprises an amino acid sequence comprising at least three of the amino acid sequences of SEQ ID NOs: 672-675. In another 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 to a sequence selected from any one of SEQ ID NOs: 676-734. In certain embodiments, 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 selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7.

[0013] In certain embodiments, AF1 has a higher isoelectric point (pI) relative to the isoelectric point (pI) of the antigen-binding fragment consisting of the sequence set forth in SEQ ID NO: 52. In one embodiment, AF1 is incorporated into a polypeptide to form an anti-EGFR bispecific antibody, where the polypeptide exhibits a higher pI relative to a control bispecific antibody, the polypeptide comprising AF1 and a reference antigen-binding fragment that binds to cluster of differentiation 3 T-cell receptor (CD3), the control bispecific antigen-binding fragment being identical to the polypeptide except for replacing AF1 with SEQ ID NO: 52. In another embodiment, AF1 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units higher than the pI of the antigen-binding fragment consisting of the sequence set forth in SEQ ID NO: 52. In certain embodiments, AF1 exhibits a pI that is 5.4 to 6.6, inclusive. In other embodiments, AF1 exhibits a pI of about 5.4 to about 5.6, or about 5.5 to about 5.7, or about 5.6 to about 5.8, or about 5.7 to about 5.9, or about 5.8 to about 6.0, or about 5.9 to about 6.1, or about 6.0 to about 6.2, or about 6.1 to about 6.3, or about 6.2 to about 6.4, or about 6.3 to about 6.5, or about 6.4 to about 6.6. In further embodiments, AF1 exhibits a pI of about 5.4, about 5.5, or about 5.6, or about 5.7, or about 5.8, or about 5.9, or about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6.

[0014] In certain embodiments, AF1 specifically binds to human or cynomolgus monkey (cyno) EGFR. In other embodiments, AF1 specifically binds to human and cynomolgus monkey (cyno) EGFR. In some embodiments, AF1 has a K of about 0.1 nM to about 100 nM as determined in an in vitro antigen binding assay involving EGFR or an epitope thereof. d It specifically binds to EGFR.

[0015] In another embodiment, the polypeptide further comprises a second antigen-binding fragment (AF2) that specifically binds to cluster of differentiation 3 T-cell receptor (CD3). In certain embodiments, (1) the AF2 fragment is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, linear antibody, single domain antibody, and single-chain variable fragment (scFv), or (2) AF1 and AF2 are configured as (Fab')2 or a single-chain diabody.

[0016] In some embodiments, AF2 is fused to AF1 by a flexible peptide linker. In certain embodiments, the flexible linker comprises two or three types of amino acids selected from the group consisting of glycine, serine, and proline.

[0017] In some embodiments, AF2 comprises a variable heavy (VH) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 766 or SEQ ID NO: 769. In certain embodiments, AF2 comprises a variable light (VL) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of any one of SEQ ID NOs: 765, 767, 768, 770, or 771. In other embodiments, AF2 comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of any one of SEQ ID NOs: 776-780.

[0018] In certain embodiments, AF2 comprises a light chain complementarity determining region (CDR-L) and a heavy chain complementarity determining region (CDR-H), and the antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3, having the amino acid sequences of SEQ ID NOs: 742, 743, and 744, respectively. In some embodiments, the CDR-L comprises a. CDR-L1 having the amino acid sequence of SEQ ID NO: 735 or 736, b. CDR-L2 having the amino acid sequence of SEQ ID NO: 738 or 739, and c. CDR-L3 having the amino acid sequence of SEQ ID NO: 740.

[0019] In other embodiments, AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and AF2 comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 746; b. FR-L2 having the amino acid sequence of SEQ ID NO: 747; c. FR-L3 having the amino acid sequence of any one of SEQ ID NOs: 748 to 751; d. FR-L4 having the amino acid sequence of SEQ ID NO: 754; e. FR-H1 having the amino acid sequence of SEQ ID NO: 755 or SEQ ID NO: 756; f. FR-H2 having the amino acid sequence of SEQ ID NO: 759; g. FR-H3 having the amino acid sequence of SEQ ID NO: 760; and h. FR-H4 having the amino acid sequence of any one of SEQ ID NOs: 764. In another embodiment, the antigen-binding fragment comprises a. FR-L1 having the amino acid sequence of SEQ ID NO: 746, b. FR-L2 having the amino acid sequence of SEQ ID NO: 747, c. FR-L3 having the amino acid sequence of SEQ ID NO: 748, d. FR-L4 having the amino acid sequence of SEQ ID NO: 754, e. FR-H1 having the amino acid sequence of SEQ ID NO: 755, f. FR-H2 having the amino acid sequence of SEQ ID NO: 759, g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 764. In yet another embodiment, the antigen-binding fragment comprises a. FR-L1 having the amino acid sequence of SEQ ID NO: 746, b. FR-L2 having the amino acid sequence of SEQ ID NO: 747, c. FR-L3 having the amino acid sequence of SEQ ID NO: 749, d. FR-L4 having the amino acid sequence of SEQ ID NO: 754, e. FR-H1 having the amino acid sequence of SEQ ID NO: 756, f. FR-H2 having the amino acid sequence of SEQ ID NO: 759, g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 764.In certain embodiments, the antigen-binding fragment comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 746; b. FR-L2 having the amino acid sequence of SEQ ID NO: 747; c. FR-L3 having the amino acid sequence of SEQ ID NO: 750; d. FR-L4 having the amino acid sequence of SEQ ID NO: 754; e. FR-H1 having the amino acid sequence of SEQ ID NO: 756; f. FR-H2 having the amino acid sequence of SEQ ID NO: 759; g. FR-H3 having the amino acid sequence of SEQ ID NO: 760; and h. FR-H4 having the amino acid sequence of SEQ ID NO: 764. In yet another embodiment, the antigen-binding fragment comprises a. FR-L1 having the amino acid sequence of SEQ ID NO: 746, b. FR-L2 having the amino acid sequence of SEQ ID NO: 747, c. FR-L3 having the amino acid sequence of SEQ ID NO: 751, d. FR-L4 having the amino acid sequence of SEQ ID NO: 754, e. FR-H1 having the amino acid sequence of SEQ ID NO: 756, f. FR-H2 having the amino acid sequence of SEQ ID NO: 759, g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 764.

[0020] In some embodiments, the polypeptide further comprises a second release segment peptide (RS2) and / or a second extended recombinant polypeptide (XTEN2), wherein RS2 is a substrate for cleavage by a mammalian protease. In some embodiments, the sequences of RS1 and RS2 are identical. In other embodiments, the sequences of RS1 and RS2 are not identical.

[0021] In some embodiments, the polypeptide has the following structural arrangement, from N-terminus to C-terminus: XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-diabody-RS1-XTEN1, or XTEN1-RS1-diabody-RS2-XTEN2, wherein the diabody comprises the VL and VH of AF1 and AF2, wherein AF2 specifically binds CD3 and AF1 specifically binds EGFR, and wherein XTEN1 and XTEN2 are of the same or different amino acid length or sequence.

[0022] In certain embodiments, RS2 is a substrate for a protease selected from legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other embodiments, RS2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 53-671. In certain embodiments, RS1 and RS2 are each substrates for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence.

[0023] In some embodiments, XTEN2 is characterized in that a. it has at least about 36 amino acids, b. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), and c. it has at least 4-6 different amino acids selected from G, A, S, T, E, and P. In certain embodiments, 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 selected from SEQ ID NOs: 676-734. In other embodiments, 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 selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7. In certain embodiments, XTEN2 comprises an amino acid sequence comprising at least three of the amino acid sequences of SEQ ID NOs: 672-675.

[0024] In some embodiments, the T of AF2 m is the T of an antigen-binding fragment consisting of the sequence of SEQ ID NO: 781, as determined by an increase in melting temperature in an in vitro assay. m at least 2°C higher, or at least 3°C ​​higher, or at least 4°C higher, or at least 5°C higher, or at least 6°C higher, or at least 7°C higher, or at least 8°C higher, or at least 9°C higher, or at least 10°C higher.

[0025] In some embodiments, AF2 binds to a CD3 complex subunit selected from any one of CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta epsilon. In one embodiment, AF2 specifically binds to human or cynomolgus monkey (cyno) CD3. In yet another embodiment, AF2 specifically binds to human and cynomolgus monkey (cyno) CD3.

[0026] In other embodiments, AF2 has a dissociation constant (K) of about 10 nM to about 400 nM as determined in an in vitro antigen binding assay. d In certain embodiments, AF2 specifically binds to human or cyno CD3 with a dissociation constant (K) of about 10 nM to about 400 nM, or about 50 nM to about 350 nM, or about 100 nM to 300 nM, as determined in an in vitro antigen binding assay. d In certain embodiments, AF2 specifically binds to human or cyno CD3 with a dissociation constant (K) weaker than about 3 nM, or 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 400 nM, as determined in an in vitro antigen binding assay. d In another embodiment, AF2 specifically binds to human or cyno CD3 with a respective dissociation constant (K d ), specifically binds to human or cyno CD3 with a binding affinity 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 than an antibody-binding fragment consisting of the amino acid sequence of SEQ ID NO: 781. In yet another embodiment, the binding affinity of AF1 to EGFR is at least 10-fold greater, or at least 100-fold greater, or at least 1000-fold greater than the binding affinity of AF2 to CD3, as measured in an in vitro antigen binding assay.

[0027] In certain embodiments, AF2 exhibits an isoelectric point (pI) of 6.6 or less. In other embodiments, AF2 exhibits a pI of 5.5 to 6.6 (inclusive). In other embodiments, AF2 exhibits a pI of about 5.5 to 6.6, or about 5.6 to about 6.4, or about 5.8 to about 6.2, or about 6.0 to about 6.2. In some embodiments, AF2 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the pI of a reference antigen-binding fragment consisting of the sequence set forth in SEQ ID NO:781. In other embodiments, AF2 is within 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 AF1. In certain embodiments, AF2 exhibits a pI that is within at least about 0.1 to about 1.5, or at least about 0.3 to about 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 to about 0.9 pH units of the pI of AF1.

[0028] In another aspect, the present disclosure provides a bispecific antigen-binding unit comprising: a. a first antigen-binding fragment (AF1), where AF1 specifically binds to EGFR; and b. a second antigen-binding fragment (AF2), where AF2 specifically binds to cluster of differentiation 3 T-cell receptor (CD3), wherein the difference between the isoelectric point (pI) of the second antigen-binding fragment and the pI of the first antigen-binding fragment is 0 to about 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, as determined by in vitro assay. In some embodiments, AF1 comprises a variable heavy (VH) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NOs: 28-32. In other embodiments, AF1 comprises a variable light (VL) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NOs: 25-27. In certain embodiments, AF1 comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequence of any one of SEQ ID NOs: 37-51. In other embodiments, (1) each of the AF1 and AF2 fragments is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, linear antibody, single domain antibody, and single chain variable fragment (scFv), or (2) AF1 and AF2 are configured as (Fab')2 or single chain diabodies.

[0029] In some embodiments, AF1 of the bispecific antigen binding unit comprises a light chain complementarity determining region (CDR-L), a heavy chain complementarity determining region (CDR-H), a light chain framework region (FR-L), and a heavy chain framework region (FR-H), and AF1 comprises FR-H1, FR-H2, FR-H3, and FR-H4.

[0030] In other embodiments, AF1 further comprises CDR-H3, wherein CDRH3 has the amino acid sequence of SEQ ID NO: 6. In certain embodiments, AF1 further comprises CDR-H1, CDR-H2, and CDR-H3, which have the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. In certain embodiments, CDR-L comprises CDR-L1, CDR-L2, and CDR-L3, which comprise the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively.

[0031] In certain embodiments, FR-H1 has the amino acid sequence of any one of SEQ ID NOs: 14-16, FR-H2 has the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19, FR-H3 has the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, and FR-H4 has the amino acid sequence of any one of SEQ ID NOs: 22-24. In some embodiments, FR-H comprises FR-H1 having the amino acid sequence of SEQ ID NO: 14, FR-H2 having the amino acid sequence of SEQ ID NO: 18, FR-H3 having the amino acid sequence of SEQ ID NO: 20, and FR-H4 having the amino acid sequence of SEQ ID NO: 22 or 23. In other embodiments, FR-H comprises FR-H1 having the amino acid sequence of SEQ ID NO: 15, FR-H2 having the amino acid sequence of SEQ ID NO: 19, FR-H3 having the amino acid sequence of SEQ ID NO: 21, and FR-H4 having the amino acid sequence of SEQ ID NO: 24. In yet another embodiment, FR-H includes FR-H1 having the amino acid sequence of SEQ ID NO: 16, FR-H2 having the amino acid sequence of SEQ ID NO: 19, FR-H3 having the amino acid sequence of SEQ ID NO: 20, and FR-H4 having the amino acid sequence of SEQ ID NO: 22 or 23.

[0032] In certain embodiments, FR-L1 exhibits at least 90% or at least 95% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 7, FR-L2 exhibits at least 90% or at least 95% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 8, FR-L3 exhibits at least 90% or at least 95% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 9-11, and FR-L4 exhibits at least 90% or at least 95% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 13. In other embodiments, FR-L comprises FR-L1 having the amino acid sequence of SEQ ID NO: 7, FR-L2 having the amino acid sequence of SEQ ID NO: 8, FR-L3 having the amino acid sequence of SEQ ID NO: 9, and FR-L4 having the amino acid sequence of SEQ ID NO: 13. In yet another embodiment, the FR-L comprises FR-L1 having the amino acid sequence of SEQ ID NO: 7, FR-L2 having the amino acid sequence of SEQ ID NO: 8, FR-L3 having the amino acid sequence of SEQ ID NO: 10, and FR-L4 having the amino acid sequence of SEQ ID NO: 13. In another embodiment, the FR-L comprises FR-L1 having the amino acid sequence of SEQ ID NO: 7, FR-L2 having the amino acid sequence of SEQ ID NO: 8, FR-L3 having the amino acid sequence of SEQ ID NO: 11, and FR-L4 having the amino acid sequence of SEQ ID NO: 13.

[0033] In certain embodiments, AF2 of the bispecific antigen binding unit comprises a light chain complementarity determining region (CDR-L) and a heavy chain complementarity determining region (CDR-H), and the antigen binding unit comprises CDR-H1, CDR-H2, and CDR-H3, which have the amino acid sequences of SEQ ID NOs: 742, 743, and 744, respectively. In some embodiments, AF2 comprises a variable heavy (VH) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of SEQ ID NO: 766 or SEQ ID NO: 769. In other embodiments, AF2 comprises a variable light (VL) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of any one of SEQ ID NOs: 765, 767, 768, 770, or 771. In certain embodiments, AF2 comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of any one of SEQ ID NOs: 776-780.

[0034] In other embodiments, the CDR-L of AF2 includes CDR-L1 having the amino acid sequence of SEQ ID NO: 735 or 736, CDR-L2 having the amino acid sequence of SEQ ID NO: 738 or 739, and CDR-L3 having the amino acid sequence of SEQ ID NO: 740.

[0035] In other embodiments, AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and AF2 comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 746; b. FR-L2 having the amino acid sequence of SEQ ID NO: 747; c. FR-L3 having the amino acid sequence of any one of SEQ ID NOs: 748 to 751; d. FR-L4 having the amino acid sequence of SEQ ID NO: 754; e. FR-H1 having the amino acid sequence of SEQ ID NO: 755 or SEQ ID NO: 756; f. FR-H2 having the amino acid sequence of SEQ ID NO: 759; g. FR-H3 having the amino acid sequence of SEQ ID NO: 760; and h. FR-H4 having the amino acid sequence of any one of SEQ ID NOs: 764. In certain embodiments, AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen binding unit comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 746, b. FR-L2 having the amino acid sequence of SEQ ID NO: 747, c. FR-L3 having the amino acid sequence of SEQ ID NO: 748, d. FR-L4 having the amino acid sequence of SEQ ID NO: 754, e. FR-H1 having the amino acid sequence of SEQ ID NO: 755, f. FR-H2 having the amino acid sequence of SEQ ID NO: 759, g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 764. In other embodiments, AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen binding unit comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 746, b. FR-L2 having the amino acid sequence of SEQ ID NO: 747, c. FR-L3 having the amino acid sequence of SEQ ID NO: 749, d. FR-L4 having the amino acid sequence of SEQ ID NO: 754, e. FR-H1 having the amino acid sequence of SEQ ID NO: 756, f. FR-H2 having the amino acid sequence of SEQ ID NO: 759, g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 764.In another embodiment, AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen binding unit comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 746, b. FR-L2 having the amino acid sequence of SEQ ID NO: 747, c. FR-L3 having the amino acid sequence of SEQ ID NO: 750, d. FR-L4 having the amino acid sequence of SEQ ID NO: 754, e. FR-H1 having the amino acid sequence of SEQ ID NO: 756, f. FR-H2 having the amino acid sequence of SEQ ID NO: 759, g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 764. In certain embodiments, AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen binding unit comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 746, b. FR-L2 having the amino acid sequence of SEQ ID NO: 747, c. FR-L3 having the amino acid sequence of SEQ ID NO: 751, d. FR-L4 having the amino acid sequence of SEQ ID NO: 754, e. FR-H1 having the amino acid sequence of SEQ ID NO: 756, f. FR-H2 having the amino acid sequence of SEQ ID NO: 759, g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 764.

[0036] In some embodiments, AF2 is fused to AF1 by a flexible peptide linker. In certain embodiments, the flexible linker comprises two or three types of amino acids selected from the group consisting of glycine, serine, and proline.

[0037] In certain embodiments, the bispecific antigen binding unit further comprises a first release segment peptide (RS1) and a second release segment peptide (RS2), each of which is a substrate for cleavage by a mammalian protease. In one embodiment, RS1 and RS2 are identical. In another embodiment, RS1 and RS2 are different. In some embodiments, RS1 and RS2 are substrates for a protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other embodiments, RS1 and RS2 each comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 53-671. In another embodiment, RS1 and RS2 each comprise an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which is set forth in Table 5.

[0038] In some embodiments, the bispecific antigen-binding unit further comprises a first extended recombinant polypeptide (XTEN1) and a second extended recombinant polypeptide, wherein each of XTEN1 and XTEN2: a. has at least about 36 amino acids; and b. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues in the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), and is characterized by at least 4-6 different amino acids selected from cG, A, S, T, E, and P. In one embodiment, XTEN1 and XTEN2 are identical. In another embodiment, XTEN1 and XTEN2 are different.

[0039] In certain embodiments, XTEN1 and XTEN2 each comprise an amino acid sequence comprising at least three of the amino acid sequences of SEQ ID NOs: 672-675. In yet other embodiments, XTEN1 and XTEN2 each comprise an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 676-734. In other embodiments, each of XTEN1 and XTEN2 is selected from the group consisting of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, A and 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 selected from the sequences of E300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7.

[0040] In some embodiments, the bispecific antigen binding unit has the following structural arrangement from N-terminus to C-terminus: XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-diabody-RS1-XTEN1, or XTEN1-RS1-diabody-RS2-XTEN2, wherein the diabody comprises the VL and VH of AF1 and AF2.

[0041] In some embodiments, AF1 specifically binds to human or cynomolgus monkey (cyno) EGFR. In other embodiments, AF1 specifically binds to human and cynomolgus monkey (cyno) EGFR. In certain embodiments, AF2 binds to a CD3 complex subunit selected from any one of CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta epsilon. In another embodiment, AF2 specifically binds to human or cynomolgus monkey (cyno) CD3. In yet another embodiment, AF2 specifically binds to human and cynomolgus monkey (cyno) CD3.

[0042] In one embodiment, AF1 has a K of about 0.1 nM to about 100 nM as determined in an in vitro antigen binding assay involving EGFR or an epitope thereof. d It specifically binds to EGFR.

[0043] In other embodiments, AF1 has a dissociation constant (K) of about 0.1 nM to about 100 nM, or about 0.5 nM to about 50 nM, or about 1.0 nM to 20 nM, or about 2.0 nM to about 10 nM, as determined in an in vitro antigen binding assay. d In some embodiments, AF2 specifically binds to EGFR with a dissociation constant (K) of about 10 nM to about 400 nM, or about 50 nM to about 350 nM, or about 100 nM to 300 nM, as determined in an in vitro antigen binding assay. dIn certain embodiments, AF2 specifically binds to human or cyno CD3 with a dissociation constant (K) weaker than about 3 nM, or 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 400 nM, as determined in an in vitro antigen binding assay. d In yet another embodiment, AF2 specifically binds to human or cyno CD3 with a respective dissociation constant (K d In some embodiments, AF2 specifically binds to human or cyno CD3 with a binding affinity 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 than an antibody-binding fragment consisting of the amino acid sequence of SEQ ID NO: 781, as determined by in vitro antigen-binding assays. d ) that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold weaker than the binding affinity of AF1.

[0044] In one aspect, the present disclosure provides pharmaceutical compositions, comprising the polypeptide disclosed herein and one or more pharmaceutically suitable excipients.In some embodiments, pharmaceutical compositions are formulated for intradermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intraperitoneal, intrathecal or intramuscular administration.In another embodiment, pharmaceutical compositions are in liquid form or frozen form.In certain embodiments, pharmaceutical compositions are in a pre-filled syringe for single injection.In another embodiment, pharmaceutical compositions are formulated as lyophilized powder that is reconstituted before administration.

[0045] In yet another aspect, the disclosure provides a polypeptide as disclosed herein in the preparation of a medicament for treating a disease in a subject in need thereof. In some embodiments, the disease is selected from the group consisting of anaplastic and medullary thyroid cancer, appendix cancer, allenoblastoma, biliary tract cancer, bladder cancer, breast cancer, cancers of the bile duct, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignant tumor with malignant ascites, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, follicular cancer, gallbladder cancer, gastric cancer, and the like. cancer), gastrointestinal stromal tumor (GIST), GE-linked cancer, genitourinary cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocellular carcinoma, HR+ and HER2+ breast cancer, Hürthle cell carcinoma, inflammatory breast cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroendocrine carcinoma, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary carcinoma, parathyroid cancer, peritoneal dissemination, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, gastric cancer cancer), testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial cancer, uterine cancer, uterine serous cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0046] In a related aspect, the present disclosure provides a method of treating a disease in a subject, comprising administering one or more therapeutically effective doses of the pharmaceutical compositions disclosed herein to a subject in need thereof. In some embodiments, the subject is selected from the group consisting of a mouse, a rat, a monkey, and a human.

[0047] In certain embodiments, the disease is anaplastic and medullary thyroid cancer, appendix cancer, allenoblastoma, biliary tract cancer, bladder cancer, breast cancer, cancers of the bile duct, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignant tumor with malignant ascites, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, follicular cancer, gallbladder cancer, gastric cancer, cancer), gastrointestinal stromal tumor (GIST), GE-linked cancer, genitourinary cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocellular carcinoma, HR+ and HER2+ breast cancer, Hürthle cell carcinoma, inflammatory breast cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroendocrine carcinoma, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary carcinoma, parathyroid cancer, peritoneal dissemination, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, gastric cancer The cancer is selected from the group consisting of breast cancer, testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial cancer, uterine cancer, uterine serous cancer, vaginal cancer, vulvar cancer, and Wilms' tumor. In other embodiments, the pharmaceutical composition is administered to the subject in one or more therapeutically effective doses administered twice weekly, once weekly, once every two weeks, once every three weeks, once every four weeks, or once monthly. In certain embodiments, the pharmaceutical composition is administered to the subject in one or more therapeutically effective doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. In some embodiments, the dose is administered intradermally, subcutaneously, intravenously, intraarterially, intraperitoneally, intraperitoneally, intrathecally, or intramuscularly.

[0048] In certain aspects, the disclosure provides an isolated nucleic acid, wherein the nucleic acid comprises (a) a polynucleotide encoding a polypeptide disclosed herein, or (b) the complement of the polynucleotide of (a).

[0049] In a related aspect, the present disclosure provides an expression vector comprising a polynucleotide sequence disclosed herein and a recombinant regulatory sequence operably linked to the polynucleotide sequence.

[0050] In yet another aspect, the present disclosure provides an isolated host cell comprising an expression vector disclosed herein. In some embodiments, the host cell is a prokaryotic organism. In certain embodiments, the host cell is E. coli or a mammalian cell.

[0051] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0052] Various features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and accompanying drawings that set forth illustrative embodiments, in which the principles of the invention are utilized. [Figure 1] Figure 1 shows the individual components of the bispecific antigen-binding fragment composition. Figure 1A shows an antigen-binding fragment with affinity for a target cell marker. Figure 1B shows an antigen-binding fragment with affinity for an effector cell. Figure 1C and Figure 1D show XTEN polypeptides of different lengths. Figure 1E shows a cleavable release segment. [Figure 2]

[0033] Figure 2A shows two different forms of the polypeptide compositions described herein. Figure 2A shows, on the left, a release segment and an antigen-binding fragment for an effector cell fused to an XTEN, while the arrow indicates the action of a protease to cleave the release segment, which results in the release of the XTEN from the antigen-binding fragment of the polypeptide on the right, allowing the antigen-binding fragment to regain its potential binding affinity (e.g., its maximum potential binding affinity) because it is no longer shielded by the XTEN. Figure 2B shows, on the left, a bispecific composition having an antigen-binding fragment for an effector cell fused to an antigen-binding fragment with binding affinity for a target cell marker. The release segment and XTEN are also fused to an antigen-binding fragment with affinity for the effector cell, while the arrow indicates the action of a protease to cleave the release segment, which results in the release of the XTEN and the fused antigen-binding fragment from the polypeptide on the right, allowing them to regain their potential maximum binding affinity because they are no longer shielded by the XTEN. [Figure 3] Two different forms of bispecific antigen-binding polypeptides are shown: on the left, a bispecific composition having an antigen-binding fragment for an effector cell is fused to an antigen-binding fragment with binding affinity for a target cell marker with a release segment (the scissors indicating susceptibility to protease cleavage) and an XTEN is fused to the antigen-binding fragment with binding affinity for the effector cell, while on the right, a bispecific composition having an antigen-binding fragment for an effector cell is fused to an antigen-binding fragment with binding affinity for a target cell marker and a release segment and an XTEN are fused to the antigen-binding fragment with binding affinity for the target cell marker. [Figure 4]

[0039] Figure 4A shows a bispecific composition having an scFv antigen-binding fragment for an effector cell fused to an scFv antigen-binding fragment with binding affinity for a target cell marker having a release segment (the scissors indicate susceptibility to protease cleavage), and an XTEN fused to each antigen-binding fragment. Figures 4B and 4C are variations of Figure 4A in which the antigen-binding fragments are in a diabody configuration and the release segment (the scissors indicate susceptibility to protease cleavage) and XTEN are fused to the antigen-binding fragment for the effector cell marker or target cell marker, respectively. [Figure 5] Schematic diagram of a bispecific antigen-binding polypeptide near tumor tissue (top) and normal tissue (bottom). The bispecific antigen-binding polypeptide is preferentially cleaved in tumor tissue, releasing one or more XTEN moieties compared to normal tissue. The cleaved bispecific antigen-binding polypeptide can bind to T cells and tumor cells expressing tumor-specific markers. [Figure 6] 1 shows the amino acid sequence of controlled release segment RSR-1517 (SEQ ID NO: 53) with the peptide cleavage sites of the listed proteases indicated. DETAILED DESCRIPTION OF THE INVENTION

[0053] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of the claims and their equivalents be covered thereby.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, suitable methods and materials are described below. In case of conflict, the present patent specification, including definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the present invention.

[0055] definition In the context of this application, the following terms have the meanings ascribed to them unless otherwise specified.

[0056] As used throughout this specification and claims, the terms "a," "an," and "the" are used in the sense of meaning "at least one," "at least the first," "one or more," or "a plurality" of the referenced components or steps, unless an upper limit is specifically stated thereafter. Thus, "release segment," as used herein, means "at least the first release segment," but includes multiple release segments. As with the amounts of any single agent, operable limits and parameters of combinations will be known to those of skill in the art in light of this disclosure.

[0057] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified by, for example, disulfide bond formation, glycosylation, lipid formation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0058] The term "monomer" as applied to a polypeptide refers to the state of a polypeptide that is a single, contiguous amino acid sequence that is not substantially associated with one or more additional polypeptides of the same or different sequence.

[0059] As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including, but not limited to, both the D or L optical isomers, as well as amino acid analogs and peptidomimetics. Amino acids can be designated using standard one-letter or three-letter codes.

[0060] The term "naturally occurring L-amino acids" or "L-amino acids" refers to the L optical isomeric forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine ​​(C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).

[0061] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, VHH antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably herein. Full-length antibodies can be, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies. Antibodies represent a large family of molecules that includes several types of molecules, such as IgD, IgG, IgA, IgM, and IgE. The term "immunoglobulin molecule" includes, for example, hybrid antibodies or engineered antibodies, as well as fragments thereof. It has been shown that the antigen-binding function of an antibody can be performed by fragments of naturally occurring antibodies or monoclonal antibodies.

[0062] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human complementarity-determining regions (CDRs) and human framework regions (FRs). In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody (which may include amino acid substitutions) and all or substantially all of the FRs correspond to those of a human antibody (which may include amino acid substitutions).

[0063] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation, with such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods, as well as other exemplary methods for producing monoclonal antibodies, are known in the art or are described herein.

[0064] As used herein, "antigen-binding fragment" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that specifically binds to (is "immunoreactive" with) an antigen. Examples include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab'), diabodies, linear antibodies (see U.S. Pat. No. 5,641,870), single-domain antibodies, single-domain camelid antibodies, single-chain fragment variable (scFv) antibody molecules, and multispecific antibodies formed from antibody fragments that retain the ability to specifically bind to an antigen. The term "antigen-binding fragment" also encompasses any polypeptide chain-containing molecular structure with a specific shape that matches, recognizes, and binds to an epitope, where one or more noncovalent interactions stabilize the complex between the molecular structure and the epitope. An antigen-binding fragment "specifically binds" or is "immunoreactive" with an antigen if it binds with greater affinity or avidity than it binds to other reference antigens, including polypeptides or other substances.

[0065] "scFv" or "single-chain fragment variable" are used interchangeably herein to refer to an antibody fragment format that contains the variable regions of the heavy (VH) and light (VL) chains of an antibody, or two copies of a VH or VL chain, linked together by a short, flexible peptide linker that enables the scFv to form the desired structure for antigen binding. scFvs are fusion proteins of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins, and can be readily expressed in a functional form in E. coli or other host cells.

[0066] "Diabodies" refer to small antibody fragments prepared by constructing scFv fragments with a short linker (approximately 5-10 residues) between the VH and VL domains, thereby achieving interchain rather than intrachain pairing of the V domains, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are more fully described, for example, in US7,635,475.

[0067] The term "bispecific antigen-binding fragment" should be understood as an antigen-binding fragment that has binding specificities for at least two different antigens.

[0068] The terms "antigen," "target antigen," and "immunogen" are used interchangeably herein and refer to a structure or binding determinant to which an antibody, antibody fragment, or antibody fragment-based molecule binds or has specificity. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound, or a portion thereof. An antigen is also a ligand of an antibody or antibody fragment that has binding affinity for the antigen. Non-limiting exemplary antigens described herein include CD3 and EGFR (and portions thereof) from humans, non-human primates, mice, and other homologs thereof.

[0069] The term "CD3 antigen-binding fragment" refers to an antigen-binding fragment that can bind to CD3 or a member of the CD3 complex with sufficient affinity such that the antigen-binding fragment is useful as a diagnostic and / or therapeutic agent in targeting Cluster of Differentiation 3 (CD3).

[0070] An "EGFR antigen-binding fragment" refers to an antigen-binding fragment capable of binding epidermal growth factor receptor. EGFR is a member of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4).

[0071] "Target tissue" or "target cell" refers to a tissue or cell bearing an EGFR antigen that is the cause of or part of a disease state, such as, but not limited to, cancer or a related condition. Diseased target tissues or cell sources include body organs, tumors, cancerous cells or cell populations, or cells that form the matrix or are found associated with cancerous cell populations, bone, skin, or cells that produce cytokines or factors that contribute to the disease state.

[0072] The term "epitope" refers to the specific site on an antigen molecule to which an antibody, antibody fragment, or binding domain binds. An epitope is the ligand of an antibody or antibody fragment.

[0073] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., between an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K d As used herein, "greater binding affinity" can be expressed as a lower K d means a value, e.g., 1 x 10 -9 M is 1 x 10 -8 M. An antibody that binds to an antigen of interest, e.g., a tumor-associated EGFR antigen, is one that binds to the antigen with sufficient affinity and does not significantly cross-react with other proteins so that the antibody is useful as a diagnostic and / or therapeutic agent in targeting cells or tissues that express the antigen.

[0074] "Dissociation constant" or "K d " are used interchangeably and refer to the affinity between a ligand "L" and a protein "P", i.e., how strongly the ligand binds to a particular protein. This is expressed by the formula K d = [L][P] / [LP], where [P], [L], and [LP] represent the molar concentrations of the protein, ligand, and complex, respectively.

[0075] The terms "hypervariable region," "HVR," or "CDR," as used herein, interchangeably refer to regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops and / or are involved in antigen recognition. Generally, antibodies contain six hypervariable regions: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Several CDR delineations are used and encompassed herein; for example, CDR-L1 refers to the first hypervariable CDR region of the light chain, CDR-H2 refers to the second hypervariable CDR region of the heavy chain, and so on. Kabat complementarity-determining regions (CDRs) are the most commonly used, based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0076] "Isoelectric point" or "pI" are used interchangeably herein and refer to the pH at which a particular molecule has no net charge or is electrically neutral on statistical average. The formal term for expressing isoelectric point is pH, which results in units of pH. For example, an antigen-binding fragment with a pI of 6.3 would have a neutral charge in a solution at pH 6.3. Isoelectric point can be determined mathematically, and several algorithms for estimating the isoelectric point of peptides and proteins, including the Henderson-Hasselbalch equation, have different pK values. Isoelectric point can also be determined experimentally by in vitro assays such as capillary isoelectric focusing.

[0077] "Framework" or "FR" residues are those variable domain residues in an antigen-binding fragment other than the hypervariable region residues as defined herein, which generally are located between or adjacent to the CDRs. Several FR delineations are used and encompassed herein, e.g., FR-L1 refers to the first FR region of the light chain, FR-H2 refers to the second FR region of the heavy chain, and so on.

[0078] The term "release segment" or "RS" refers to a cleavage sequence within a subject composition that can be recognized and cleaved by one or more proteases, affecting the release of the antigen-binding fragment and XTEN from the composition. As used herein, "mammalian protease" refers to a protease that is normally present in bodily fluids, cells, and tissues, and may be found at higher levels in certain target tissues or cells in a mammal, such as diseased tissues (e.g., tumors). RS sequences can be engineered to be cleaved by a variety of mammalian proteases or multiple mammalian proteases present in or near target tissues in a subject, or introduced in an in vitro assay. Other equivalent proteases (endogenous or exogenous) that can recognize a defined cleavage site can be utilized. It is specifically contemplated that the RS sequence can be tailored and customized to the protease utilized and can incorporate linker amino acids to connect to adjacent polypeptides.

[0079] The term "cleavage site" refers to a position between adjacent amino acids in a peptide or polypeptide that can be broken or cleaved by an enzyme such as a protease, i.e., cleavage of the peptide bond between adjacent amino acids.

[0080] The term "internal," when referring to a first polypeptide linked to a second polypeptide, encompasses the linkage or fusion of an additional component that links the N-terminus of the first or second polypeptide to the C-terminus of the second or first polypeptide, respectively, as well as the insertion of the first polypeptide into the sequence of the second polypeptide. For example, when an RS component is "linked" within a chimeric polypeptide construct, the RS may be linked to the N-terminus, C-terminus, or inserted between any two amino acids of the XTEN polypeptide.

[0081] "Activity," as applied to the forms of compositions provided herein, refers to actions or effects, including, but not limited to, antigen binding, antagonist activity, agonist activity, cellular or physiological response, cell lysis, cell death, or effects generally known in the art for the effector component of the composition, whether measured by in vitro, ex vivo, or in vivo assays, or by clinical effect.

[0082] As used herein, "effector cells" include any eukaryotic cells that can affect target cells. For example, effector cells can induce loss of membrane integrity, nuclear condensation, karyorrhexis, apoptosis, lysis, and / or death of target cells. In another example, effector cells can induce target cell division, growth, differentiation, or otherwise alter the signal transduction of target cells. Non-limiting examples of effector cells include plasma cells, T cells, CD4 cells, CD8 cells, B cells, cytokine-induced killer cells (CIK cells), pluripotent stem cells, dendritic cells, regulatory T cells (RegT cells), helper T cells, myeloid cells, macrophages, and NK cells.

[0083] "Effector cell antigen" refers to a molecule expressed by an effector cell, including, but not limited to, a cell surface molecule such as a protein, glycoprotein, or lipoprotein. Exemplary effector cell antigens include the CD3 complex or T cell receptor (TCR), CD4, CD8, CD25, CD38, CD69, CD45RO, CD57, CD95, CD107, and CD154 proteins, as well as effector molecules such as cytokines associated with, bound to, expressed within, or expressed and released by effector cells. Effector cell antigens can function as binding counterparts for the binding domains of the subject chimeric polypeptide constructs.

[0084] As used herein, "CD3" or "cluster of differentiation 3" refers to the T cell surface antigen CD3 complex, which includes all known CD3 subunits, e.g., CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta, either individually or in combination. The extracellular domains of CD3 epsilon, gamma, and delta contain immunoglobulin-like domains and are therefore considered part of the immunoglobulin superfamily. CD3 includes, for example, the 207-amino acid long human CD3 epsilon protein (NCBI Reference SEQ ID NO: NP_000724) and the 182-amino acid long human CD3 gamma protein (NCBI Reference SEQ ID NO: NP_000064).

[0085] As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay as described herein or otherwise known in the art.

[0086] A "host cell" includes an individual cell or cell culture that can be or has been a recipient of a subject vector into which exogenous nucleic acid, such as those described herein, has been introduced. A host cell includes the progeny of a single host cell. The progeny are not necessarily completely identical (in terms of morphology of total DNA complement and genome) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a vector of the invention.

[0087] "Isolated," as used to describe various polypeptides disclosed herein, refers to a polypeptide that has been identified, separated, and / or recovered from components of its natural environment or from a more complex mixture (such as during protein purification). Contaminant components of its natural environment are typically materials that would interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As will be apparent to those skilled in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof does not require "isolation" to distinguish it from its naturally occurring counterpart. In addition, an "enriched," "separated," or "diluted" polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume generally exceeds its naturally occurring counterpart. Generally, a polypeptide produced by recombinant means and expressed in a host cell is considered "isolated."

[0088] An "isolated nucleic acid" is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the natural source of the polypeptide-encoding nucleic acid. For example, an isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Thus, an isolated polypeptide-encoding nucleic acid molecule is distinguished from a specific polypeptide-encoding nucleic acid molecule because it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule also includes, for example, a polypeptide-encoding nucleic acid molecule contained in cells that normally express the polypeptide, where the nucleic acid molecule is in a chromosomal or extrachromosomal location different from its location in natural cells.

[0089] A "chimeric" protein or polypeptide comprises at least one fusion polypeptide that contains at least one region in a position within the sequence that is different from the position where it occurs in nature. The regions may normally be present in separate proteins and are brought together in the fusion polypeptide, or may normally be present in the same protein but are arranged in a new configuration in the fusion polypeptide. Chimeric proteins can be made, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

[0090] "Fused" and "fusion" are used interchangeably herein and refer to the linking together of two or more peptide or polypeptide sequences by recombinant means. A "fusion protein" or "chimeric protein" comprises a first amino acid sequence linked to a second amino acid sequence that is not naturally linked in nature.

[0091] "XTENized" is used to refer to a peptide or polypeptide that has been modified by the attachment or fusion of one or more XTEN polypeptides (described below) to the peptide or polypeptide, whether by recombinant or chemical cross-linking means.

[0092] "Operably linked" means that the linked DNA sequences are in reading phase or in frame. An "in-frame fusion" refers to the joining of two or more open reading frames (ORFs) to form a longer, contiguous ORF in a manner that maintains the reading frame of the original ORFs. For example, a promoter or enhancer is operably linked to a coding sequence for a polypeptide if it affects the transcription of the polypeptide sequence. The resulting recombinant fusion protein is thus a single protein containing two or more segments corresponding to the polypeptides encoded by the original ORFs (segments that are not normally so joined in nature).

[0093] A "linear sequence" or "sequence," in the context of a polypeptide, is the order of amino acids in the polypeptide from amino terminus to carboxyl terminus (N-terminus to C-terminus) in which adjacent residues in the sequence are contiguous in the primary structure of the polypeptide. A "subsequence" is the linear sequence of a portion of a polypeptide that is known to contain additional residues in one or both directions.

[0094] "Heterologous" means derived from a genetically distinct entity from the rest of the entity to which it is being compared. For example, a glycine-rich sequence removed from a native coding sequence and operably linked to a coding sequence other than the native sequence is a heterologous glycine-rich sequence. The term "heterologous" as applied to polynucleotides, polypeptides, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from the rest of the entity to which it is being compared.

[0095] The terms "polynucleotide," "nucleic acid," "nucleotide," and "oligonucleotide" are used interchangeably. They refer to nucleotides of any length, including single or multiple nucleic acids, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides are coding or non-coding regions of a gene or gene fragment, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component.

[0096] The term "complement of a polynucleotide" means a polynucleotide molecule having a complementary base sequence and reverse orientation compared to a reference sequence such that it can hybridize with complete fidelity to the reference sequence.

[0097] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of recombination steps, which may include cloning, restriction, and / or ligation steps, as well as other procedures that result in the expression of a recombinant protein in a host cell.

[0098] The terms "gene" and "gene fragment" are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that can encode a specific protein after being transcribed and translated. A gene or gene fragment can be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame that can cover the entire coding region or a segment thereof. A "fusion gene" is a gene composed of at least two heterologous polynucleotides linked together.

[0099] As used herein, a "coding region" or "coding sequence" refers to a portion of a polynucleotide consisting of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not typically translated into an amino acid, it can be considered part of the coding region; however, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. The boundaries of a coding region are typically determined by a 5'-terminal start codon, which encodes the amino terminus of the resulting polypeptide, and a 3'-terminal translation stop codon, which encodes the carboxyl terminus of the resulting polypeptide. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. A single vector can then contain only a single coding region, or can contain two or more coding regions; for example, a single vector can separately encode binding domain-A and binding domain-B, as described below. In addition, the vectors, polynucleotides, or nucleic acids of the invention can encode heterologous coding regions, either fused or unfused to the nucleic acid encoding the binding domain of the invention, including, but not limited to, specialized elements or motifs such as secretory signal peptides or heterologous functional domains.

[0100] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence refers to the sequence following the transcription start point. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0101] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence. In certain embodiments, the upstream nucleotide sequence refers to a sequence located 5' to a coding region or a transcription start site. For example, most promoters are located upstream of the transcription start site.

[0102] "Homology" or "homology" refers to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences. When using a program such as BestFit to determine sequence identity, similarity, or homology between two different amino acid sequences, default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity, or homology scores. Preferably, homologous polynucleotides hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity compared to their respective sequences. Homologous polypeptides preferably have sequence identities that are at least 70%, preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95-99% identical when optimally aligned against sequences of comparable length.

[0103] "Ligation," as applied to polynucleic acids, refers to the process of forming phosphodiester bonds between two nucleic acid fragments or genes, joining them together. In order to ligate DNA fragments or genes together, the ends of the DNA must be compatible with each other. In some cases, the ends are directly compatible after endonuclease digestion. However, it may be necessary to first convert the staggered ends typically produced after endonuclease digestion to blunt ends to make them compatible for ligation.

[0104] The terms "stringent conditions" or "stringent hybridization conditions" include reference to conditions under which a polynucleotide hybridizes to its target sequence to a detectably greater extent than other sequences (e.g., at least twice background). Generally, the stringency of hybridization is expressed, in part, in terms of the temperature and salt concentration under which the wash step is carried out. Typically, stringent conditions are those in which the salt concentration is less than about 1.5 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short polynucleotides (e.g., 10 to 50 nucleotides) and at least about 60°C for long polynucleotides (e.g., more than 50 nucleotides). For example, "stringent conditions" may include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, followed by three washes (15 minutes each) in 0.1×SSC / 1% SDS at 60°C-65°C. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentrations may vary from about 0.1 to 2×SSC, with SDS present at about 0.1%. Such wash temperatures are typically selected to be about 5°C-20°C lower than the thermal melting point of the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Equations for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual," 3rd edition, Cold Spring Harbor Laboratory Press, 2001. Typically, a blocking reagent is used to block nonspecific hybridization. Such a blocking reagent contains, for example, about 100-200 μg / ml of sheared and denatured salmon sperm DNA.Organic solvents such as formamide at concentrations of about 35-50% v / v may also be used under certain circumstances, such as RNA:DNA hybridization. Useful variations on these washing conditions will be readily apparent to those of skill in the art.

[0105] The terms "percent identity," "percent sequence identity," and "% identity" as applied to polynucleotide sequences refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such algorithms insert gaps in a standardized, reproducible manner within the sequences being compared to optimize the alignment between the two sequences, thus achieving a more meaningful comparison of the two sequences. Percent identity can be measured over the length of the entire defined polynucleotide sequence, or over a shorter length, e.g., over the length of a fragment taken from a larger, defined polynucleotide sequence, e.g., a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210, or at least 450 contiguous residues. It is understood that such lengths are exemplary only, and that any fragment length supported by the sequences set forth herein in the tables, figures, or sequence listing can be used to describe the length over which percent identity can be measured. The sequence identity percentage is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of matched positions (where identical residues occur in both polypeptide sequences), dividing the number of matched positions by the total number of positions within the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the sequence identity percentage. When sequences of different lengths are compared, the shortest sequence defines the length of the comparison window. Conservative substitutions are not taken into account when calculating sequence identity.

[0106] With respect to the polypeptide sequences specified herein, the terms "percent identity," "percent sequence identity," and "% identity" are defined as the percentage of amino acid residues in a query sequence that are identical to the amino acid residues of a second reference polypeptide sequence or a portion thereof of equivalent length, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve optimal alignment across the entire length of the sequences being compared. Percent identity can be measured over the length of the entire defined polypeptide sequence, or over a shorter length, such as a fragment taken from a larger defined polypeptide sequence, for example, a fragment of at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. It will be understood that such lengths are exemplary only, and that any fragment length supported by the sequences set forth herein in the tables, figures, or sequence listing can be used to describe the length over which percent identity can be measured.

[0107] "Repetitiveness," as used in the context of polynucleotide sequences, refers to the degree of internal homology in a sequence, e.g., the frequency of identical nucleotide sequences of a given length. Repetitiveness can be measured, for example, by analyzing the frequency of identical sequences.

[0108] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, e.g., an RNA or a polypeptide. This includes, but is not limited to, transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), short hairpin RNA (shRNA), short interfering RNA (siRNA), or other RNA products, and translation of mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products described herein further include nucleic acids that have post-transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides that have post-translational modifications, e.g., methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0109] "Vector" or "expression vector" are used interchangeably and refer to a nucleic acid molecule that preferably self-replicates in a suitable host cell, transferring an inserted nucleic acid molecule into and / or between host cells. This term includes vectors that function primarily for the insertion of DNA or RNA into a cell, replicating vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Also included are vectors that provide more than one of the above functions. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" generally refers to a suitable host cell containing an expression vector that can function to produce a desired expression product.

[0110] "Serum degradation resistance," as applied to a polypeptide, refers to the ability of the polypeptide to withstand degradation in blood or its components, typically involving proteases in serum or plasma. Serum degradation resistance can be measured by combining the protein with human (or, as appropriate, mouse, rat, dog, or monkey) serum or plasma, typically at about 37°C, for a range of days (e.g., 0.25, 0.5, 1, 2, 4, 8, or 16 days). Samples from these time points can be run in a Western blot assay, and the protein is detected using an antibody. The antibody can be directed against a tag in the protein. If the protein shows a single band in the Western blot (where the size of the protein is identical to that of the injected protein), degradation has not occurred. In this exemplary method, the time point at which 50% of the protein is degraded, as determined by Western blot or an equivalent technique, is the serum degradation half-life or "serum half-life" of the protein.

[0111] "t 1 / 2 The terms "half-life", "terminal half-life", "elimination half-life", and "circulating half-life" are used interchangeably herein and, as used herein, refer to ln(2) / K el The terminal half-life is calculated as K el is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration versus time curve. Half-life typically refers to the time required for half of the amount of an administered substance deposited in a living organism to be metabolized or eliminated by normal biological processes. When a clearance curve for a given polypeptide is constructed as a function of time, the curve is usually biphasic, with a rapid α-phase and a longer β-phase. The typical half-life of a human antibody in humans is 21 days. Half-life can be measured using timed samples from any body fluid, but is most typically measured in plasma samples.

[0112] The term "molecular weight" generally refers to the sum of the atomic weights of the constituent atoms in a molecule. Molecular weight can be theoretically determined by summing the atomic masses of the constituent atoms in a molecule. When applied to polypeptides, molecular weight is calculated based on amino acid composition by adding the molecular weights of each type of amino acid in the composition or by estimating it from comparison with molecular weight standards in an SDS electrophoresis gel. The calculated molecular weight of a molecule may differ from the molecule's "apparent molecular weight," which generally refers to the molecular weight of the molecule as determined by one or more analytical techniques. "Apparent molecular weight factor" and "apparent molecular weight" are related terms, and when used in the context of polypeptides, these terms refer to a measure of the relative increase or decrease in apparent molecular weight exhibited by a particular amino acid or polypeptide sequence. Apparent molecular weight can be determined, for example, by comparison with globular protein standards measured in "apparent kD" using size exclusion chromatography (SEC) or similar methods. The apparent molecular weight factor is the ratio between the apparent molecular weight and the "molecular weight," the latter calculated by adding the molecular weights of each type of amino acid in the composition as described above or by estimating it from comparison with molecular weight standards in an SDS electrophoresis gel. The determination of apparent molecular weight and apparent molecular weight factor is described in US Pat. No. 8,673,860.

[0113] "Defined media," as the media's components are known, refers to a medium that contains the nutritional and hormonal requirements necessary for the survival and / or growth of cells in culture. Traditionally, defined media are formulated with the addition of nutritional and growth factors necessary for growth and / or survival. Typically, defined media provide at least one component from one or more of the following categories: a) all essential amino acids and a basic set of usually 20 amino acids plus cysteine; b) an energy source (usually in the form of a carbohydrate such as glucose); c) vitamins and / or other organic compounds required at low concentrations; d) free fatty acids; and e) trace elements (trace elements are defined as inorganic compounds or naturally occurring elements required at very low concentrations, typically in the micromolar range). Defined media may also be optionally supplemented with one or more components from any of the following categories: a) one or more mitogens; b) salts and buffers, e.g., calcium, magnesium, and phosphate; c) nucleosides and bases, e.g., adenosine and thymidine, hypoxanthine; and d) protein and tissue hydrolysates.

[0114] The term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of a naturally occurring polypeptide disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of naturally occurring polypeptides, peptides, small organic molecules, and the like. Methods for identifying agonists of naturally occurring polypeptides can include contacting the naturally occurring polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the naturally occurring polypeptide.

[0115] As used herein, "treatment" or "treating," or "alleviating," or "ameliorating," are used interchangeably herein. These terms refer to an approach to achieving beneficial or desired results, including, but not limited to, therapeutic benefit and / or preventative benefit. Therapeutic benefit refers to the eradication or amelioration of the underlying disease being treated. Therapeutic benefit is also achieved by the eradication or amelioration of one or more physiological symptoms or improvement of one or more clinical parameters associated with the underlying disease, such that an improvement is observed in a subject, even though the subject may still be suffering from the underlying disease. In the case of preventative benefit, the composition may be administered to a subject at risk of developing a particular disease, or to a subject who reports one or more physiological symptoms of the disease despite not having been diagnosed with the disease.

[0116] As used herein, "therapeutic effect" or "therapeutic benefit" refers to a physiological effect, including, but not limited to, the reduction, amelioration, or prevention of disease, or the improvement of one or more clinical parameters associated with an underlying disease in a subject, or refers to an enhancement of the physical or mental health of a subject resulting from the administration of a polypeptide of the present invention other than the ability to induce the production of antibodies against an antigenic epitope possessed by an otherwise biologically active protein. In the case of prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, a recurrence of a previous disease, or a condition or symptom of that disease, or to a subject who reports one or more physiological symptoms of a disease despite not having been diagnosed with the disease.

[0117] As used herein, the terms "therapeutically effective amount" and "therapeutically effective dose" refer to an amount of a drug or biologically active protein, either alone or as part of a composition, that, when administered to a subject in single or repeated doses, can produce some detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease state or condition. Such an effect need not be absolute to be beneficial. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0118] As used herein, the term "therapeutically effective non-toxic dose" refers to a tolerable dose of a composition defined herein that is high enough to cause tumor or cancer cell depletion, tumor elimination, tumor shrinkage, or disease stabilization without or essentially without significant toxic effects on the subject. Such a therapeutically effective non-toxic dose may be determined by dose escalation studies as described in the art and should be below a dose that induces severe adverse side effects.

[0119] The term "therapeutic index," as used herein, refers to the ratio between the blood concentration at which a drug becomes toxic and the concentration at which the drug is effective. One exemplary ratio of the therapeutic index is the LD 50 :ED 50 where LD 50 is the dose that produces 50% mortality in the target population, and ED 50 is the dose that produces efficacy in the target population.

[0120] As used herein, the term "dosing regimen" refers to a schedule of sequentially administered multiple doses (i.e., at least two or more) of a composition, wherein the doses are administered in therapeutically effective amounts to produce a sustained beneficial effect on any symptom, aspect, measured parameter, endpoint, or characteristic of a disease state or condition in a subject.

[0121] As used herein, "administration" refers to a method of administering a dosage of a compound (e.g., an anti-CD3 antibody of the invention) or composition (e.g., a pharmaceutical composition comprising an anti-CD3 antibody of the invention) to a subject.

[0122] A "subject" is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject or individual is a human.

[0123] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Examples of cancer include carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, genitourinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous carcinoma, endometrial cancer, cervical cancer, colorectal cancer, epithelial intraperitoneal malignant tumor with malignant ascites, uterine cancer, peritoneal mesothelioma, kidney cancer, lung cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, cancer), small intestine cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, salivary gland cancer, thyroid cancer, epithelial carcinoma, adenocarcinoma, sarcoma of any origin, primary hematologic malignancy (including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, myeloproliferative neoplastic disorder, or myelodysplastic disorder), myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpasture's syndrome.

[0124] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

[0125] As used herein, "tumor-specific marker" refers to an antigen present on or in cancer cells that may, but is not necessarily, found in greater abundance in or on cancer cells compared to normal cells or tissues.

[0126] I). General techniques The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, within the skill of the art. Sambrook J et al., “Molecular Cloning: A Laboratory Manual,” 3rd edition, Cold Spring Harbor Laboratory Press, 2001, “Current protocols in molecular biology”, FMAsubel, et al. eds., 1987, the series “Methods in Enzymology,” Academic Press, San Diego, CA., “PCR 2: a practical approach”, MJ MacPherson, BD Hames and GRTaylor. eds., Oxford University Press, 1995, “Antibodies, a laboratory manual” Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988, “Goodman & Gilman's The Pharmacological Basis of Therapeutics,” 11th Edition, McGraw-Hill, 2005, and Freshney, RI, “Culture of Animal Cells: A Manual of Basic Technique,” ​​4th edition,John See Wiley & Sons, Somerset, NJ, 2000, the contents of which are incorporated herein by reference in their entireties.

[0127] Host cells can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Minimum Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma), are suitable for culturing eukaryotic cells. Additionally, animal cells can be grown in defined media that lack serum but are supplemented with hormones, growth factors, or any other factors necessary for the survival and / or growth of a particular cell type. Defined media that support cell survival maintain the viability, morphology, metabolic capacity, and potentially the differentiation potential of cells, while defined media that promote cell growth provide all the chemicals necessary for cell proliferation or multiplication. The general parameters governing mammalian cell survival and growth in vitro are well established in the art. Physicochemical parameters that can be controlled in different cell culture systems include pH, pO2, temperature, and osmolality. The nutritional requirements of cells are typically provided in standard media formulations developed to provide an optimal environment. Nutrients can be divided into several categories: amino acids and their derivatives, carbohydrates, sugars, fatty acids, complex lipids, nucleic acid derivatives, and vitamins. In addition to nutrients for maintaining cellular metabolism, most cells also require one or more hormones from at least one of the following groups to grow in serum-free media: steroids, prostaglandins, growth factors, pituitary hormones, and peptide hormones (Sato, GH, et al., "Growth of Cells in Hormonally Defined Media," Cold Spring Harbor Press, NY, 1982). In addition to hormones, cells may require transport proteins, such as transferrin (plasma iron transport protein), ceruloplasmin (copper transport protein), and high-density lipoprotein (lipid carrier), for in vitro survival and growth. The optimal set of hormones or transport proteins varies for each cell type. Most of these hormones or transport proteins are added exogenously, or in rare cases, mutant cell lines have been discovered that do not require specific factors.Those skilled in the art will know other factors necessary to maintain a cell culture without undue experimentation.

[0128] Growth media for growing prokaryotic host cells include nutrient broth (liquid nutrient medium) or LB medium (Luria Bertani). Suitable media include defined and non-defined media. Generally, media contain a carbon source, such as glucose, necessary for bacterial growth, water, and salts. Media may also contain an amino acid source and a nitrogen source, such as beef or yeast extract (in non-defined media) or known amounts of amino acids (in defined media). In some embodiments, the growth medium is LB broth, such as LB Miller broth or LB Lennox broth. LB broth contains peptone (an enzymatic digestion product of casein), yeast extract, and sodium chloride. In some embodiments, a selective medium containing an antibiotic is used. Only desired cells resistant to the antibiotic grow in this medium.

[0129] II).EGFR antigen binding composition In a first aspect, the present disclosure provides a polypeptide comprising a first antigen-binding fragment (AF1) that binds to epidermal growth factor (EGFR) or an epitope thereof. The antigen-binding fragment that binds to the EGFR antigen is particularly useful for pairing with a second antigen-binding fragment (AF2) that has binding affinity for the CD3 antigen (or other antigen) of effector cells in a composition specifically designed to result in cell killing of diseased cells or tissues bearing the EGFR antigen. Binding specificity can be determined by complementarity-determining regions (CDRs), or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, binding specificity is determined by light chain CDRs and heavy chain CDRs. A given combination of heavy chain CDRs and light chain CDRs provides a given binding pocket that confers higher affinity and / or specificity for EGFR compared to other reference antigens.

[0130] The antigen-binding fragments contemplated by the present disclosure can be derived from naturally occurring antibodies or fragments thereof, non-naturally occurring antibodies or fragments thereof, humanized antibodies or fragments thereof, synthetic antibodies or fragments thereof, hybrid antibodies or fragments thereof, or engineered antibodies or fragments thereof. Methods for generating antibodies against a given target marker are well known in the art. For example, monoclonal antibodies can be produced using the hybridoma method described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (U.S. Patent No. 4,816,567). The structure of antibodies and their fragments, the variable regions of heavy and light chains (VH and VL), single-chain variable regions (scFv), complementarity-determining regions (CDRs), and domain antibodies (dAbs) are well understood. Methods for generating polypeptides having desired EGFR antigen-binding fragments are well known in the art.

[0131] The various EGFR-binding antigen-binding fragments of the present disclosure are specifically modified to enhance their stability in the polypeptide embodiments described herein compared to EGFR antibodies and antigen-binding fragments known in the art. Protein aggregation of monoclonal antibodies continues to pose a significant challenge to their developability and remains a key area of ​​focus in antibody production. Antibody aggregation can be triggered by partial unfolding of their domains, leading to monomer-monomer association followed by nucleation and aggregate growth. While the aggregation propensity of antibodies and antibody-based proteins can be influenced by external experimental conditions, it is highly dependent on intrinsic antibody properties determined by their sequence and structure. While it is well known that proteins are marginally stable in their folded state, it is less understood that most proteins are inherently prone to aggregation in their unfolded or partially unfolded state, and that the resulting aggregates can be highly stable and long-lived. Reduced aggregation propensity has also been shown to be accompanied by increased expression titers, indicating that reduced protein aggregation can be beneficial throughout the development process and lead to a more efficient path to clinical trials. For therapeutic proteins, aggregates are a significant risk factor for adverse immune responses in patients and can occur through a variety of mechanisms. Controlling aggregation can improve protein stability, manufacturability, wear rate, safety, formulation, potency, immunogenicity, and solubility. Intrinsic protein properties, such as size, hydrophobicity, electrostatics, and charge distribution, play an important role in protein solubility. It has been shown that poor solubility of therapeutic proteins due to surface hydrophobicity can make formulation development more challenging and lead to poor in vivo biodistribution, undesirable pharmacokinetic behavior, and immunogenicity. Reducing the overall surface hydrophobicity of candidate monoclonal antibodies can also provide benefits and cost savings related to purification and administration regimens. Individual amino acids can be identified through structural analysis as contributing to an antibody's aggregation potential and can be located in both CDRs and framework regions.In particular, residues may be predicted to have a high risk of causing hydrophobicity problems for a given antibody. In one embodiment, the present disclosure provides an antigen-binding fragment having the ability to specifically bind to EGFR, wherein the antigen-binding fragment has at least one amino acid substitution of a hydrophobic amino acid in a framework region relative to a parent antibody or antibody fragment, wherein the hydrophobic amino acid is selected from isoleucine, leucine, or methionine. In another embodiment, the EGFR antigen-binding fragment has at least two amino acid substitutions of a hydrophobic amino acid in one or more framework regions, wherein the hydrophobic amino acid is selected from isoleucine, leucine, or methionine.

[0132] In the context of a subject antigen-binding fragment, the isoelectric point (pI) is the pH at which the antibody fragment has no net charge. If the pH is below the pI of an antibody fragment, it has a net positive charge. A greater positive charge tends to correlate with increased blood clearance and tissue retention, and generally a shorter half-life. If the pH is above the pI of an antibody fragment, it has a negative charge. A negative charge generally results in decreased tissue uptake and a longer half-life. This charge on framework residues can be manipulated through mutation. These considerations informed the design of the various sequences of the antigen-binding fragments of the embodiments described herein, in which individual amino acid substitutions were made to the parent antibody used as a starting point. The isoelectric point of a polypeptide can be determined mathematically or experimentally in in vitro assays. The isoelectric point (pI) is the pH at which a protein has a net charge of zero and can be calculated using the charges of specific amino acids in the protein sequence. An estimate of the charge, called the acid dissociation constant or pKa value, is used to calculate the pI. The pI can be determined in vitro by methods such as capillary isoelectric focusing (see Datta-Mannan, A., et al. The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies. mAbs 7:1084 (2015); Li, B., et al. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecular charge. mAbs 6, 1255-1264 (2014)) or other methods known in the art.

[0133] In some aspects of any of the embodiments disclosed herein, a subject polypeptide comprising AF1 comprises a light chain complementarity determining region (CDR-L) and a heavy chain complementarity determining region (CDR-H) listed in Table 1, wherein AF1 binds to EGFR or an epitope thereof. Additionally or alternatively, a subject AF1 of the present disclosure may comprise a CDR-L or CDR-H having at least 60% identity with any of the CDR-L or CDR-H listed in Table 1. In some aspects, a subject AF1 of the present disclosure may comprise a CDR-L or CDR-H and may exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with any of the SEQ ID NOs listed in Table 1. Furthermore, a subject AF1 of the embodiments may further comprise a light chain framework region (FR-L) and a heavy chain framework region (FR-H) listed in Table 2. In some aspects, a subject AF1 of the present disclosure may comprise an FR-L or FR-H that exhibits at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more sequence identity to any of the SEQ ID NOs listed in Table 2. In one embodiment, AF1 of any of the subject composition embodiments described herein is a chimeric or humanized antigen-binding fragment. In one embodiment, AF1 of any of the subject composition embodiments described herein is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, a linear antibody, and a single-chain variable fragment (scFv). AF1 having a CDR-H and a CDR-L may be configured in a (CDR-H)-(CDR-L) or (CDR-H)-(CDR-L) orientation from the N-terminus to the C-terminus.

[0134] In one embodiment, the disclosure provides a polypeptide comprising AF1, wherein AF1 comprises a CDR-L and a CDR-H, and a heavy chain framework region (FR-H), wherein AF1 (a) specifically binds to EGFR, and (b) comprises FR-H1, FR-H2, FR-H3, and FR-H4, wherein FR-H1 has the amino acid sequence of any one of SEQ ID NOs: 14-16, FR-H2 has the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19, FR-H3 has the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, and FR-H4 has the amino acid sequence of any one of SEQ ID NOs: 22-24. In another embodiment, a polypeptide of a subject composition embodiment described herein comprises AF1, wherein AF1 comprises CDR-H3, wherein CDR-H3 has the amino acid sequence of SEQ ID NO: 6. In another embodiment, AF1 comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0135] In another embodiment, the present disclosure provides a polypeptide comprising AF1, wherein AF1 has a higher isoelectric point (pI) than the isoelectric point (pI) of an antigen-binding fragment consisting of the sequence set forth in SEQ ID NO: 52, as demonstrated by in vitro assays. In one embodiment, AF1 is incorporated into a polypeptide to form an anti-EGFR bispecific antibody, wherein the polypeptide exhibits a higher pI relative to a control bispecific antibody, the polypeptide comprising AF1 and a reference antigen-binding fragment that binds to cluster of differentiation 3 T-cell receptor (CD3), the control bispecific antigen-binding fragment being identical to the polypeptide except that AF1 is replaced with SEQ ID NO: 52. In the foregoing embodiment, AF1 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units higher than the pI of the antigen-binding fragment consisting of the sequence set forth in SEQ ID NO: 52. In the foregoing embodiment, the in vitro assay for determining pI can be capillary isoelectric focusing or other assays known in the art.

[0136] In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 comprises a CDR-L, a CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H), wherein AF1 (a) is configured to specifically bind to EGFR, (b) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, and (c) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. , FR-H2, FR-H3, and FR-H4, each of which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NOs: 14-16, SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, and SEQ ID NOs: 22-24, respectively, and further comprises FR-L, which FR-L exhibits (a) an amino acid sequence identical to or identical to the amino acid sequence of SEQ ID NO: 7. (b) FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO:8; (c) FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO:9; and (d) FR-L4 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 13.

[0137] In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 comprises a CDR-L, a CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H), wherein AF1 (a) is configured to specifically bind to EGFR, (b) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, and (c) comprises FR-H1, FR-H2, and CDR-H3. 2, FR-H3, and FR-H4, each of which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequences of SEQ ID NOs: 14-16, SEQ ID NOs: 18 and 19, SEQ ID NOs: 18 and 19, SEQ ID NOs: 20 and 21, and SEQ ID NOs: 22-24, respectively; and (d) FR-L, which further comprises (i) the amino acid sequence of SEQ ID NO: 7. (ii) FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 8; (iii) FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 1; 9, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NO: 10; and (iv) FR-L4 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NO: 13.

[0138] In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 comprises a CDR-L, a CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H), wherein AF1 (a) is configured to specifically bind to EGFR, (b) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, and (c) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. (i) R-H2, FR-H3, and FR-H4, each of which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 14-16, SEQ ID NO: 18 and 19, SEQ ID NO: 20 and 21, and SEQ ID NO: 22-24, respectively; and (d) FR-L, which (i) has at least one amino acid sequence identical to or identical to the amino acid sequence of SEQ ID NO: 7. (ii) FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NO: 8; (iii) FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NO: 11; and (iv) FR-L4 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 13.

[0139] In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 comprises a CDR-L, a CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H), wherein AF1 (a) is configured to specifically bind to EGFR, (b) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, and (c) comprises a CDR-L, a CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H). (d) FR-L1, FR-L2, FR-L3, and FR-L4, each of which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9-11, and SEQ ID NO:13; and (d) FR-H1, FR-H2, FR-H3, and FR-H4, each of which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequences of SEQ ID NO:14. FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 18; FR-H2 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 2 0, and FR-H4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of SEQ ID NO: 22 or 23, and FR-H5 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of SEQ ID NO: 24 or 25.

[0140] In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 comprises a CDR-L, a CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H), wherein AF1 (a) is configured to specifically bind to EGFR, (b) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, and (c) comprises FR-L1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. , FR-L2, FR-L3, and FR-L4, each of which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9-11, and SEQ ID NO:13, respectively; and (d) FR-H1, FR-H2, FR-H3, and FR-H4, FR-H1 exhibiting at least the amino acid sequence of SEQ ID NO:15. FR-H1 has the sequence of at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, FR-H1, and FR-H2 has the amino acid sequence of SEQ ID NO: 19 having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, FR-H FR-H3 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO:21, and FR-H4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO:24.

[0141] In yet another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 comprises a CDR-L, a CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H), wherein AF1 (a) is configured to specifically bind to EGFR, (b) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively, and (c) comprises FR-L1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. , FR-L2, FR-L3, and FR-L4, each of which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9-11, and SEQ ID NO:13; and (d) FR-H1, FR-H2, FR-H3, and FR-H4, wherein FR-H1 has the amino acid sequence of SEQ ID NO:16. FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 19, and FR-H2 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 20. FR-H3 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 22 or 23, and FR-H4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 22 or 23.

[0142] In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 is configured to specifically bind to EGFR, and wherein AF1 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NOs: 28-32.

[0143] In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 is configured to specifically bind to EGFR, and wherein AF1 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NOs:25-27.

[0144] In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NOs: 28-32, and a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NOs: 25-27. AF1 can be configured in a VL-VH or VH-VL orientation and is fused by a linker peptide.

[0145] In yet another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of any one of SEQ ID NOs: 37-51.

[0146] It will be understood that the use of the term "antigen-binding fragment" with respect to the composition embodiments disclosed herein is not limited and is intended to include portions or fragments of antibodies that retain the ability to bind to an antigen that is the ligand of the corresponding intact antibody. In such embodiments, the antigen-binding fragment may be, but is not limited to, CDRs and intervening framework regions, variable or hypervariable regions of the antibody light and / or heavy chains (VL, VH), variable fragments (Fv), Fab' fragments, F(ab')2 fragments, Fab fragments, single-chain antibodies (scAbs), VHH camelid antibodies, single-chain variable fragments (scFv), linear antibodies, single-domain antibodies, complementarity-determining regions (CDRs), domain antibodies (dAbs), BHH-type or BNAR-type single-domain heavy chain immunoglobulins, single-domain light chain immunoglobulins, or other polypeptides known in the art, including fragments of antibodies capable of binding to antigen. The VL and VH of the two antigen-binding fragments can also be configured in a single-chain diabody configuration, i.e., the VL and VH of AF1 and AF2 are configured with a linker of appropriate length to allow configuration as a diabody.

[0147] In certain embodiments, the VL and VH of the antigen-binding fragment are fused by a relatively long linker of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids that have flexible properties when linked 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 selected from the sequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 790), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 791), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 792), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 793).

[0148] In yet another embodiment, AFl of any of the subject composition embodiments described herein specifically binds to human or cynomolgus monkey (cyno) EGFR. In another embodiment, AFl of any of the subject composition embodiments described herein specifically binds to human and cynomolgus monkey (cyno) EGFR.

[0149] In another aspect, the present disclosure provides AF1 having specific binding affinity for EGFR for incorporation into a subject composition, wherein one or more individual amino acids in the framework regions have been modified to increase the pI of AF1 relative to the parent antigen-binding fragment in order to enhance the stability of the bispecific polypeptide into which it is incorporated. In one embodiment, the polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 exhibits a pI of about 5.4, about 5.5, or about 5.6, or about 5.6, or about 5.7, or about 5.8, or about 5.9, or about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6, as demonstrated by in vitro assay. In another embodiment, the polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 exhibits a pI of 5.4 to 6.6 (inclusive), as demonstrated by in vitro assay. In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 exhibits a pI of about 5.4 to 6.6, or about 5.6 to about 6.4, or about 5.8 to about 6.2, or about 6.0 to about 6.2, or about 6.1 to about 6.3, or about 6.2 to about 6.4, or about 6.3 to about 6.5, or about 6.4 to about 6.6, as determined computationally or demonstrated by in vitro assays.

[0150] In another aspect, the present disclosure provides AF1 having specific binding affinity for EGFR antigen for incorporation into a subject composition having a binding affinity for EGFR antigen within a set range. In one embodiment, the polypeptide of any of the subject composition embodiments described herein comprises AF1, wherein AF1 has a K of about 0.1 nM to about 100 nM as determined in an in vitro antigen binding assay involving an EGFR antigen. d In another embodiment, AF1 specifically binds to EGFR with a binding affinity (K in an in vitro binding assay) of less than about 0.1 nM, or less than about 0.5 nM, or less than about 1.0 nM, or less than about 10 nM, or less than about 50 nM, or less than about 100 nM. d In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF1 and AF2, wherein the binding affinity of AF1 to EGFR is at least 10-fold higher, or at least 100-fold higher, or at least 1000-fold higher than the binding affinity of AF2 to CD3, as measured in an in vitro antigen binding assay. d It will be understood that a value such as a binding affinity of 1 nM is a binding affinity greater than 10 nM.The binding affinity of the subject composition to target ligand can be assayed using binding assay or competitive binding assay, for example, Biacore assay using chip-bound receptor or binding protein, or the ELISA assay described in U.S. Patent No. 5,534,617, the assay described in the Examples herein, radioreceptor assay, or other assays known in the art.Then, binding affinity constant can be determined using standard methods such as Scatchard analysis described by van Zoelen, et al., Trends Pharmacol Sciences (1998) 19) 12): 487, or other methods known in the art.

[0151] In another aspect, the present disclosure provides AFl having specific binding affinity for EGFR for incorporation into a subject composition, wherein one or more individual amino acids in the framework regions have been modified to reduce the hydrophobicity of the antigen-binding framework relative to the parent antigen-binding fragment in order to enhance the stability of the bispecific polypeptide into which it is incorporated. In one embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AFl, wherein AFl specifically binds to EGFR, and wherein AFl has at least one amino acid substitution of a hydrophobic amino acid in the framework region relative to the amino acid sequence of SEQ ID NO: 52, wherein the hydrophobic amino acid is selected from isoleucine, leucine, or methionine, and the replacement amino acid is selected from arginine, threonine, or glutamine. In another embodiment, AFl has at least two amino acid substitutions of a hydrophobic amino acid in one or more framework regions relative to the amino acid sequence of SEQ ID NO: 52, wherein the hydrophobic amino acid is selected from isoleucine, leucine, or methionine, and the replacement amino acid is selected from arginine, threonine, or glutamine. [Table 1] [Table 2] [Table 3] [Table 4-1] [Table 4-2]

[0152] III) Release segment In another aspect, the present disclosure relates to release segment (RS) peptides suitable for inclusion in the subject compositions described herein that are substrates for one or more mammalian proteases associated with or produced by cells found in or near diseased tissue. Such proteases include, but are not limited to, classes of proteases such as metalloproteases, cysteine ​​proteases, aspartic acid proteases, and serine proteases. RSs are useful, among other things, to impart a prodrug format to the subject compositions that can be activated by cleavage of the RS by a mammalian protease. As described herein, RSs are incorporated into the subject composition embodiments described herein to link the incorporated antigen-binding fragments to XTENs (the configuration of which is described more fully below), such that upon cleavage of the RS by the action of one or more proteases for which the RS is a substrate, the antigen-binding fragments and XTENs are released from the composition, and the antigen-binding fragments no longer shielded by the XTENs increase their binding availability to their respective ligands. In a specific aspect, the RS serves as a substrate for a protease found in close association with or co-localized with diseased tissues or cells, such as, but not limited to, tumors, cancer cells, and inflamed tissues; upon cleavage of the RS, antigen-binding fragments that would otherwise be shielded by the XTEN of the subject compositions (and therefore have lower binding affinity for their respective ligands) are released from the composition and regain increased ability to bind to target and / or effector cell ligands. In another embodiment, the RS of the subject polypeptide compositions comprises an amino acid sequence that is a substrate for a cellular protease located within the target cell. In another specific aspect of the subject compositions described herein, RSs that are substrates for two or three classes of proteases are designed with sequences that can be cleaved at different positions in the RS sequence by different proteases; representative examples are shown in FIG. 6.Thus, an RS that is a substrate for two, three, or more classes of proteases will have two, three, or multiple distinct cleavage sites within the RS sequence, but cleavage by a single protease will nevertheless result in the release of an antigen-binding fragment and an XTEN from a composition comprising the RS.

[0153] In one embodiment, the disclosure provides an activatable polypeptide comprising one or more release segments, wherein the release segments are substrates for cleavage by one or more mammalian proteases. In another embodiment, the disclosure provides a polypeptide comprising a first release segment (RS1) sequence, wherein RS1 is a substrate for cleavage by a mammalian protease, wherein RS1 is a substrate for a protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other instances, the polypeptide of any of the subject composition embodiments described herein comprises a first release segment (RS1) sequence, wherein RS1 is selected from the group consisting of meprin, neprilysin (CD10), PSMA, BMP-1, A disintegrin and metalloproteinase (ADAM), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM with thrombospondin motifs (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3 (MMP-3, stromelysin 1), matrix metalloproteinase-4 (MMP-4, stromelysin 1), matrix metalloproteinase-5 (MMP-5, stromelysin 1), matrix metalloproteinase-6 (MMP-6, stromelysin 1), matrix metalloproteinase-7 (MMP-7, stromelysin 1), matrix metalloproteinase-8 (MMP-8, stromelysin 1), matrix metalloproteinase-9 (MMP-8, stromelysin 1), matrix metalloproteinase-10 (MMP-8, stromelysin 1), matrix metalloproteinase-11 (MMP-8, stromelysin 1), matrix metalloproteinase-12 (MMP-8, stromelysin 1), matrix metalloproteinase-13 (MMP-8, stromelysin 1), matrix metalloproteinase-14 (MMP-8, stromelysin 1), matrix metalloproteinase-15 (MMP-8, stromelysin 1), matrix metalloproteinase-16 (MMP-8, stromelysin 1), matrix metalloproteinase-17 (MMP-8, strome matrix metalloproteinase-7 (MMP-7, matrilysin 1), matrix metalloproteinase-8 (MMP-8, collagenase 2), matrix metalloproteinase-9 (MMP-9, gelatinase B), matrix metalloproteinase-10 (MMP-10, stromelysin 2), matrix metalloproteinase-11 (MMP-11, stromelysin 3), matrix metalloproteinase-12 (MMP- 12, macrophage elastase), matrix metalloproteinase-13 (MMP-13, collagenase 3), matrix metalloproteinase-14 (MMP-14, MT1-MMP), matrix metalloproteinase-15 (MMP-15, MT2-MMP), matrix metalloproteinase-19 (MMP-19), matrix metalloproteinase-23 (MMP-23, CA-MMP),Matrix metalloproteinase-24 (MMP-24, MT5-MMP), matrix metalloproteinase-26 (MMP-26, matrilysin 2), matrix metalloproteinase-27 (MMP-27, CMMP), legumain, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X, cathepsin D, cathepsin E, secretase, urokinase (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, prostate-specific antigen (PSA, KLK3), human neutrophil elastase or a substrate for cleavage by one or more mammalian proteases selected from the group consisting of: tase (HNE), elastase, tryptase, type II transmembrane serine protease (TTSP), DESC1, hepsin (HPN), matriptase, matriptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast activation protein (FAP), kallikrein-related peptidase (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14.

[0154] In another embodiment, the disclosure provides a polypeptide comprising a first release segment (RS1) sequence for incorporation into a subject polypeptide composition described herein, wherein RS1 is a substrate for cleavage by one or more mammalian proteases, and 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 selected from SEQ ID NOs: 53-671. In another embodiment, RS1 comprises an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which is set forth in Table 5. As described in more detail below in the description of the composition and properties of the subject polypeptide compositions, the release segment is fused between the antigen-binding fragment and the XTEN polypeptide such that upon cleavage of the release segment, the XTEN is released from the composition.

[0155] In other embodiments, the present disclosure provides a polypeptide comprising a first release segment (RS1) sequence and a second release segment (RS2) for incorporation into a subject polypeptide composition described herein, wherein RS1 and RS2 are identical. In another embodiment, the present disclosure provides a polypeptide comprising a first release segment (RS1) sequence and a second release segment (RS2) for incorporation into a subject polypeptide composition, wherein RS1 and RS2 are different. In some instances of the foregoing embodiments, RS1 and RS2 are each substrates for cleavage by a mammalian protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In another embodiment, the disclosure provides a polypeptide comprising RS1 and RS2 sequences for incorporation into a subject polypeptide composition described herein, wherein RS1 and RS2 are each substrates for cleavage by one or more mammalian proteases, and wherein RS1 and RS2 each comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 53-671. In another embodiment, RS1 and RS2 each comprise an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which is set forth in Table 5. As described in more detail below in the sections relating to the description of the composition and properties of the subject polypeptide compositions, the release segments are fused between the antigen-binding fragment and the XTEN polypeptide such that upon cleavage of each release segment, the adjacent XTEN is released from the composition. [Table 5-1] Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 Table 5-9 Table 5-10 Table 5-11 Table 5-12 Table 5-13 Table 5-14 Table 5-15 Table 5-16 Table 5-17 Table 5-18 [Table 5-19] [Table 5-20]

[0156] In another aspect, the release segments (either RS1 and / or RS2) for incorporation into the polypeptides of any of the subject composition embodiments described herein can be designed to be selectively susceptible to different cleavage rates and cleavage efficiencies for the various proteases for which they are substrates. Because a given protease may be found at different concentrations in diseased tissues, including, but not limited to, tumors, blood cancers, or inflamed tissues or sites, compared to healthy tissues or circulating blood, the present disclosure provides RSs in which the individual amino acid sequences are engineered to have higher or lower cleavage efficiencies for a given protease compared to the cleavage rate of the release segment in healthy tissues or circulating blood, such that the released antigen-binding fragments have a higher ability to bind to ligands in diseased tissues compared to the prodrug form that remains in the circulating blood, ensuring that the polypeptide is preferentially converted from the prodrug form to the active form (i.e., by separation and release of the antigen-binding fragment and XTEN from the polypeptide after cleavage of the release segment) when in the vicinity of target cells or tissues and their co-localized proteases. Such selective design can improve the therapeutic index of the resulting composition and reduce side effects compared to conventional therapeutic agents that do not incorporate such site-specific activation.

[0157] As used herein, cleavage efficiency is defined as the log2 value of the ratio of the percentage of test substrates containing cleaved release segments to the percentage of cleaved control substrate RSR-1517 (AC1611) when subjected to a protease enzyme in a biochemical assay (described further in the Examples) in which the reaction is performed, where the initial substrate concentration is 6 μM, the reaction is stopped (e.g., by adding EDTA) after incubation at 37° C. for 2 hours, and the amount of digestion product and uncleaved substrate are analyzed using non-reducing SDS-PAGE to establish the ratio of the percentage of cleaved release segments. Cleavage efficiency is calculated as follows:

number

[0158] Thus, a cleavage efficiency of -1 means that the amount of cleavage of the test substrate was 50% of the amount of cleavage of the control substrate, and a cleavage efficiency of +1 means that the amount of cleavage of the test substrate was 200% of the amount of cleavage of the control substrate. A higher cleavage rate by the test protease compared to the control results in a higher cleavage efficiency, and a slower cleavage rate by the test protease compared to the control results in a lower cleavage efficiency. As detailed in the Examples, when tested in in vitro biochemical assays for cleavage rates by individual proteases, the control RS sequence AC1611 (RSR-1517), having the amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO: 53), was established as having an appropriate baseline cleavage efficiency by the proteases legumain, MMP-2, MMP-7, MMP-9, MMP-14, uPA, and matriptase. By selectively substituting amino acids at individual positions in the RS peptide, a library of RSs was generated and evaluated against a panel of seven proteases (more fully detailed in the Examples), resulting in profiles that were used to establish guidelines for appropriate amino acid substitutions to achieve RSs with desired cleavage efficiencies. While substitutions using the hydrophilic amino acids A, E, G, P, S, and T are preferred in generating RSs with desired cleavage efficiencies, other L-amino acids can be substituted at a given position to adjust cleavage efficiency, as long as the release segment retains at least some susceptibility to cleavage by the protease.

[0159] IV). XTEN Polypeptides In another aspect, the present disclosure relates to polypeptides comprising at least a first extended recombinant polypeptide (XTEN) incorporated into the subject composition embodiments described herein, thereby serving both to increase the mass and size of the construct and to significantly reduce the ability of the antigen-binding fragment to bind to a ligand when the molecule is in its intact, uncleaved state, as described more fully below. In some embodiments, the present disclosure provides polypeptides comprising a single XTEN fused to the end of an RS located between the antigen-binding fragment and the XTEN. In other embodiments, the present disclosure provides polypeptides comprising a first XTEN and a second XTEN (XTEN1 and XTEN2) fused to the N- and C-termini of RS1 and RS2, respectively, located between each antigen-binding fragment and the XTEN.

[0160] Without being bound by theory, the incorporation of XTEN can be incorporated into the design of the subject compositions to impart certain properties: 1) providing the polypeptide composition with XTEN that shields the antigen-binding fragments and reduces their binding affinity for target cell markers and effector cell antigens when the composition is in its intact prodrug form; ii) providing the polypeptide composition with XTEN that provides an enhanced half-life when administered to a subject; iii) contributing to the solubility and stability of the intact composition, thereby enhancing the pharmaceutical properties of the subject compositions; and iv) providing the polypeptide composition with XTEN that reduces extravasation in normal tissues and organs, but allows some extravasation in diseased tissues (e.g., tumors) that have larger pore sizes in the vasculature and may nevertheless be released from the tissue by the action of certain mammalian proteases, thereby allowing the antigen-binding fragments of the composition to more easily penetrate diseased tissues, e.g., tumors, and bind to and link together target cell markers on effector cells and tumor cells. To meet these needs, the present disclosure provides compositions comprising one or more XTENs, wherein the XTENs provide increased mass and hydrodynamic radius to the resulting compositions. The XTEN polypeptides of the embodiments not only provide increased mass and hydrodynamic radius to the compositions, but their flexible, unstructured nature can also provide a shielding effect for the antigen-binding fragments of the compositions, thereby providing a particular advantage in the design of the subject compositions in that they reduce binding to antigens in normal tissues or the vasculature of normal tissues that do not express, or express at reduced levels of, target cell markers and / or effector cell antigens. Additionally, the incorporation of XTENs into the subject compositions can enhance the solubility and proper folding of single-chain antibody-binding fragments during expression and recovery.

[0161] XTENs are polypeptides having non-naturally occurring, substantially non-repetitive sequences that have some degree of secondary or tertiary structure under physiological conditions, as well as one or more additional properties described in the following paragraphs. In some embodiments, the present disclosure provides polypeptides comprising one or more XTENs having at least about 36, 72, 96, 100, 144, 200, 288, 292, 293, 300, 576, 584, 800, 864, 867, 868, 900, or at least about 1000 or more amino acids. In one embodiment, the disclosure provides a polypeptide comprising XTEN1, wherein the XTEN1 is characterized by having at least about 36 amino acid residues, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues in the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), with at least 4-6 different amino acids selected from G, A, S, T, E, and P. In some embodiments, the disclosure provides a polypeptide comprising XTEN1 having at least about 36 to about 1000, or at least 100 to about 900, or at least about 144 to about 868, or at least about 288 to 868 amino acid residues. In other cases, the disclosure provides polypeptides comprising XTEN1 having at least about 36 to about 1000, or at least 100 to about 900, or at least about 144 to about 868, or at least about 288 to 868 amino acid residues, wherein 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues are selected from four to six types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P).In other instances, the disclosure provides a polypeptide comprising XTEN1, wherein the XTEN1 is characterized by having at least about 36 to about 1000 amino acid residues, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues in the XTEN1 sequence are selected from six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P).

[0162] In another embodiment, the disclosure provides a polypeptide of any of the embodiments described herein comprising XTEN1, wherein the XTEN1 is characterized by having at least about 36 to about 1000, or at least about 100 to about 900, or at least 144 to about 868 amino acid residues, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from at least three of the sequences set forth in SEQ ID NOs: 672-675. In some cases, the XTEN1 sequence can be constructed by any combination of 12-amino acid units of SEQ ID NOs: 672-675 in 12 amino acid increments, such that any length of at least 36 amino acids or greater (e.g., 36, 48, 60, 72, 84, 96 amino acids, etc.) can be achieved. In other cases, the polypeptide of any of the subject composition embodiments described herein can include XTEN1, wherein the XTEN1 is characterized by having at least about 36 to about 1000, or at least about 100 to about 900, or at least 144 to about 868 amino acid residues, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from the sequences of SEQ ID NOs: 676-734. In another embodiment, the XTEN of any of the subject composition embodiments described herein can have an affinity tag of the sequence EPEA (SEQ ID NO: 796), HHHHHH (SEQ ID NO: 794), HHHHHHHH (SEQ ID NO: 795), or added to the N- or C-terminus of the XTEN of the composition to facilitate purification of the composition to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% purity by chromatographic methods known in the art, such as IMAC chromatography or C-tagXL chromatography, or the methods described in the Examples below.

[0163] In another embodiment, the disclosure provides a polypeptide comprising XTEN1, wherein the XTEN1 is selected from the group consisting of AE36 (comprising a sequence selected from any three of the sequences of SEQ ID NOs: 672-675), AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288, 1, AE288_2, AE288_3, AE292, AE293, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7.

[0164] In some aspects of any of the embodiments disclosed herein, the subject polypeptide comprises XTEN1 and XTEN2. The composition of polypeptides comprising, among other components, XTEN1 and XTEN2 is described herein below. In one embodiment, the disclosure provides a polypeptide comprising XTEN1 and XTEN2, wherein XTEN2 is characterized by having at least about 36 to about 1000 amino acid residues, and wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues in the XTEN2 sequence are selected from at least three of the sequences set forth in SEQ ID NOs: 672-675. In another embodiment, the disclosure provides a polypeptide comprising XTEN1 and XTEN2, wherein XTEN1 and XTEN2 each have at least about 36 to about 1000 amino acid residues, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues in the XTEN2 sequence are selected from the sequences of SEQ ID NOs: 676-734. In another embodiment, the polypeptide of any of the subject composition embodiments described herein can comprise XTEN1 and XTEN2, each of which can be selected from the group consisting of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288, 1, AE288_2, AE288_3, AE292, AE293, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7. In some cases of the foregoing embodiments of this paragraph, XTEN1 and XTEN2 are identical. In other cases of the foregoing embodiments of this paragraph, XTEN1 and XTEN2 of the foregoing embodiments of this paragraph have different amino acid sequences.In some cases, XTEN1 of any of the polypeptide composition embodiments having two XTENs is fused to the C-terminus of the polypeptide and is selected from the group consisting of AE293, AE300, AE584, and AEAE868. In other cases, XTEN2 of any of the polypeptide composition embodiments having two XTENs is fused to the N-terminus of the polypeptide and is selected from the group consisting of AE144_7A, AE292, AE576, and AE864. In other cases, XTEN1 of any of the polypeptide composition embodiments having two XTENs is fused to the C-terminus of the polypeptide and is selected from the group consisting of AE293, AE300, AE584, and AEAE868, and XTEN2 is fused to the N-terminus and is selected from the group consisting of AE144_7A, AE292, AE576, and AE864. [Table 6] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 7-11]

[0165] The present disclosure contemplates compositions of any of the embodiments described herein, including XTENs of intermediate lengths relative to those in Table 7, as well as XTENs of lengths greater than those in Table 7, e.g., those in which the 12 amino acid motif of Table 6 is added to the N- or C-terminus of an XTEN in Table 7.

[0166] In another embodiment, the present disclosure contemplates a polypeptide composition of any of the embodiments described herein comprising XTEN1 and XTEN2, which can further comprise a His-tag of HHHHHH (SEQ ID NO: 794) or HHHHHHHH (SEQ ID NO: 795) at the N-terminus of the polypeptide composition and / or a His-tag of the sequence EPEA (SEQ ID NO: 796) at the C-terminus, respectively, to facilitate production of the composition to a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% by chromatographic methods known in the art, including, but not limited to, IMAC chromatography, C-tagXL affinity matrix, and other such methods (e.g., including but not limited to, those described in the following embodiments).

[0167] Additional examples of XTEN sequences that can be used in accordance with the present disclosure are described in U.S. Patent Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1, or These are described in International Patent Publication Nos. 2010 / 091122A1, 2010 / 144502A2, 2010144508A1, 2011 / 028228A1, 2011 / 028229A1, 2011 / 028344A2, 2014 / 011819A2, or 2015 / 023891.

[0168] V).CD3 cell antigen-binding fragment In another aspect, the present disclosure relates to an antigen-binding fragment (AF2) having specific binding affinity for an effector cell antigen, which can be incorporated into any of the subject composition embodiments described herein. In some cases, the effector cell antigen is expressed on the surface of an effector cell selected from plasma cells, T cells, B cells, cytokine-induced killer cells (CIK cells), mast cells, dendritic cells, regulatory T cells (RegT cells), helper T cells, myeloid cells, and NK cells.

[0169] Various AF2s that bind to effector cell antigens are particularly useful for pairing with antigen-binding fragments that have binding affinity for EGFR antigens associated with diseased cells or tissues in a format that results in cell killing of the diseased cells or tissues. Binding specificity can be determined by the complementarity-determining regions, or CDRs, such as light chain or heavy chain CDRs. In many cases, binding specificity is determined by the light chain and heavy chain CDRs. A given combination of heavy chain and light chain CDRs provides a given binding pocket that confers higher affinity and / or specificity for the effector cell antigen compared to other reference antigens. The resulting bispecific composition, in which a first antigen-binding fragment (AF1) for EGFR is linked to a second antigen-binding fragment (AF2) with binding specificity for the effector cell antigen via a short, flexible peptide linker, is a bispecific in which each antigen-binding fragment has specific binding affinity for its respective ligand. It will be appreciated that in such compositions, AF1 directed against the EGFR of the diseased tissue is used in combination with AF2 directed toward an effector cell marker to bring effector cells into close proximity with cells of the diseased tissue, resulting in cytolysis of the cells of the diseased tissue. Furthermore, AF1 and AF2 are incorporated into a specifically designed polypeptide comprising a cleavable release segment and XTEN to confer prodrug properties to the composition that are activated by release of the fused AF1 and AF2 upon cleavage of the release segment when in the vicinity of diseased tissue that has a protease capable of cleaving the release segment at one or more positions in the release segment sequence.

[0170] In one embodiment, AF2 of the subject compositions has binding affinity for an effector cell antigen expressed on the surface of T cells. In another embodiment, AF2 of the subject compositions has binding affinity for CD3. In another embodiment, AF2 of the subject compositions has binding affinity for members of the CD3 complex, including all known CD3 subunits of the CD3 complex, e.g., CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta, either individually or in combination. In another embodiment, AF2 has binding affinity for CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, or CD3 beta.

[0171] The antigen-binding fragments contemplated by the present disclosure can be derived from naturally occurring antibodies or fragments thereof, non-naturally occurring antibodies or fragments thereof, humanized antibodies or fragments thereof, synthetic antibodies or fragments thereof, hybrid antibodies or fragments thereof, or engineered antibodies or fragments thereof. Methods for generating antibodies against a given target marker are well known in the art. For example, monoclonal antibodies can be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (U.S. Patent No. 4,816,567). The structure of antibodies and their fragments, including antibody heavy and light chain variable regions (VH and VL), single-chain variable regions (scFv), complementarity-determining regions (CDRs), and domain antibodies (dAbs), are well understood. Methods for generating polypeptides having desired antigen-binding fragments with binding affinity for a given antigen are known in the art.

[0172] It will be understood that the use of the term "antigen-binding fragment" with respect to the composition embodiments disclosed herein is intended to include portions or fragments of antibodies that retain the ability to bind to an antigen that is the ligand of the corresponding intact antibody. In such embodiments, the antigen-binding fragment may be, but is not limited to, CDRs and intervening framework regions, variable or hypervariable regions of the antibody light and / or heavy chains (VL, VH), variable fragments (Fv), Fab' fragments, F(ab')2 fragments, Fab fragments, single-chain antibodies (scAbs), VHH camelid antibodies, single-chain variable fragments (scFv), linear antibodies, single-domain antibodies, complementarity-determining regions (CDRs), domain antibodies (dAbs), BHH-type or BNAR-type single-domain heavy chain immunoglobulins, single-domain light chain immunoglobulins, or other polypeptides known in the art, including fragments of antibodies capable of binding to antigen. An antigen-binding fragment having a CDR-H and a CDR-L can be configured in the (CDR-H)-(CDR-L) or (CDR-H)-(CDR-L) orientation from N-terminus to C-terminus. The VL and VH of two antigen-binding fragments can also be configured in a single-chain diabody configuration, i.e., the VL and VH of AF1 and AF2 are configured with a linker of appropriate length to allow for configuration as a diabody.

[0173] The various CD3-binding AF2s disclosed herein have been specifically modified to enhance their stability in the polypeptide embodiments described herein. Protein aggregation of antibodies continues to pose a significant challenge to their developability and remains a key area of ​​focus in antibody production. Antibody aggregation can be triggered by partial unfolding of their domains, leading to monomer-monomer association followed by nucleation and aggregate growth. While the aggregation propensity of antibodies and antibody-based proteins can be influenced by external experimental conditions, it is strongly dependent on intrinsic antibody properties determined by their sequence and structure. While it is well known that proteins are marginally stable in their folded state, it is less understood that most proteins are inherently prone to aggregation in their unfolded or partially unfolded state, and that the resulting aggregates can be highly stable and long-lived. Reduced aggregation propensity has also been shown to be accompanied by increased expression titers, indicating that reduced protein aggregation can be beneficial throughout the development process and lead to a more efficient path to clinical trials. For therapeutic proteins, aggregates are a significant risk factor for adverse immune reactions in patients and can occur through a variety of mechanisms. Controlling aggregation can improve protein stability, manufacturability, wear rate, safety, formulation, potency, immunogenicity, and solubility. Intrinsic protein properties, such as size, hydrophobicity, electrostatics, and charge distribution, play an important role in protein solubility. It has been shown that low solubility of therapeutic proteins due to surface hydrophobicity can make formulation development more challenging and lead to poor in vivo biodistribution, undesirable pharmacokinetic behavior, and immunogenicity. Reducing the overall surface hydrophobicity of candidate monoclonal antibodies can also provide benefits and cost savings related to purification and administration regimens. Individual amino acids can be identified through structural analysis as contributing to antibody aggregation potential and can be located in both CDRs and framework regions. Residues, in particular, can be predicted to be at high risk of causing hydrophobicity problems for a given antibody.In one embodiment, the present disclosure provides an AF2 having the ability to specifically bind to CD3, wherein AF2 has at least one amino acid substitution in a framework region of a hydrophobic amino acid selected from isoleucine, leucine, or methionine relative to a parent antibody or antibody fragment. In another embodiment, CD3 AF2 has at least two amino acid substitutions in one or more framework regions of a hydrophobic amino acid selected from isoleucine, leucine, or methionine.

[0174] The isoelectric point (pI) is the pH at which an antibody or antibody fragment has no net charge. If the pH is below the pI of an antibody or antibody fragment, it has a net positive charge. A greater positive charge tends to correlate with increased blood clearance and tissue retention, generally resulting in a shorter half-life. If the pH is above the pI of an antibody or antibody fragment, it has a negative charge. A negative charge generally results in decreased tissue uptake and a longer half-life. This charge on framework residues can be manipulated through mutation. These considerations informed the design of the AF2 sequence of the embodiments described herein, in which individual amino acid substitutions were made to the parent antibody used as a starting point. The isoelectric point of a polypeptide can be determined mathematically (e.g., computationally) or experimentally in in vitro assays. The isoelectric point (pI) is the pH at which a protein has a net charge of zero and can be calculated using the charges of specific amino acids in the protein sequence. An estimate of the charge, called the acid dissociation constant or pKa value, is used to calculate the pI. The pI can be determined in vitro by methods such as capillary isoelectric focusing (see Datta-Mannan, A., et al. The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies. mAbs 7:1084 (2015); Li, B., et al. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecular charge. mAbs 6, 1255-1264 (2014)) or other methods known in the art. In some embodiments, the isoelectric points of AF1 and AF2 are designed to be within a certain range of each other, thereby promoting stability.

[0175] In one embodiment, the present disclosure provides AF2 for use in any of the polypeptide embodiments described herein comprising a CDR-L and a CDR-H, wherein AF2 (a) specifically binds to the cluster of differentiation 3 T-cell receptor (CD3), and (b) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 742, 743, and 744, respectively. In another embodiment, the disclosure provides AF2 for use in any of the polypeptide embodiments described herein, comprising a CDR-L and a CDR-H, wherein AF2 (a) specifically binds to the cluster of differentiation 3 T-cell receptor (CD3); (b) comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 742, 743, and 744, respectively; and (c) comprises a CDR-L, wherein the CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 735 or 736, CDR-L2 having the amino acid sequence of SEQ ID NO: 738 or 739, and CDR-L3 having the amino acid sequence of SEQ ID NO: 740.In another embodiment, the aforementioned AF2 embodiment of this paragraph further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 comprises a FR-L1 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 746 and a FR-L1 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 747. FR-L2 exhibits 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical thereto; FR-L3 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical thereto with the amino acid sequence of any one of SEQ ID NOs: 748 to 751; and FR-L4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical thereto with the amino acid sequence of SEQ ID NO: 754. FR-L4 exhibiting 7%, 98%, 99% sequence identity or being identical thereto; FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto with the amino acid sequence of SEQ ID NO: 755 or SEQ ID NO: 756; and FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the amino acid sequence of SEQ ID NO: 759. FR-H2 exhibiting or being identical to the amino acid sequence of SEQ ID NO: 760; FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or being identical to the amino acid sequence of SEQ ID NO: 760; and FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or being identical to the amino acid sequence of SEQ ID NO: 764.In another embodiment, AF2 for use in any of the polypeptide embodiments described herein comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 comprises FR-L1 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 746 and a heavy chain framework region (FR-H) that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 747. FR-L2 exhibiting 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; and FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; and FR-L5 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 755; FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 759. FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 760; and FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 764.In another embodiment, AF2 for use in any of the polypeptide embodiments described herein comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 comprises FR-L1 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 746, and a heavy chain framework region (FR-H) that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 746. FR-L2, which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 747; and FR-L3, which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 749. L3 and FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NO: 754; and FR-L4 and FR-L5 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NO: 755. FR-H1, which is identical thereto, exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 759, or FR-H2, which is identical thereto, exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 760. or is identical thereto, and FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 764.In another embodiment, AF2 of the subject polypeptide embodiments described herein comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 comprises a FR-L1 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 746 and a FR-L1 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 747. FR-L2 exhibiting 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; and FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; and FR-L5 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 755; FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 759; or is identical thereto; FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 760; and FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 764.In another embodiment, AF2 of the subject polypeptide embodiments described herein comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 comprises a FR-L1 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 746 and a FR-L1 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 747. FR-L2 exhibiting 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; and FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; and FR-L5 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical thereto; FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 756; FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 759; or is identical thereto; FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 760; and FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to the amino acid sequence of SEQ ID NO: 764.

[0176] In another embodiment, the disclosure provides AF2 for use in any of the polypeptide embodiments described herein, wherein AF2 comprises a variable heavy (VH) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of SEQ ID NO: 766 or SEQ ID NO: 769. In another embodiment, the disclosure provides AF2 for use in any of the polypeptide embodiments described herein, wherein AF2 comprises a variable light (VL) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of any one of SEQ ID NOs: 765, 767, 768, 770, or 771. In another embodiment, the disclosure provides AF2 for use in any of the polypeptide embodiments described herein, wherein AF2 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NO: 766 or SEQ ID NO: 769, and a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of any one of SEQ ID NOs: 765, 767, 768, 770, or 771.

[0177] In another embodiment, the disclosure provides AF2 for use in any of the polypeptide embodiments described herein, wherein AF2 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of any one of SEQ ID NOs: 776-780.

[0178] In another aspect, the present disclosure provides AF2 antigen-binding fragments that bind to the CD3 protein complex and have enhanced stability compared to CD3-binding antibodies or antigen-binding fragments known in the art. Additionally, the CD3 antigen-binding fragments of the present disclosure are designed to confer greater stability to chimeric bispecific antigen-binding fragment compositions into which they are incorporated, resulting in improved expression and recovery, increased shelf life, and enhanced stability of the fusion protein when administered to a subject. In one approach, the CD3 AF2 of the present disclosure is designed to have greater thermal stability compared to certain CD3-binding antibodies and antigen-binding fragments known in the art. As a result, CD3 AF2s utilized as components of chimeric bispecific antigen-binding fragment compositions into which they are incorporated exhibit favorable pharmaceutical properties, including high thermal stability and low aggregation tendency, resulting in improved expression and recovery during manufacturing and storage, and promoting a long serum half-life. Biophysical properties such as thermal stability are often limited by antibody variable domains, whose intrinsic properties vary widely. High thermal stability is often associated with high expression levels and other desirable properties, such as low susceptibility 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 measured by the "melting temperature" (T), defined as the temperature at which half of the molecule is denatured. m The melting temperature of each heterodimer indicates its thermal stability. mIn vitro assays for determining the melting point of a heterodimer are known in the art. The melting point of a heterodimer can be measured using techniques such as differential scanning calorimetry (Chen et al. (2003) Pharm Res 20:1952-60, Ghirlando et al. (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of a heterodimer can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9) or as described in the Examples below.

[0179] Thermal denaturation curves of the CD3-binding fragments of the present disclosure and anti-CD3 bispecific antibodies comprising the anti-CD3 binding fragments and a reference binding fragment show that the constructs of the present disclosure are more resistant to thermal denaturation than an antigen-binding fragment consisting of the sequence set forth in SEQ ID NO: 781 or a control bispecific antibody comprising SEQ ID NO: 781 and a reference antigen-binding fragment that binds to an EGFR embodiment described herein. In one embodiment, the polypeptide of any of the subject composition embodiments described herein comprises an anti-CD3 AF2 of an embodiment described herein, wherein the T of AF2 is m is the T of an antigen-binding fragment consisting of the sequence of SEQ ID NO: 781, as determined by an increase in melting temperature in an in vitro assay. m at least 2°C higher, or at least 3°C ​​higher, or at least 4°C higher, or at least 5°C higher, or at least 6°C higher, or at least 7°C higher, or at least 8°C higher, or at least 9°C higher, or at least 10°C higher.

[0180] In another embodiment, the polypeptide of any of the subject composition embodiments described herein has a dissociation constant (K) of about 10 nM to about 400 nM, or about 50 nM to about 350 nM, or about 100 nM to 300 nM, as determined in an in vitro antigen binding assay involving human or cyno CD3 antigen. dIn another embodiment, the polypeptide of any of the subject composition embodiments described herein comprises AF2 that specifically binds to human or cyno CD3 with 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 about 400 nM, as determined in an in vitro antigen binding assay. d ) containing AF2, which specifically binds to human or cyno CD3. For clarity, a K of 400 d An antigen-binding fragment having a K d In another embodiment, the polypeptides of any of the subject composition embodiments described herein bind to their ligands more weakly than those having a respective dissociation constant (K d AF2 specifically binds to human or cyno CD3 with a binding affinity 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 than an antibody-binding fragment consisting of the amino acid sequence of SEQ ID NO: 781, as determined by a dissociation constant (K dThe present invention provides a bispecific polypeptide comprising an AF2 that exhibits a binding affinity for CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold weaker than the binding affinity of an AF1 EGFR embodiment incorporated into the subject polypeptide, as determined by ELISA. The binding affinity of the subject compositions for the target ligand can be assayed using a binding or competitive binding assay, such as a Biacore assay using a chip-bound receptor or binding protein, or an ELISA assay as described in U.S. Pat. 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 standard methods, such as Scatchard analysis as described by van Zoelen, et al., Trends Pharmacol Sciences (1998) 19) 12):487, or other methods known in the art.

[0181] In a related aspect, the present disclosure provides AF2 incorporated into chimeric bispecific polypeptide compositions that bind to CD3 and are designed to have an isoelectric point (pI) that confers enhanced stability to the disclosed compositions relative to corresponding compositions comprising CD3-binding antibodies or antigen-binding fragments known in the art. In one embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF2 that binds to CD3, wherein the AF2 exhibits a pI that is between 6.0 and 6.6, inclusive. In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF2 that binds to CD3, wherein the AF2 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the pI of a reference antigen-binding fragment consisting of the sequence set forth in SEQ ID NO:781. In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises a CD3-binding AF2 fused to an AF1 that binds an EGFR antigen, wherein AF2 exhibits a pI that is within at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units of the pI of AF1 that binds the EGFR antigen or an epitope thereof. In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises a CD3-binding AF2 fused to an AF1 that binds an EGFR antigen, wherein AF2 exhibits a pI that is within at least about 0.1 to about 1.5, or at least about 0.3 to about 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 to about 0.9 pH units of the pI of AF1. By designing the pI of these two antigen-binding fragments to be within such ranges, it is specifically intended that the resulting fused antigen-binding fragments will confer a greater degree of stability to the chimeric bispecific antigen-binding fragment composition into which they are incorporated, resulting in improved expression and enhanced recovery of the fusion protein in a soluble, non-aggregated form, increased shelf life of the formulated chimeric bispecific polypeptide composition, and enhanced stability when the composition is administered to a subject.Separately, having AF2 and AF1 within a relatively narrow pI range can allow for the selection of a buffer or other solution in which both AF2 and AF1 are stable, thereby promoting the overall stability of the composition.

[0182] In certain embodiments, the VL and VH of the antigen-binding fragment are fused together by a relatively long linker of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids that has flexible properties when linked together. In one embodiment, the VL and VH of any of the scFv embodiments described herein are linked together by a relatively long linker of hydrophilic amino acids selected from the sequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 790), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 791), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 792), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 793). In another embodiment, AF1 and AF2 are linked together by a short linker of hydrophilic amino acids having 3, 4, 5, 6, or 7 amino acids. In one embodiment, the short linker sequence is selected from the group consisting of the sequences SGGGGS (SEQ ID NO:797), GGGGS (SEQ ID NO:798), GGSGGS (SEQ ID NO:799), GGS, or GSP. In another embodiment, the present disclosure provides a composition comprising a single-chain diabody in which, after folding, the first domain (VL or VH) pairs with the last domain (VH or VL) to form one scFv, and these two domains pair in the middle to form the other scFv, where the first and second domains and the third and last domains are fused together by one of the aforementioned short linkers, and the second and third variable domains are fused by one of the aforementioned relatively long linkers. As will be understood by those skilled in the art, the selection of short and relatively long linkers is to prevent incorrect pairing of adjacent variable domains, thereby facilitating the formation of a single-chain diabody configuration comprising the VL and VH of the first antigen-binding fragment and the second antigen-binding fragment. [Table 8] [Table 9] [Table 10] [Table 11-1] [Table 11-2]

[0183] VI. Bispecific antigen-binding compositions—construction and functional properties In another aspect, the present disclosure relates to novel chimeric bispecific antigen-binding compositions that bind to an antigen or epitope of the CD3 protein complex of effector cells (e.g., T cells) and an EGFR antigen associated with diseased cells or tissues. They may therefore be referred to as T cell engagers. As described in more detail below, bispecific antigen-binding compositions that offer advantages over bispecific T cell engagers and related compounds known in the art are comprised of activatable prodrug forms. The compositions of the present disclosure have properties including enhanced stability during their manufacture and purification, enhanced stability and increased half-life in the circulation when administered to a subject, the ability to be activated at the intended site of therapy but not in normal, healthy tissues, and, upon activation by proteolytic cleavage of the release segment and release of the fused AF1 and AF2, exhibit binding affinities for target and effector cells at least comparable to those of corresponding conventional bispecific IgG antibodies. Upon binding of effector and target cells via fused AF1 and AF2, an immunological synapse is formed that leads to activation of the effector cell and promotes the subsequent destruction of the target cell by apoptosis or cytolysis.

[0184] The various bispecific antigen-binding compositions of the present disclosure described herein are specifically designed to be in prodrug form, in that the XTEN component shields the antigen-binding fragments, reducing their ability to bind to their ligands until they are released from the composition by protease cleavage at one of the protease cleavage sites located within the release segment. Proteases known to be associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine ​​proteases, aspartic acid proteases, and metalloproteases, including, but not limited to, the specific proteases described herein. This prodrug property of the bispecific antigen-binding compositions improves the specificity of the compositions for diseased tissues or cells compared to bispecific T cell engager therapeutics that are not in prodrug form. In contrast, by specifically activating the bispecific antigen-binding composition in the microenvironment of target cells or diseased tissues where EGFR antigens and proteases capable of cleaving the release segment are highly expressed, the bispecific antigen-binding fragment and its XTEN construct are released upon cleavage of the release segment, and the fused AF1 and AF2 can crosslink cytotoxic effector cells with cells expressing the EGFR antigen in a highly specific manner, thereby directing the cytotoxic activity of T cells toward the target cells. After protease cleavage, the fused AF1 and AF2 are no longer shielded and effectively regain their full ability to bind to target cells bearing the EGFR antigen and effector cells such as cytotoxic T cells by binding to the CD3 antigen, thereby forming part of a T cell receptor complex and triggering T cell activation that mediates the subsequent lysis of target cells expressing the specific EGFR antigen. Thus, it is contemplated that the bispecific antigen-binding composition will exhibit potent, specific, and efficient target cell killing. In such cases, cells are selectively eliminated, thereby reducing the possibility of toxic side effects.

[0185] The design of the subject compositions having first and second antigen-binding fragments (AF1 and AF2, respectively) was driven by consideration of at least three properties: 1) the composition has a bispecific antigen-binding fragment capable of binding to and linking together effector and target cells bearing the EGFR antigen, resulting in the formation of an immunological synapse; 2) the composition has an XTEN that i) shields both of these antigen-binding fragments, reducing their ability to bind to target and effector cell ligands when the composition is in an intact prodrug form; ii) provides an enhanced half-life when administered to a subject; iii) reduces extravasation of the intact composition from the blood circulation in normal tissues and organs compared to diseased tissues (e.g., tumors); and iv) confers an increased safety profile compared to conventional bispecific cytotoxic antibody therapeutics; and 3) the RS is activated when cleaved by one or more mammalian proteases near the diseased tissue, thereby releasing the bispecific antigen-binding fragments, allowing them to fully regain their binding affinity for the target ligand. The design of the subject compositions utilizes the properties of the XTEN and release segment (RS) components and their positioning relative to the bispecific antigen-binding fragment to achieve the aforementioned properties, as evidenced by the results of the illustrative examples below.

[0186] In one embodiment, the present disclosure provides a bispecific antigen-binding composition having two antigen-binding fragments, AF1 and AF2, of any of the antigen-binding fragment embodiments described herein, wherein AF2 is fused to AF1 by a flexible peptide linker. In one embodiment, the bispecific antigen-binding fragment composition comprises a first antigen-binding fragment (AF1), where AF1 specifically binds to EGFR or an epitope thereof, and a second antigen-binding fragment (AF2), where AF2 specifically binds to cluster of differentiation 3 T-cell receptor (CD3), wherein the difference between the isoelectric point (pI) of the second antigen-binding fragment and the pI of the first antigen-binding fragment is 0 to about 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, as determined computationally or in an in vitro assay. In one embodiment of the bispecific antigen-binding composition, AF1 has a K of about 0.1 nM to about 100 nM, or about 0.5 nM to about 50 nM, or about 1 nM to about 20 nM, or about 2 nM to about 10 nM, as determined by an in vitro antigen binding assay involving EGFR or an epitope thereof. d In another embodiment of the bispecific antigen-binding composition, AF2 specifically binds to EGFR with a dissociation constant (K) of about 0.1 nM to about 100 nM, or about 0.5 nM to about 50 nM, or about 1.0 nM to 20 nM, or about 2.0 nM to about 10 nM, as determined in an in vitro antigen-binding assay. d In another embodiment of the bispecific antigen-binding composition, AF2 specifically binds to human or cyno CD3 with a dissociation constant (K) of about 10 nM to about 400 nM, as determined in an in vitro antigen-binding assay. d In yet another embodiment of the bispecific antigen-binding composition, AF2 specifically binds to human or cyno CD3 with a dissociation constant (K) of about 10 nM to about 400 nM, or about 50 nM to about 350 nM, or about 100 nM to 300 nM, as determined in an in vitro antigen-binding assay. dIn another embodiment of the bispecific antigen-binding composition, AF2 specifically binds to human or cyno CD3 with a dissociation constant (K) weaker than about 3 nM, or 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 400 nM, as determined in an in vitro antigen-binding assay. d In another embodiment of the bispecific antigen-binding composition, AF2 specifically binds to human or cyno CD3 with a respective dissociation constant (K d In another embodiment of the bispecific antigen-binding composition, AF2 specifically binds to human or cyno CD3 with a binding affinity 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 than an antibody-binding fragment consisting of the amino acid sequence of SEQ ID NO: 781, as determined by in vitro antigen-binding assays. d ) that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold weaker than the binding affinity of AF1. For clarity, a K of 400 d An antigen-binding fragment having a K d binds to its ligand more weakly than one with

[0187] In another embodiment of a bispecific antigen-binding composition of any of the subject embodiments described herein having two antigen-binding fragments (AF1 and AF2), a single RS, and a single XTEN, the polypeptide, in its uncleaved state, can have, from N-terminus to C-terminus, the structural arrangement of AF2-AF1-RS1-XTEN1, AF1-AF2-RS1-XTEN1, XTEN1-RS1-AF2-AF1, XTEN1-RS1-AF1-AF2, or diabody-RS1-XTEN1, or XTEN1-RS1-diabody, where the diabody comprises the VL and VH of AF1 and AF2.

[0188] In another aspect, it is a feature of various designed compositions of any of the embodiments described herein that, when the RS of the bispecific antigen-binding composition is cleaved by a mammalian protease in the environment of a target cell and converted from a prodrug form to an activated or apoprotein form, upon cleavage and release of the bispecific antigen-binding fragment and XTEN from the composition, the fused AF1 and AF2 bind to and link together an effector cell targeted by AF2 (e.g., a CD3-bearing T cell) and a diseased cell (e.g., a tumor or cancer cell) bearing the EGFR antigen on the target cell targeted by AF1, whereupon the effector cell is activated. In one embodiment, the RS of the bispecific antigen-binding composition is cleaved, releasing the antigen-binding fragments, and subsequent simultaneous binding to the effector cell and the target cell results in at least 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, or 1000-fold activation of the effector cell, as assessed by the production of cytokines, cytolytic proteins, or lysis of the target cell, as assessed in an in vitro cell-based assay. In another embodiment, simultaneous binding of the released antigen-binding fragments to a CD3 antigen-bearing T cell and a target cell bearing the EGFR antigen forms an immunological synapse, and this binding leads to the release of T cell-derived effector molecules that can lyse the diseased cell.Non-limiting examples of in vitro assays for measuring effector cell activation and / or cell lysis include cell membrane integrity assays, mixed cell culture assays, FACS-based propidium iodide assays, trypan blue influx assays, photometric enzyme release assays, ELISA, radiometric 51Cr release assays, fluorometric europium release assays, Calcein AM release assays, photometric MTT assays, XTT assays, WST-1 assays, Alamar Blue assays, radiometric 3H-Thd incorporation assays, clonogenic assays measuring mitotic activity, fluorometric Rhodamine 123 assays measuring mitochondrial transmembrane gradients, FACS-based phosphatidylserine exposure-monitored apoptosis assays, ELISA-based TUNEL assays, caspase activity assays, and cell morphology assays, or other assays known in the art for assaying cytokines, cytolytic proteins, or cell lysis, or methods described in the Examples below.

[0189] In other embodiments, the present disclosure provides bispecific antigen-binding compositions having two antigen-binding fragments of any of the embodiments described herein, two RSs of any of the embodiments described herein, and two XTENs of any of the embodiments described herein. The design of these compositions was driven by the consideration that the addition of a second XTEN further reduces the binding affinity of the uncleaved composition to the ligands of each of the AF1 and AF2 antibody fragments, further reducing unintended binding of the composition to healthy tissues or cells when administered to a subject, thereby further improving the therapeutic index of the subject composition compared to compositions having only one RS and one XTEN. The addition of a second RS and a second XTEN resulted in a surprising reduction in the binding affinity of the uncleaved intact polypeptide to the ligands of each of the AF1 and AF2 antibody fragments when assayed in vitro compared to compositions having a single RS and XTEN, and also resulted in reduced toxicity in animal models of disease when administered at therapeutically effective doses, as described in the Examples below. In embodiments of a composition having two antigen-binding fragments, two RSs, and two XTENs, the composition, in its uncleaved state, can have, from N-terminus to C-terminus, the structural arrangement: XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-diabody-RS1-XTEN1 (wherein the diabody comprises the VL and VH of AF1 and AF2), or XTEN1-RS1-diabody-RS2-XTEN2 (wherein the diabody comprises the VL and VH of AF1 and AF2).

[0190] Without being bound by any particular theory, it is believed that by using the above-mentioned bispecific antigen-binding composition format, upon cleavage of RS, the released fusion AF1 and AF2 can kill target cells by recruiting cytotoxic effector cells without the need for pre-stimulation and / or costimulation. Furthermore, the independence of effector cells from pre-stimulation and / or costimulation may substantially contribute to the extremely high cytotoxicity mediated by the released fusion AF1 and AF2 antigen-binding fragments. In some embodiments, the released AF1 and AF2, in which AF1 remains fused to AF2 by a linker peptide, are engineered with binding specificities such that they have the ability to bind to and link together in proximity a pre-selected EGFR antigen with AF1 having binding specificity for an EGFR antigen associated with cytotoxic effector cells (e.g., T cells, NK cells, cytokine-induced killer cells (CIK cells), tumor cells, cancer cells, or cells associated with diseased tissue), thereby creating an immunological synapse and selective, directed, and localized effect of the released cytokine and effector molecule on the target disease or cancer cells, resulting in damage or destruction of the disease or cancer cells and providing a therapeutic benefit to the subject. The released AF2 that binds to the effector cell antigen can modulate one or more functions of the effector cell, and can enhance the activity of the target tumor. The effector cell antigen exerts or contributes to a cytolytic effect on tumor cells. The effector cell antigen may be expressed by an effector cell or other cells. In one embodiment, the effector cell antigen is expressed on the cell surface of an effector cell. Non-limiting examples of effector cell antigens are CD3, CD4, CD8, CD16, CD25, CD38, CD45RO, CD56, CD57, CD69, CD95, CD107, and CD154. Thus, one skilled in the art will understand that the configuration of the subject compositions is intended to selectively or disproportionately deliver the active form of the composition to target tumor tissue or cancer cells relative to healthy tissue or cells in the subject to which the composition is administered, resulting in a therapeutic benefit. As is apparent from the foregoing, the present disclosure provides a large family of polypeptides in configurations designed to provide desired properties.

[0191] It is an object of the present disclosure that the design of subject bispecific antigen binding compositions in which the shielding effect is conferred by the XTEN in the intact circulating composition, and which concomitantly reduces the likelihood of binding to effector cells and target tissues, will result in reduced production of Th1 T cell-associated cytokines or other pro-inflammatory mediators during systemic exposure when administered to a subject, such that the overall side effect and safety profile (e.g., therapeutic index) is improved compared to bispecific antigen binding compositions that are not linked to a shielding moiety, such as an XTEN. As key components of cellular immunity, the production of IL-2, TNF-alpha, and IFN-gamma is a hallmark of Th1 responses (Romagnani ST-cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol. 2000. 85(1):9-18), particularly in anti-CD3 stimulated T cells (Yoon, SH. Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro. Exp Mol Med. 2009. 41(3):161-170). IL-4, IL-6, and IL-10 are also pro-inflammatory cytokines important in the cytotoxic response to bispecific antibody compositions (Zimmerman, Z., et al. Unleashing the clinical power of T cells: CD19 / CD3 bi-specific T cell engager (BiTE®) antibody composition blinatumomab as a potential therapy. Int. Immunol. (2015) 27(1):31-37).In one embodiment, an uncleaved, intact bispecific antigen-binding composition of the embodiments described herein may exhibit at least a 3-fold, or at least a 4-fold, or at least a 5-fold, or at least a 6-fold, or at least a 7-fold, or at least a 8-fold, or at least a 9-fold, or at least a 10-fold, or at least a 20-fold, or at least a 30-fold, or at least a 50-fold, or at least a 100-fold, or at least a 1000-fold decrease in the likelihood of resulting in the production of Th1 and / or pro-inflammatory cytokines when the uncleaved, intact polypeptide is contacted with effector and target cells in an in vitro cell-based cytokine stimulation assay compared to the Th1 and / or cytokine levels stimulated by the corresponding released AF1 and AF2 (which remain fused together after release by RS proteolysis) of the corresponding protease-treated composition in an in vitro cell-based cytokine stimulation assay performed under comparable conditions, e.g., at an equivalent molar concentration. Non-limiting examples of Th1 and / or pro-inflammatory cytokines are IL-2, IL-4, IL-6, IL-10, TNF-alpha, and IFN-gamma. In one aforementioned embodiment, Th1 cytokine production is assayed in an in vitro assay comprising effector cells, such as PBMCs or CD3+ T cells, and target cells bearing the EGFR antigen disclosed herein. In another embodiment, cytokines can be assessed from blood, body fluid, or tissue samples collected from a subject to which a polypeptide composition has been administered. In the aforementioned embodiments, the subject can be a mouse, rat, monkey, or human. However, an advantage of the subject bispecific antigen-binding compositions of the embodiments described herein is that the cytolytic properties of the compositions do not require cytokine pre-stimulation; rather, it has been found that the formation of an immunological synapse between effector cells bound to target cells by antigen-binding fragments is sufficient to affect cytolysis or apoptosis in the target cells. Nevertheless, the production of pro-inflammatory cytokines is a useful marker for assessing the efficacy or effectiveness of a subject polypeptide composition, whether by in vitro assay or by monitoring the treatment of a tumor-bearing subject.

[0192] In the context of use of bispecific antigen-binding fragment compositions in subjects, it is an object of the present disclosure that the subject bispecific antigen-binding compositions are designed to take advantage of the difference in pore size of the vasculature in tumor or inflamed tissue compared to healthy vasculature through the addition of XTEN, such that extravasation of the intact bispecific antigen-binding composition in normal tissue is reduced, but in the leaky environment of the tumor vasculature or other inflamed area, the intact construct can leak and thereby be activated by proteases in the diseased cellular environment, releasing antigen-binding fragments to effector and target cells (see, e.g., Figure 5). In the case of the RS of the bispecific antigen-binding composition, this design takes advantage of the situation where, when the bispecific antigen-binding composition is in the vicinity of diseased tissue, e.g., a tumor, that produces one or more proteases, RS sequences that are sensitive to one or more proteases expressed by the tumor can be cleaved by the proteases (described more fully above). The action of protease cleaves the release segment (RS) of the composition, separating the antigen-binding fragment from XTEN, resulting in components with reduced molecular weight and hydrodynamic radius, particularly for the released fusion AF1 and AF2.As can be understood, the reduced molecular weight and hydrodynamic radius of the composition also gives the released fusion AF1 and AF2 the properties of moving more freely in solution, moving through smaller pore spaces in tissues and tumors, leaking more easily through the larger pores of tumor vasculature, penetrating more easily into tumors, and increasing the ability to bind to and link effector cells and tumor cells together.Such properties can be measured by different assays.Therefore, those skilled in the art will understand that in the context of treating a subject using the subject composition, the bispecific antigen-binding composition exists in a prodrug form, and is converted into a more active form when entering a certain cellular environment by the action of proteases co-localized with affected tissues or cells. Upon release from the composition by the action of proteases in the target tissue, AF2, which has binding specificity for an effector cell antigen, and the linked AF1, which has binding specificity for an antigen on a target cell, fully regain the ability to bind to and link effector and target cells together, forming an immunological synapse.Formation of an immunological synapse activates effector cells, and various signaling pathways activate new gene transcription and release the effector molecule contents of their vesicles by exocytosis. Different cytokines and lymphokines are released depending on the effector cell type; for example, type 1 helper T cells (Th1) release cytokines such as IFN-gamma, IL-2, and TNF-alpha, whereas type 2 helper T cells (Th2) release cytokines such as IL-4, IL-5, IL-10, and IL-13 that stimulate B cells, and cytotoxic T lymphocytes (CTLs) release cytotoxic molecules such as perforin and granzymes (collectively "effector molecules") that kill targets. It is specifically contemplated that simultaneous binding and linking of effector cells and target tumor cells together by the released bispecific antigen-binding fragments of the bispecific antigen-binding composition will result in tumor cells being acted upon by the effector molecules released by the effector cells into the immunological synapse between the cells at very low effector:target (E:T) ratios, resulting in tumor cell damage, perforin-mediated lysis, granzyme B-induced cell death, and / or apoptosis. Thus, in another aspect, without being bound by theory, it is a feature of the designed compositions that when the activatable bispecific antigen-binding fragment composition is administered to a subject with a tumor, the prodrug form remains in the circulation in normal tissues but can leak into the more permeable vasculature of the tumor such that the prodrug form of the construct is activated by proteases co-localized with the tumor and the released antigen-binding fragment binds to and links together effector cells (e.g., T cells) and tumor cells expressing the EGFR antigen targeted by AF1 of the composition, whereupon the effector cells are activated, resulting in tumor cell lysis.In other words, in some cases, the more permeable vasculature in tumor tissue allows the bispecific antigen-binding polypeptide to leak into that tissue, where tumor-associated proteases can act on the release segment (RS), cleave it, releasing the binding moiety, and then bind to effector cells and tumor-associated cells, linking them together. In normal tissue, extravasation may be blocked by a tighter vasculature barrier, or if the bispecific antigen-binding polypeptide leaks to some extent, the bispecific antigen-binding polypeptide may remain primarily in the "pro" form because insufficient proteases may be present in healthy tissue to release the binding moiety, with the net effect being that no immunological synapse is formed. In some cases, the released fusion AF1 and AF2 in the subject's tumor, bound to both tumor cells and effector cells, exhibit at least a 10-fold, or at least a 30-fold, or at least a 100-fold, or at least a 200-fold, or at least a 300-fold, or at least a 400-fold, or at least a 500-fold, or at least a 1000-fold increase in the ability to activate effector cells compared to the corresponding uncleaved intact bispecific antigen-binding composition. In other cases, the released fusion AF1 and AF2 in the subject's tumor, bound to both tumor cells and effector cells, exhibit at least a 10-fold, or at least a 30-fold, or at least a 100-fold, or at least a 200-fold, or at least a 300-fold, or at least a 400-fold, or at least a 500-fold, or at least a 1000-fold increase in the ability to lyse tumor cells compared to the corresponding uncleaved intact bispecific antigen-binding composition in the tumor. In the foregoing embodiments, effector cell activation and / or cytotoxicity can be assayed by conventional methods known in the art, such as measuring cell counts of activated effector cells, assaying cytokines, measuring tumor size, or histopathology. In the foregoing embodiments, the subject can be a mouse, rat, dog, monkey, or human.Specifically, it is specifically contemplated that the subject compositions are designed such that, when administered to a subject with a disease having an EGFR antigen to which AF2 can bind, the bispecific antigen-binding composition exhibits an enhanced therapeutic index and reduced incidence of side effects compared to conventional bispecific antibodies known in the art, achieved by a combination of steric hindrance and the shielding effect of XTEN on the binding affinity for the antigen-binding fragment in a prodrug form, but is capable of releasing the bispecific AF1 and AF2 (achieved by inclusion of a cleavage sequence in the RS) near or within a target tissue (e.g., a tumor) that produces a protease for which the RS is a substrate.

[0193] VII. Methods and Uses of Bispecific Antigen-Binding Compositions In another aspect, the present disclosure provides activatable bispecific antigen-binding compositions and pharmaceutical compositions comprising the bispecific antigen-binding compositions, which are particularly useful in the prevention, treatment, and / or amelioration of medical conditions, such as certain cancers, tumors, or inflammatory diseases. For use in treating a disease, the bispecific antigen-binding compositions of the invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical professionals.

[0194] In particular, several therapeutic strategies have been used to design polypeptide compositions for use in methods for treating subjects with cancerous diseases, including regulating T cell responses by targeting TcR signaling using the VL and VH portions of anti-human CD3 monoclonal antibodies, which are widely used clinically in immunosuppressive regimens. The CD3-specific monoclonal OKT3 was the first such monoclonal approved for human use (Sgro, Toxicology 105 (1995), 23-29) and is widely used clinically as an immunosuppressant during transplantation (Chatenoud L: Immunologic monitoring during OKT3 therapy. Clin Transplant 7:422-430, 1993). Furthermore, anti-CD3 monoclonal antibodies can induce partial T cell signaling and clonal anergy (Smith, J. Exp. Med. 185 (1997), 1413-1422). OKT3 reacts with and blocks the function of the CD3 complex in the membrane of T cells, which associates with the antigen-recognition structure of T cells (TCR), essential for signal transduction. These and other such CD3-specific antibodies can induce various T cell responses, including cytokine production (Von Wussow, Human gamma interferon production by leukocytes induced with monoclonal antibodies recognizing T cells. J. Immunol. 127:1197-1200 (1981)), proliferation, and the induction of suppressor T cells. In cancer, attempts have been made to lyse cancer cells using cytotoxic T cells. Without being bound by theory, it is believed that cytotoxic T cells require direct cell-to-cell contact to bring about target cell lysis, and the TCR on the cytotoxic T cell must recognize and engage with the appropriate antigen on the target cell. This creates an immunological synapse that then initiates a signaling cascade within the cytotoxic T cell, leading to T cell activation and the production of a variety of cytotoxic cytokines and effector molecules.Perforin and granzymes are highly toxic molecules stored in preformed granules present in activated cytotoxic T cells. After target cell recognition, the cytoplasmic granules of the engaged cytotoxic T cells migrate toward the cytotoxic T cell membrane, eventually fusing with it and releasing their contents in a directed manner into the immunological synapse, forming pores in the target cell membrane and disrupting the tumor cell plasma membrane. The created pores act as entrances for granzymes, a family of serine proteases that induce apoptosis in tumor cells.

[0195] The subject bispecific antigen binding compositions described herein, in which AF2, which has specific binding affinity for CD3 on T cells, is tightly fused to AF1, which has specific binding affinity for the EGFR antigen, are T cell engagers that, upon release from the intact prodrug form of the composition by cleavage of the release segment, fully regain their ability to bind to T cells and target cells, form an immunological synapse that promotes T cell activation and the subsequent destruction of tumor cells by apoptosis or cytolysis.

[0196] The present disclosure contemplates the use of engineered bispecific antigen-binding compositions that target a wide range of malignant cells, such as tumors, in addition to effector cells to initiate target cell lysis and provide beneficial therapeutic outcomes in that bispecific antigen-binding compositions can be designed such that one antigen-binding fragment binds and associates with CD3 to activate cytotoxic T cells, while the second antigen-binding fragment targets the EGFR marker characteristic of a particular malignancy, bringing them together to create an immunological synapse. A particular advantage of this design is that the physical association of the cytotoxic effector cells and EGFR-bearing cells eliminates the need for antigen processing, MHCI / β2-microglobulin, and costimulatory molecules. Due to the range of EGFR-bearing cells, the resulting compositions will be appreciated for their utility against a variety of cancers, including solid and hematologic tumors. In one embodiment, the present disclosure provides a method for treating a subject with a tumor. The tumors to be treated may contain tumor cells originating from cells selected from the group consisting of stromal cells, fibroblasts, myofibroblasts, glial cells, epithelial cells, adipocytes, lymphocytes, vascular cells, smooth muscle cells, mesenchymal cells, breast tissue cells, prostate cells, kidney cells, brain cells, colon cells, ovarian cells, uterine cells, bladder cells, skin cells, stomach cells, urogenital tract cells, cervical cells, uterine cells, small intestine cells, liver cells, pancreatic cells, gallbladder cells, bile duct cells, esophageal cells, salivary gland cells, lung cells, and thyroid cells. A further advantage of the composition is that the cytotoxic effector cells are not consumed during the damage / destruction of the crosslinked target cancer cells; after causing lysis of one target cell, the activated effector cells can be released and migrate through the local tissue toward other target cancer cells, bind to EGFR antigens, and initiate additional cell lysis. In addition, it is contemplated that in a localized environment such as a solid tumor, the release of effector cell molecules such as perforin and granzymes will result in damage to tumor cells adjacent to, but not bound by, a given molecule of the bispecific binding domain, resulting in stasis of tumor growth or regression.

[0197] Thus, it will be appreciated that the present disclosure has utility in that, after a therapeutically effective dose of a pharmaceutical composition comprising the bispecific antigen-binding composition described herein is administered to a subject with a cancer or tumor bearing the EGFR antigen, the composition can be acted upon by a protease associated with or co-localized with the cancer or tumor cells to release the fused AF1 and AF2, thereby enabling an immunological synapse to be created by linkage between the EGFR-bearing cell and an effector cell, resulting in the release of effector molecules from the effector cell that can lyse the target cell, resulting in apoptosis, cytolysis, or death of the target cancer or tumor cell. Furthermore, one skilled in the art will appreciate that the formation of an immunological synapse by binding of the released binding domains to effector cells and target cancer cells can result in the use of a bispecific antigen-binding composition to provide a beneficial therapeutic effect that is sustained and more systemic than "single killing."

[0198] In one aspect, the present disclosure relates to methods of treating a disease in a subject, such as a subject with cancer. In some embodiments, the present disclosure provides a method of treating a disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising any of the bispecific antigen-binding compositions of the embodiments described herein. The therapeutically effective amount of the pharmaceutical composition may vary depending on factors such as the individual's medical condition, age, sex, and weight, as well as the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the subject composition are outweighed by the therapeutically beneficial effects. A prophylactically effective amount refers to the amount of pharmaceutical composition needed for the period of time necessary to achieve the desired prophylactic result.

[0199] The therapeutically effective dose of the bispecific antigen-binding compositions described herein generally provides therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the bispecific antigen-binding compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal experiments, LD50 (lethal dose for 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects can be determined as the LD 50 / ED 50 The therapeutic index can be expressed as the ratio of ED to ED. Bispecific antigen-binding compositions exhibiting large therapeutic indices are preferred. In one aspect, the bispecific antigen-binding molecules according to the present invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal trials can be used to formulate a range of doses suitable for use in humans. Dosages are determined to be those that result in little or no toxicity, such as the ED 50 The blood concentration range is preferably within the range including the range of 0.1 to 1.0 mg / kg of the bispecific antigen-binding composition. The dosage can vary within this range depending on various factors, such as the dosage form used, the route of administration utilized, the condition of the subject, etc. The exact formulation, route of administration, and dosage can be chosen by the individual physician in consideration of the patient's condition (see, for example, Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Ch. 1, p. 1). Those skilled in the art will readily recognize that in many cases, a bispecific antigen-binding composition may provide only a partial benefit rather than a cure. In some embodiments, physiological changes that have some benefit are also considered therapeutically beneficial. Thus, in some embodiments, the amount of a bispecific antigen-binding composition that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount." The subject, patient, or individual in need of treatment is typically a mouse, rat, dog, monkey, or human.

[0200] The bispecific antigen-binding compositions of the present invention may be administered in combination with one or more other agents during treatment. For example, any of the bispecific antigen-binding molecules of the embodiments described herein may be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain aspects, the additional therapeutic agent is an immunomodulatory agent, an immuno-oncology antibody, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, a cell apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis inducers. In certain aspects, the additional therapeutic agent is an anti-cancer agent, such as a microtubule-disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis activator, or an anti-angiogenic agent.

[0201] In one embodiment of the method of treating a disease in a subject, the disease for treatment is anaplastic and medullary thyroid cancer, appendix cancer, allenoblastoma, biliary tract cancer, bladder cancer, breast cancer, cancers of the bile duct, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignant tumor with malignant ascites, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, follicular cancer, gallbladder cancer, gastric cancer, cancer), gastrointestinal stromal tumor (GIST), GE-linked cancer, genitourinary cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocellular carcinoma, HR+ and HER2+ breast cancer, Hürthle cell carcinoma, inflammatory breast cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroendocrine carcinoma, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary carcinoma, parathyroid cancer, peritoneal dissemination, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, gastric cancer cancer), testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial cancer, uterine cancer, uterine serous cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0202] A therapeutically effective amount can have a beneficial effect in helping to treat (e.g., cure or reduce the severity of) or prevent (e.g., reduce the likelihood of recurrence) cancer or tumors. In another embodiment of the method for treating a disease in a subject, the pharmaceutical composition is administered to the subject as two or more therapeutically effective doses administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, or once a month. In another embodiment of the method, the pharmaceutical composition is administered to the subject as two or more therapeutically effective doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. In another embodiment of the method, a first low priming dose is administered to the subject, followed by one or more higher maintenance doses over a dosing schedule of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. The initial priming dose administered is selected from the group consisting of at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, and at least about 0.1 mg / kg, and the one or more subsequent maintenance doses administered are selected from the group consisting of at least about 0.02 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.22 mg / kg, mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg, or at least 5.0 mg / kg.In another embodiment of this method, the pharmaceutical composition is administered to the subject intradermally, subcutaneously, intravenously, intraarterially, intraperitoneally, intraperitoneally, intrathecally, or intramuscularly. In another embodiment of this method, the pharmaceutical composition is administered to the subject as one or more therapeutically effective bolus doses or by infusion for 5 minutes to 96 hours, as tolerated for maximum safety and efficacy. In another embodiment of this method, the pharmaceutical composition is administered to the subject as one or more therapeutically effective bolus doses or by infusion for 5 minutes to 96 hours, the dose being at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.14 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.25 mg / kg, at least about 0.30 mg / kg, at least about 0.31 mg / kg, at least about 0.32 mg / kg, at least about 0.33 mg / kg, at least about 0.34 mg / kg, at least about 0.35 mg / kg, at least about 0.36 mg / kg, at least about 0.37 mg / kg, at least about 0.38 mg / kg, at least about 0.40 mg / kg, at least about 0.41 mg / kg, at least about 0.42 mg / kg, at least about 0.43 mg / kg, at least about 0.44 mg / kg, at least about 0.45 mg / kg, at least about 0.46 mg / kg, at least about 0.47 mg / kg, at least about 0.48 mg / kg, at least about 0.22 mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg, or at least about 5.0 mg / kg. In another embodiment of this method, the pharmaceutical composition is administered to the subject as one or more therapeutically effective bolus doses or by infusion over a period of from 5 minutes to 96 hours, and wherein administration to the subject results in a C plasma concentration of uncleaved intact bispecific antigen-binding composition in the subject of at least about 0.1 ng / mL to at least about 2 μg / mL or more that is maintained for at least about 3 days, at least about 7 days, at least about 10 days, at least about 14 days, or at least about 21 days.A therapeutically effective dose may be at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.14 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.22 mg / kg, at least about 0.2 4 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg. In one embodiment, the initial dose is selected from the group consisting of at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, and at least about 0.1 mg / kg, and subsequent doses are selected from the group consisting of at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.14 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.2 2 mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg.In the foregoing embodiments, administration to a subject results in a plasma concentration of the polypeptide in the subject of at least about 0.1 ng / mL to at least about 2 ng / mL or more for at least about 3 days, at least about 7 days, at least about 10 days, at least about 14 days, or at least about 21 days. In the foregoing embodiments of the method, the subject can be a mouse, rat, monkey, or human.

[0203] VIII). Nucleic acid sequence In some embodiments, the invention provides an isolated polynucleotide sequence encoding the AF1 sequence, or the AF2 sequence, or the release segment sequence (RS1 and RS2), or the XTEN sequence of any of the embodiments described herein, or any combination of these component embodiments described herein, or the complement of that polynucleotide sequence. In one embodiment, the invention provides an isolated polynucleotide sequence encoding the polypeptide or bispecific antigen-binding composition of any of the embodiments described herein, or the complement of that polynucleotide sequence. In one embodiment, the invention provides an isolated polynucleotide sequence encoding the polypeptide or bispecific antigen-binding composition having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a polynucleotide sequence set forth in Table 12.

[0204] In another aspect, the present disclosure relates to methods for generating polynucleotide sequences encoding the polypeptide or bispecific antigen-binding composition of any of the embodiments described herein, or sequences complementary to the polynucleotide sequences (including homologous variants thereof), as well as methods for expressing the proteins expressed by the polynucleotide sequences. Generally, the methods involve generating a polynucleotide sequence encoding the proteinaceous polypeptide or bispecific antigen-binding composition of any of the embodiments described herein and incorporating the encoding gene into a suitable expression vector in a host cell. To produce the encoded polypeptide or bispecific antigen-binding composition of any of the embodiments described herein, the method involves transforming a suitable host cell with the expression vector and culturing the host cell under conditions that cause or allow the resulting polypeptide or bispecific antigen-binding composition of any of the embodiments described herein to be expressed in the transformed host cell, thereby producing the polypeptide or bispecific antigen-binding composition, which is recovered by the methods described herein or by standard protein purification methods known in the art. Standard recombinant techniques in molecular biology are used to generate the polynucleotides and expression vectors of the present disclosure.

[0205] According to the present disclosure, a nucleic acid sequence (or its complement) encoding a polypeptide or bispecific antigen-binding composition of any of the embodiments described herein is used to generate a recombinant DNA molecule that directs expression in a suitable host cell. Several cloning strategies are suitable for practicing the present disclosure, many of which are used to generate constructs comprising genes encoding the compositions of the present disclosure, or their complements. In one embodiment, a cloning strategy is used to generate a gene encoding a construct comprising nucleotides encoding the polypeptide or bispecific antigen-binding composition that is used to transform a host cell to express the composition. In the foregoing embodiments described in this paragraph, the gene may comprise nucleotides encoding the antigen-binding fragment, release segment, and XTEN in the configurations disclosed herein.

[0206] In one approach, a construct containing a DNA sequence encoding a polypeptide or bispecific antigen-binding composition construct is first prepared. Exemplary methods for preparing such constructs are described in the Examples. This construct is then used to create an expression vector suitable for transforming a host cell, such as a prokaryotic or eukaryotic host (e.g., mammalian) cell, to express and recover the polypeptide construct. If desired, the host cell is E. coli. In another embodiment, the host cell is selected from BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, NIH3T3 cells, COS, HeLa, CHO, or yeast cells. Exemplary methods for creating an expression vector, transforming a host cell, and expressing and recovering XTEN are described in the Examples.

[0207] Genes encoding polypeptide or bispecific antigen-binding composition constructs can be generated in one or more steps, either entirely synthetically or synthetically, combined with enzymatic processes such as restriction enzyme-mediated cloning, PCR, and overlap extension, including methods more fully described in the Examples. The methods disclosed herein can be used, for example, to ligate polynucleotide sequences encoding various component (e.g., binding domain, linker, release segment, and XTEN) genes of desired length and sequence. Genes encoding polypeptide compositions are assembled from oligonucleotides using standard gene synthesis techniques. Gene design can be performed using algorithms that optimize codon usage and amino acid composition appropriate for the E. coli or mammalian host cell utilized for polypeptide or bispecific antigen-binding composition production. In one method of the present disclosure, a library of polynucleotides encoding the components of the construct is generated and then assembled, as described above. The resulting genes are then constructed, host cells are transformed with the resulting genes, and the polypeptide compositions are produced and recovered, and their properties evaluated, as described herein.

[0208] The resulting polynucleotides encoding the polypeptide or bispecific antigen-binding composition sequences can then be individually cloned into expression vectors. Nucleic acid sequences can be inserted into vectors by a variety of procedures. Generally, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components uses standard ligation techniques known to those skilled in the art. Such techniques are well known in the art and are fully described in the scientific and patent literature. Various vectors are publicly available. Vectors can be in the form of, for example, a plasmid, cosmid, viral particle, or phage that can be conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it will be introduced. Thus, vectors can be autonomously replicating vectors, i.e., vectors that exist as extrachromosomal entities and whose replication is independent of chromosomal replication, such as plasmids. Alternatively, the vector may be a vector that, when introduced into a host cell, is integrated into the host cell genome and replicates together with the chromosome into which it is integrated. Once introduced into a suitable host cell, the expression of the antigen-binding fragment or bispecific antigen-binding composition can be determined using any nucleic acid or protein assay known in the art. For example, the presence of transcribed mRNA of the light chain CDR or heavy chain CDR, antigen-binding fragment, or bispecific antigen-binding composition can be detected and / or quantified by conventional hybridization assays (e.g., Northern blot analysis), amplification procedures (e.g., RT-PCR), SAGE (U.S. Patent No. 5,695,937), and array-based technologies (see, e.g., U.S. Patent Nos. 5,405,783, 5,412,087, and 5,445,934) using probes complementary to any region of the antigen-binding unit polynucleotide.

[0209] The present disclosure provides for the use of plasmid expression vectors containing replication and control sequences compatible with and recognized by a host cell and operably linked to a gene encoding a polypeptide for controlled expression of the polypeptide. The vector typically carries a replication site and a sequence encoding a protein capable of providing phenotypic selection in transformed cells. Such vector sequences are well known for various bacteria, yeast, and viruses. Useful expression vectors that can be used include, for example, chromosomal segments, non-chromosomal segments, and synthetic DNA sequences. An "expression vector" refers to a DNA construct containing a DNA sequence operably linked to a suitable control sequence capable of effecting expression of the DNA encoding a polypeptide in a suitable host. The vector must be replicable and viable in the selected host cell. Low-copy or high-copy vectors may be used as desired.

[0210] Suitable vectors include, but are not limited to, SV40 and pcDNA and derivatives of known bacterial plasmids, e.g., col EI, pCR1, pBR322, pMal-C2, pET, pGEX (described in Smith, et al., Gene 57:31-40 (1988)), pMB9, and their derivatives, plasmids such as RP4, phage DNA, e.g., numerous derivatives of phage I, such as NM98 9, and other phage DNA, e.g., M13 and filamentous single-stranded phage DNA; yeast plasmids, e.g., 2 micron plasmid or derivatives of 2 micron plasmid, and centromeric and integrative yeast shuttle vectors; vectors useful in eukaryotic cells, e.g., vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, e.g., phage DNA or plasmids modified to use expression control sequences, and the like. Yeast expression systems that may also be used in the present disclosure include, but are not limited to, the non-fusion pYES2 vector (Invitrogen), the fusion pYESHisA, B, C (Invitrogen), the pRS vector, and the like. Control sequences in the vector include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.Suitable promoters for use in expression vectors using prokaryotic hosts include, for example, the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, tryptophan (trp) promoter systems [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters, such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)], all operably linked to DNA encoding the XTEN polypeptide. Promoters for use in bacterial systems may also contain a Shine-Dalgarno (SD) sequence operably linked to DNA encoding the polypeptide.

[0211] Expression of the vector can also be determined by examining the antigen-binding fragment or component of the expressed bispecific antigen-binding composition. A variety of techniques for protein analysis are available in the art. These include, but are not limited to, radioimmunoassays, ELISA (enzyme-linked immunoradiometric assays), "sandwich" immunoassays, immunoradiometric assays, in situ immunoassays (e.g., using colloidal gold, enzyme, or radioisotope labels), Western blot analysis, immunoprecipitation assays, immunofluorescence assays, and SDS-PAGE.

[0212] IX) Methods of Producing Polypeptides and Bispecific Antigen-Binding Compositions In another aspect, the present disclosure provides methods of producing a subject composition. In one embodiment, the method comprises culturing host cells containing a nucleic acid construct encoding a polypeptide or bispecific antigen-binding composition of any of the embodiments described herein under conditions that promote expression of the polypeptide or bispecific antigen-binding composition, and then recovering the polypeptide or bispecific antigen-binding composition using standard purification methods (e.g., column chromatography, HPLC, etc.) to recover the composition, wherein at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the binding fragments of the expressed polypeptide or bispecific antigen-binding composition are correctly folded. In another embodiment of the method of making, the expressed polypeptide or bispecific antigen-binding composition is recovered, wherein at least or at least 90%, or at least 95%, or at least 97%, or at least 99% of the polypeptide or bispecific antigen-binding composition is recovered in monomeric, soluble form.

[0213] In another aspect, the disclosure relates to methods for producing polypeptides and bispecific antigen-binding compositions at high fermentative expression levels of functional proteins using E. coli or mammalian host cells, and methods for providing expression vectors encoding constructs useful in the methods for generating cytotoxically active polypeptide construct compositions at high expression levels. In one embodiment, the method includes 1) preparing a polynucleotide encoding a polypeptide of any of the embodiments disclosed herein, 2) cloning the polynucleotide into an expression vector, which can be a plasmid or other vector under the control of transcription and translation sequences suitable for high-level protein expression in a biological system, 3) transforming a suitable host cell with the expression vector, and 4) culturing the host cell in a conventional nutrient medium under conditions suitable for expression of the polypeptide composition. If desired, the host cell is E. coli. By this method, expression of the polypeptide results in a fermentation titer of at least 0.05 g / L, or at least 0.1 g / L, or at least 0.2 g / L, or at least 0.3 g / L, or at least 0.5 g / L, or at least 0.6 g / L, or at least 0.7 g / L, or at least 0.8 g / L, or at least 0.9 g / L, or at least 1 g / L of host cell expression product, and at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the expressed protein is correctly folded. As used herein, the term "correctly folded" means that the antigen-binding fragment component of the composition has the ability to specifically bind to its target ligand.In another embodiment, the disclosure provides a method for producing a polypeptide or bispecific antigen-binding composition, comprising culturing host cells comprising a vector encoding a polypeptide comprising a polypeptide or bispecific antigen-binding composition under conditions effective to express the polypeptide product at a concentration of greater than about 10 milligrams per gram (mg / g) of the polypeptide, 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 dry weight host cells in a fermentation reaction when the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, wherein the antigen-binding fragment of the expressed protein is correctly folded. In another embodiment, the disclosure provides a method for producing a polypeptide or bispecific antigen-binding composition, comprising culturing host cells comprising a vector encoding the composition under conditions effective to express the polypeptide product in a fermentation reaction at a concentration of greater than about 10 milligrams per gram (mg / g) of the polypeptide, 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 dry weight host cells 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.

[0214] The following are examples of compositions and composition evaluations of the present disclosure. It will be understood that various other embodiments can be practiced in light of the summary provided above. [Example]

[0215] Example 1: Construction of a bispecific antigen-binding polypeptide with two release segments. To generate a plasmid from which individual scFvs could be removed by restriction digestion, pCW1700, encoding the anti-EpCAM-anti-CD3 (UCHT1) bispecific tandem scFv along with the release segment RSR2486, AE866 XTEN, and 6X His-tag affinity tag (SEQ ID NO: 794), was digested with SacII and BstXI to remove the 3' end of the anti-EpCAM binding domain, the linker between the anti-EpCAM and anti-CD3 domains, and the 5' end of the anti-CD3 domain. A DNA fragment encoding the same region was synthesized with a silent point mutation at the junction between the anti-EpCAM binding domain and the linker to introduce a Bsu36I site. The synthetic DNA fragment was cloned into the digested backbone using an In-Fusion kit (New England Biolabs) to construct pJB0035. pJB0035 was then digested with NheI and BsaI to remove the BSRS1 release segment sequence. Overlapping single-stranded oligonucleotides encoding RSR2486 were synthesized with single-stranded tails that annealed to the NheI and BsaI overhangs. These oligonucleotides were annealed together and ligated into digested pJB0035 to yield pCW1880, which encodes the anti-EpCAM-anti-CD3 (UCHT1) bispecific tandem scFv, RSR2486, XTEN866, and a 6X His-tag affinity tag (SEQ ID NO: 794).

[0216] To generate plasmids carrying different CD3-binding domain variants, pCW1880 was digested with Bsu36I and NheI to remove the UCHT1 anti-CD3 scFv. A DNA fragment encoding CD3.23 was synthesized. The gene fragment contained 30 nucleotides containing restriction sites at the 5' and 3' ends to serve as DNA overlaps for Gibson DNA assembly. The synthetic DNA fragment was cloned into the digested backbone using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to construct pJB0205.

[0217] To generate bispecific antigen-binding polypeptides with both N- and C-terminal XTEN, AE292 XTEN was PCR-amplified from a plasmid using primers containing the backbone DNA at the N-terminus and a 17- to 21-bp 5' region of homology to the uncleavable release segment (RSR3058, amino acid sequence TTGEAGEAAGATSAGATGP (SEQ ID NO: 111)) at the C-terminus. A second PCR product encoding the light chain of anti-EpCAM antibody 4D5MOCB and a portion of its heavy chain was amplified using primers containing RSR3058 at the N-terminus and a 16- to 21-bp 5' region of homology to the heavy chain of 4D5MOCB at the C-terminus. These PCR fragments were cloned into a BsiWI-SacII-digested backbone vector encoding the remainder of the 4D5MOCB heavy chain / anti-CD3 tandem scFv, a second copy of the non-cleavable release segment RSR3058, and the AE837 XTEN with a 6xHIS affinity tag (SEQ ID NO: 794) using the In-Fusion Plasmid Assembly Kit (Takara Bio). The final vector encodes a bispecific antigen-binding polypeptide under the control of the PhoA promoter and STII secretion leader, consisting of (N- to C-terminally) the AE292 XTEN, the non-cleavable release segment RSR3058, and the anti-EpCAM-anti-CD3 bispecific tandem scFv, with RSR3058 fused to the AE867 XTEN with a 6xHIS affinity tag (SEQ ID NO: 794). The resulting construct is pJB0084, with the DNA and encoded amino acid sequence provided in Table 12.

[0218] Using pJB0084 as a template, a bispecific antigen-binding polypeptide construct was generated encoding the AE292 XTEN, cleavable release segment RSR2295, and anti-EpCAM-anti-CD3 bispecific tandem scFv, with RSR2295 fused to the AE868 XTEN. The plasmid utilized two PCR products using pJB0084 as a template: the first encodes the AE292 XTEN with a 6xHIS affinity tag (SEQ ID NO: 794) and a 5' region of homology to the vector backbone and a 3' region encoding the first RSR2295, and the second encodes the anti-EpCAM-anti-CD3 bispecific tandem scFv with 5' and 3' regions of homology encoding the 5' and 3' release segments RSR2295 of the tandem scFv. The third fragment encoded the AE868 XTEN with a C-tag affinity tag (amino acid sequence EPEA (SEQ ID NO:796)) with a 5' homology region encoding the second RSR2295 and a 3' homology region to the backbone vector. These three PCR fragments were cloned into BsiWI-NotI-digested pJB0084 using the In-Fusion Plasmid Assembly Kit. The final vector, pJB0169, encodes a bispecific antigen-binding polypeptide molecule under the control of the PhoA promoter and STII secretion leader, consisting of (N- to C-terminus) a 6xHIS affinity tag (SEQ ID NO:794), AE292 XTEN, release segment RSR2295, anti-EpCAM-anti-CD3 bispecific tandem scFv, RSR2295, AE868 XTEN with a C-tag affinity tag, with the DNA and protein sequences in Table 12.

[0219] To introduce new CD3 scFvs with altered isoelectric points and removal of potential aggregation sites within the amino acid sequence, pJB0244 was digested with BsaI and BbvCI to remove both HER2 and CD3 scFv. DNA fragments encoding anti-EGFR scFv variants paired with CD3.33 were synthesized containing 40 bp of homology to the digested vector at both the 5' and 3' ends to facilitate Gibson DNA assembly. Plasmids pJB0358 to pJB0372 were constructed containing the structures of 15 anti-EGFR scFv variants paired with a 6xHIS affinity tag (SEQ ID NO: 794), AE292 XTEN, RSR2295, and, individually, AE868 XTEN with anti-CD3 scFv, RSR2295, and C-tag affinity tags (DNA and protein sequences in Table 12).

[0220] pAH0025 and pAH0026 were generated by first digesting pJB0368 and pJB0373 with BtsI to remove the anti-CD3 scFv. DNA fragments encoding the anti-CD3.32 scFv flanked by 40 bp homology regions to the digested backbone were ordered. These fragments were introduced into pJB0368 and pJB0373 by Gibson Assembly to generate plasmids encoding the anti-EGFR-anti-CD3 bispecific tandem scFv, RSR2295, AE868 XTEN with a 6xHIS affinity tag (SEQ ID NO: 794), 6xHIS affinity tag (SEQ ID NO: 794), and RSR2295, constructed with two distinct anti-EGFR binding domains, EGFR.23 and EGFR.2, resulting in the pAH0025 and pAH0026 constructs (DNA and protein sequences in Table 12). Similar methodology was used to generate constructs with EGFR.13, EGFR.14, EGFR.15, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR.20, EGFR.21, EGFR.22, EGFR.24, EGFR.25, EGFR.26, EGFR.27, CD3.30, CD3.31, and CD3.33 scFvs in any combination or orientation (i.e., AF1-AF2 or AF2-AF1 from N-terminus to C-terminus), the sequences of which are provided herein. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7] Table 12-8 Table 12-9 Table 12-10 Table 12-11 Table 12-12 Table 12-13 Table 12-14 Table 12-15 Table 12-16 Table 12-17 Table 12-18 Table 12-19 Table 12-20 Table 12-21 Table 12-22 Table 12-23 Table 12-24 Table 12-25 Table 12-26 Table 12-27 Table 12-28 Table 12-29 Table 12-30 Table 12-31 Table 12-32 Table 12-33 Table 12-34 Table 12-35 Table 12-36 Table 12-37 Table 12-38 Table 12-39 Table 12-40 Table 12-41 Table 12-42 Table 12-43 Table 12-44 Table 12-45 Table 12-46 Table 12-47 Table 12-48 Table 12-49 Table 12-50 Table 12-51 Table 12-52 Table 12-53 Table 12-54 Table 12-55 Table 12-56 Table 12-57 Table 12-58 Table 12-59 Table 12-60 Table 12-61 Table 12-62 Table 12-63 Table 12-64 Table 12-65 Table 12-66 Table 12-67 Table 12-68 Table 12-69 Table 12-70 Table 12-71 Table 12-72 Table 12-73 Table 12-74 Table 12-75 Table 12-76 Table 12-77 Table 12-78 Table 12-79 Table 12-80 Table 12-81 Table 12-82 Table 12-83 Table 12-84 Table 12-85 Table 12-86 Table 12-87 Table 12-88 Table 12-89

Table 12-90

Table 12-95

Table 12-100

Table 12-110

Table 12-113

Table 12-119

Table 12-120

Table 12-130

Table 12-132

Table 12-139

Table 12-148

Table 12-170

Table 12-180

Table 12-200

Table 12-209

Table 12-231

Table 12-234

[0221] Example 2: Evaluation of CD3 scFv sequence variants compared to the parent CD3 scFv The purpose of this experiment was to evaluate four CD3 sequence variants to determine whether they had enhanced properties compared to the CD3.9 parent scFv.

[0222] 1. Melting temperature (T m ) determination The melting temperature of each scFv variant was measured to determine its thermal stability. Briefly, 200 μL of 1% BSA-PBST was aliquoted into PCR tubes. The tubes were incubated at several different temperatures (50°C, 51.4°C, 53.7°C, 57.3°C, 61.7°C, 65.5°C, and 68°C) for 1 hour. 50 μL of each sample was added to the CD3 TIFF2026000901000351.tif78μ target antigen (Creative Biomart) or BSA (see reference for sticky material) was added to an ELISA plate coated with 1% BSA-PBST. The wells of the ELISA plate were pre-filled with 1% BSA-PBST (50μl / well). The plate was incubated for 1 hour at room temperature. The plate was washed three times with water and 0.05% TWEEN to remove unbound scFv. Bound scFv was detected by adding anti-YOL antibody (Thermo Scientific, no. MA180189) (1:500 dilution (0.05%) in 1% BSA-PBST), which detects the porcine alpha-tubulin motif in the linker between the heavy and light chains. The sample was incubated for 1 hour at room temperature. The plate was washed three times with water and 0.05% TWEEN to remove unbound scFv. Anti-YOL antibody was prepared using anti-rat-HRP antibody (Thermo Scientific, no. 31470) (1:7500 dilution (0.05%) in 1% BSA-PBST) [100 TIFF2026000901000352.tif) and incubated at room temperature for 1 hour. Plates were washed three times with water and 0.05% TWEEN to remove unbound antibody. Plates were developed using TMB (3,3',5,5'-tetramethylbenzidine) substrate (100 μL / well, 6 minutes at room temperature). The reaction was stopped with H2SO4 (0.5 M, 100 μL / well). Relative activity was measured as absorbance readings at 450 nM. Absorbance at each temperature was graphed. The melting temperature was determined to be the EC50 for each sample, the temperature at which scFv binding decreased to 50% of the maximum signal. The results are presented in Table 15.

[0223] Results: The assay results showed that CD3 scFvs 3.23 and 3.24 had a Tm 5°C higher than the parental CD3.9, while CD3.25 and CD3.26 (sequences shown in Table 14) scFvs had Tm comparable to the parental CD3.9. m Indicates that the [Table 14]

[0224] 2. Determination of binding affinity to CD3 The binding affinity of each scFv was measured using a ForteBio BLItz instrument. A dilution series of each scFv was prepared in PBS (300 μL / tube) starting from 1000 nM to 62.5 nM in 1:1 dilution steps for CD3.24-26 and from 400 nM to 25 nM in 1:1 dilution steps for CD3.23. Biotinylated CD3 TIFF2026000901000354.tif78μ antigen (Creative Biomart) was diluted in PBS to a final concentration of 30μg / ml. Streptavidin biosensors (ForteBio) were activated in PBS for 10 minutes. To perform the measurements, the streptavidin biosensors were applied to the BLItz instrument. A tube containing 300μL of PBS was transferred to the BLItz instrument for 30 seconds. Biotinylated CD3 Tubes containing 78 μg (30 μg / ml, 300 μL / tube) were transferred to the BLItz instrument to measure antigen capture to the sensor over 120 seconds. Tubes containing 300 μL of PBS were transferred to the BLItz instrument over 30 seconds to measure baseline signal. Tubes containing test scFv (30 μg / ml, 300 μL / tube) were transferred to the BLItz instrument to measure association of scFv to the antigen-loaded biosensor over 120 seconds. Tubes containing 300 μL of PBS were transferred to the BLItz instrument over 120 seconds to measure dissociation of scFv from the antigen-loaded biosensor. This protocol was repeated for each scFv dilution. The K for each antibody was D was determined using BLI software (ForteBio). The results are presented in Table 15.

[0225] Results: The assay results show that all CD3 sequence variants had reduced binding affinity to CD3 compared to the parent CD3.9. [Table 15]

[0226] Conclusions: Two new anti-CD3 scFvs with improved thermal stability have been identified. Each of the new scFvs contains eight to nine mutations to CD3.9, primarily located in the CDRs. Although these mutations result in reduced affinity of the scFvs for their target (CD3) compared to the parent CD3.9, bispecific T cell engagers utilizing CD3.23 remain effective in cell killing assays and in vivo.

[0227] Example 3: Fermentation and purification of stable chimeric fusion polypeptides containing bispecific antigen-binding fragments, release segments, and XTEN The following examples describe the production of chimeric bispecific antigen-binding fragment compositions.

[0228] Construct ID pJB0169 is a molecule with eight distinct domains. From N- to C-terminus, it consists of an N-terminal polyhistidine tag (His6) (SEQ ID NO: 794), an unstructured 292 amino acid chain (XTEN_AE293), a protease-cleavable release segment (RS), an anti-EGFR scFv (aEGFR.2), an anti-CD3 scFv (aCD3.9), another protease-cleavable release segment (RS), an unstructured 864 amino acid chain, and the four C-terminal residues glutamic acid, proline, glutamic acid, alanine (C-tag) (XTEN_AE868).

[0229] Expression: The molecule pJB0169 was expressed in a proprietary E. coli AmE098 strain and distributed to the periplasm via an N-terminal secretion leader sequence (MKKNIAFLLASMFVFSIATNAYA- (SEQ ID NO: 940)), which was cleaved during translocation. Fermentation cultures were grown in animal-free complex medium at 37°C and phosphate depleted after a temperature shift to 26°C. After phosphate depletion, fermentation was continued for 12 hours. During harvest, the fermentation whole broth was centrifuged to pellet the cells. At harvest, the total volume and wet cell weight (WCW, the ratio of pellet to supernatant) were recorded, and the pelleted cells were collected and frozen at -80°C.

[0230] Clarification: Frozen cell pellets of pJB0169 were resuspended 3x in lysis buffer (60 mM acetic acid, 350 mM NaCl) at pH 4.5, and cells were lysed by homogenization. The homogenate was allowed to flocculate overnight at pH 4.5 and 2-8°C. The flocculated homogenate was centrifuged, and the supernatant was retained. The supernatant was diluted approximately 3x with water and then adjusted to 7 ± 1 mS / cm with NaCl. The supernatant was then adjusted to 0.1% (m / m) diatomaceous earth and mixed using an impeller. The supernatant was filtered through a filter train ending in a 0.22 μm filter. The filtrate was adjusted to pH 7.0 using dibasic sodium phosphate.

[0231] Purification: The molecule pJB0169 was first captured from the clarified lysate and purified by Protein L chromatography (TOYOPEARL AF-rProtein L-650F). Subsequently, IMAC chromatography (GE IMAC Sepharose 6 FF) was used to select for the N-terminal His6 tag (SEQ ID NO: 794), followed by C-tag affinity chromatography (CaptureSelect C-tagXL Affinity Matrix) to select for the C-terminal EPEA tag (SEQ ID NO: 796). Anion exchange chromatography (BIA CIMmultus QA monolith) was used to remove HMWC and polish to final purity.

[0232] Analytics: The aggregation state of process intermediates was monitored by SEC-HPLC. SEC-HPLC was performed using a Phenomenex 3 μm SEC-4000 300 x 7.8 mm (P / N 00H-4514-K0) column, 20 min isocratic, at 1 mL / min, while absorbance at 220 nm was monitored. pJB0169 monomer elutes from the analytical column at 6.2 min, and HMWC elutes between 4.8 and 6.0 min. SEC-HPLC quality was measured as the relative area under the curve at 6.2 min relative to the total area under the curve between 4.8 and 6.4 min.

[0233] Results: The aggregation summary (SEC-HPLC % monomer) of construct pJB0169 after each unit operation is presented in Table 16. ≥95% monomer recovery at final polish was the quality threshold as a criterion for a molecule to be considered stable or processable. [Table 16]

[0234] Conclusion: Construct pJB0169 was purified to the target monomer quality by SEC-HPLC (>95% monomer), indicating that this construct is stable and compatible with both recovery and purification procedures.

[0235] Stability Improvement and Evaluation: New scFvs (anti-EGFR.23 and anti-CD3.32) were designed to improve stability by (1) reducing surface hydrophobicity and (2) reducing the isoelectric point difference between paired scFv molecules (fused by a short peptide linker) through amino acid substitutions at selected positions. Constructs pAH0025 and pAH0026 represent design iterations of pJB0169, with pAH0025 containing an anti-CD3.32 scFv variant and pAH0026 containing both an anti-CD3.32 scFv variant and an EGFR.23 scFv variant. Constructs pAH0025 and pAH0026 were expressed, purified, and analyzed as described above. SEC-HPLC results were monitored throughout the purification to assess relative stability compared to pJB0169 or other constructs (e.g., αEGFR.2-αCD3.23). The new design pair may be more stable than the corresponding αEGFR.2-αCD3.23 (such as a molecule consisting of, from N- to C-terminus, an N-terminal polyhistidine tag (His6) (SEQ ID NO: 794), an unstructured 292 amino acid chain (XTEN_AE292), a protease-cleavable release segment (RS), an anti-EGFR scFv (aEGFR.2), an anti-CD3 scFv (aCD3.23), another protease-cleavable release segment (RS), an unstructured 864 amino acid chain, and the four C-terminal residues glutamic acid, proline, glutamic acid, alanine (C-tag) (XTEN_AE868)). The pAH0025 and pAH0026 constructs may also be expected to show a concomitant improvement in percent monomer content as measured by SEC-HPLC after the unit operations (or a subset thereof) shown in the table below (Table 17). Any construct meeting a purity target of 95% or greater monomer will be considered stable or processable. [Table 17]

[0236] Example 4: Binding affinity of anti-EpCAM x anti-CD3 bispecific antigen-binding polypeptide compositions. The binding affinities of the anti-EpCAM x anti-CD3 bispecific antigen-binding polypeptide constructs pJB0189 and pCW1645 to human EpCAM and human CD3 were measured using flow cytometry with huEp-CHO 4-12B cells (a CHO cell line transfected with human EpCAM) and Jurkat cells.

[0237] The binding constants of anti-EpCAM x anti-CD3 bispecific antigen-binding polypeptides bound to EpCAM- and CD3-expressing cells were measured by competitive binding with fluorescently labeled protease-treated bispecific antigen-binding polypeptides. The fluorescently labeled protease-treated bispecific antigen-binding polypeptides were generated by conjugation of Alexa Fluor 647 C2 maleimide (Thermo Fisher, catalog no. A20347) to a cysteine-containing protease-treated bispecific antigen-binding polypeptide mutant (MMP-9-treated pCW1645). Binding experiments were performed on 10,000 cells in a total volume of 100 μL of binding buffer (2% FCS, 5 mM EDTA, HBSS) for 1 hour at 4°C. Cells were washed once with cold binding buffer, then resuspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Binding of fluorescently labeled protease-treated pCW1645 was observed with an apparent K of 1 nM for hEp-CHO 4-12B cells and 4 nM for CD3+ Jurkat cells. d It was found to have value.

[0238] Competitive binding experiments were performed on 10,000 hEp-CHO 4-12B cells with 1.5 nM fluorescently labeled protease-treated pCW1645 in a total volume of 100 μL of binding buffer (2% FCS, 5 mM EDTA, HBSS) for 1 hour at 4°C. Cells were washed once with cold binding buffer, then resuspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Competitive binding of fluorescently labeled protease-treated pCW1645 with truncated bispecific antigen-binding polypeptides (pJB0189 hEp.2-hCD3.9 or AC1984 hEp.2-hCD3.23) to hEp-CHO 4-12B cells yielded an apparent binding constant of 0.5 nM for hEp.2 (panitumumab).

[0239] Competitive binding experiments were performed on 10,000 Jurkat cells with 10 nM fluorescently labeled protease-treated pCW1645 in a total volume of 100 μL of binding buffer (2% FCS, 5 mM EDTA, HBSS) for 1 hour at 4°C. Cells were washed once with cold binding buffer, then resuspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Competitive binding of fluorescently labeled protease-treated pCW1645 with truncated bispecific antigen-binding polypeptides (pJB0189 hEp.2-hCD3.9 or AC1984 hEp.2-hCD3.23) to Jurkat cells yielded apparent binding constants of 75 nM for hCD3.9 and 300 nM for hCD3.23 for CD3 binding, and 0.5 nM for EpCAM binding.

[0240] Conclusion: The binding affinity of CD3.23 to CD3 on Jurkat cells is 300 nM, which is 4-fold weaker than that of CD3.9. The binding affinity of hEp.2 to EpCAM on Jurkat cells is 0.5 nM.

[0241] Example 5: Binding affinity of anti-EGFR x anti-CD3 bispecific antigen-binding polypeptide compositions. The binding affinity of the anti-EGFR x anti-CD3 bispecific antigen-binding polypeptide constructs to human EGFR and human CD3 is measured using flow cytometry using EGFR-positive human cells selected from HT-29, HCT-116, NCI-H1573, NCI-H1975, and CD3 Jurkat cells.

[0242] The binding constants of anti-EGFR x anti-CD3 bispecific antigen-binding polypeptides bound to EGFR-expressing cells and CD3-expressing cells were measured by competitive binding with fluorescently labeled protease-treated bispecific antigen-binding polypeptides. Fluorescently labeled bispecific antigen-binding polypeptides were prepared by conjugating Alexa Fluor 647 C2 maleimide (Thermo Fisher, catalog number A20347) to a cysteine-containing bispecific antigen-binding polypeptide variant (MMP-9-treated pJB0297) with hEGFR.2-hCD3.23 and two XTENs. Fluorescently labeled protease-treated bispecific antigen-binding polypeptides were prepared by conjugating Alexa Fluor 647 C2 maleimide (Thermo Fisher, catalog number A20347) to a cysteine-containing protease-treated bispecific antigen-binding polypeptide variant (MMP-9-treated pJB0297). Binding experiments are performed on 10,000 cells in a total volume of 100 μL of binding buffer (2% FCS, 5 mM EDTA, HBSS) for 1 hour at 4°C. Cells are washed once with cold binding buffer, then resuspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Binding of fluorescently labeled protease-treated pJB0297 was observed at low nM concentrations for hEGFR-bearing cells and with an apparent K of approximately 300 nM for CD3+ Jurkat cells. dBinding of fluorescently labeled pJB0297 to the two XTENs is expected to have an apparent K value approximately 10-100 times weaker than that of fluorescently labeled protease-treated bispecific antigen-binding polypeptides on hEGFR-bearing cells and CD3+ Jurkat cells. d It is expected to have a value.

[0243] Competitive binding experiments were performed using the K d The binding of fluorescently labeled protease-treated pJB0297 to 10,000 hEGFR cells was performed in a total volume of 100 μL of binding buffer (2% FCS, 5 mM EDTA, HBSS) at a concentration close to 100 μL for 1 hour at 4°C. Cells were washed once with cold binding buffer, then resuspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Competitive binding of fluorescently labeled protease-treated pJB0297 to hEGFR cells with the pJB0244 bispecific antigen-binding polypeptide is expected to have an apparent binding constant similar to the direct binding constant of fluorescently labeled pJB0297.

[0244] Competitive binding experiments were performed using a K d Fluorescently labeled protease-treated pJB0297 (at a concentration close to 100 μL) is incubated with 10,000 Jurkat cells in a total volume of 100 μL of binding buffer (2% FCS, 5 mM EDTA, HBSS) for 1 hour at 4°C. Cells are washed once with cold binding buffer, then resuspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Competitive binding of fluorescently labeled protease-treated pJB0297 to Jurkat cells with the pJB0244 bispecific antigen-binding polypeptide is expected to have an apparent binding constant similar to the direct binding constant of fluorescently labeled pJB0297, which is expected to be in the low micromolar to nanomolar range.

[0245] Example 6: Enzyme activation, storage, and digestion of RSR-1517-containing XTEN AC1611 (RSR-1517). This example demonstrates that the RSR-1517-containing XTEN construct AC1611 can be cleaved in vitro by various tumor-associated proteases, including recombinant human uPA, matriptase, legumain, MMP-2, MMP-7, MMP-9, and MMP-14. The amino acid sequence of AC1611 is presented in Table 18 below.

[0246] 1. Enzyme activation All enzymes used were obtained from R&D Systems. Recombinant human u-plasminogen activator (uPA) and recombinant human matriptase were provided as activated enzymes and stored at -80°C until use. Recombinant mouse MMP-2, recombinant human MMP-7, and recombinant mouse MMP-9 were provided as proenzymes and required activation with 4-aminophenylmercuric acetate (APMA). APMA was first dissolved in 0.1 M NaOH to a final concentration of 10 mM, and the pH was readjusted to neutral using 0.1 M HCl. Further dilutions of the APMA stock to 2.5 mM were made in 50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM CaCl2. To activate pro-MMPs, 1 mM APMA and 100 μg / mL pro-MMPs in 50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM CaCl were incubated at 37°C for 1 hour (MMP-2, MMP-7) or 24 hours (MMP-9). To activate MMP-14, 0.86 μg / mL recombinant human furin and 40 μg / mL pro-MMP-14 in 50 mM Tris (pH 9), 1 mM CaCl were incubated at 37°C for 1.5 hours. To activate legumain, 100 μg / mL prolegumain in 50 mM sodium acetate (pH 4), 100 mM NaCl was incubated at 37°C for 2 hours. 100% ultrapure glycerol was added to all activated enzymes (including uPA and MTSP1) to a final concentration of 50% glycerol, which was then stored at −20°C for several weeks.

[0247] 2. Enzyme digestion A panel of enzymes was tested to determine the cleavage efficiency of each enzyme in AC1611. Six micromolar amounts of substrate were incubated with each enzyme at the following enzyme:substrate molar ratios and conditions: uPA (1:25 in 50 mM Tris, pH 8.5), matriptase (1:25 in 50 mM Tris, pH 9, 50 mM NaCl), legumain (1:20 in 50 mM MES, pH 5, 250 mM NaCl), MMP-2 (1:1200 in 50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM CaCl), MMP-7 (1:1200 in 50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM CaCl), MMP-9 (1:1200 in 50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM CaCl), in 20 uL reactions. Reactions were incubated at 37°C for 2 hours and then stopped by adding EDTA to 20 mM for MMP reactions, heating at 85°C for 15 minutes for uPA and matriptase reactions, and adjusting the pH to 8.5 for legumain.

[0248] 3. Analysis of Cleavage Efficiency As shown in Figure 75, the reaction mixture of 2 μL of undigested substrate (12 μM) and 4 μL of digested substrate (6 μM) was loaded onto SDS-PAGE and analyzed by staining with Stains-All (Sigma Aldrich) to determine the percentage of cleavage products. ImageJ software was used to analyze the corresponding band intensities and determine the percent cleavage. Upon cleavage by various proteases at the release segment, the substrate RSR-1517-containing XTEN yielded two fragments, and the larger fragment was utilized for percent cleavage calculations (amount of reaction product divided by total initial substrate reacted), while the band intensity of the smaller product was too low to be quantified. Under current standard experimental conditions, the cleavage percentages of AC1611 are 31%, 14%, 16%, 40%, 51%, 38%, and 30% for uPA, matriptase, legumain, MMP-2, MMP-7, MMP-9, and MMP-14, respectively.

[0249] Conclusion: We selected a specific release segment, RSR-1517 (amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO: 53)), and determined its cleavage profile, defined by the percentage of cleavage, for all seven enzymes under current standard experimental conditions. This release segment has moderate cleavage efficiency for all enzymes, which allows for accurate ranking of faster or slower cleavage variants during screening, falling within the assay window. [Table 18]

[0250] Example 7: Release segment screening using RSR-1517 (AC1611) as a control In this example, uPA is chosen as an example to demonstrate how release segment screening was performed. The same procedure was applied to all seven tumor-associated proteases to define the relative cleavage profile for each substrate, which is a 7-number array that describes how well each enzyme can cleave it compared to the control substrate RSR-1517. All polypeptides in Table 19 have the amino acid sequence of AC1611, but with the release segment peptide of the indicated construct swapped to the EAGRSANHEPLGLVAT sequence of AC1611 (SEQ ID NO: 53); for example, BSRS-4 has a release segment sequence of LAGRSDNHSPLGLAGS (SEQ ID NO: 945), but otherwise has complete sequence identity with AC1611.

[0251] 1. Enzyme digestion All release segment-containing XTEN variants and the control AC1611 were diluted to 12 μM in individual Eppendorf tubes in 50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM CaCl2. A master mix of uPA was mixed 1:1 with each substrate, resulting in a total reaction volume of 20 μL, an initial substrate concentration of 6 μM, and an enzyme:substrate ratio varying from 1:20 to 1:3000 depending on the enzyme, to obtain reaction products and uncleaved substrates that could be visualized at the endpoint. All reactions were incubated at 37°C for 2 h and then stopped by adding EDTA to a final concentration of 20 mM. All products were analyzed using non-reducing SDS-PAGE followed by Stains-All. For each gel, AC1611 digestion products were always included as a staining control to normalize staining across different gels.

[0252] 2. Calculation of relative cleavage efficiency The percentage of cleavage of individual substrates was analyzed using ImageJ software and calculated as previously described. For each variant, the relative cleavage efficiency is calculated as follows:

number

[0253] Under the experimental conditions specified above, a value of +1 in the relative cleavage efficiency indicates that the substrate produced twice as much product as the AC1611 control, while a value of -1 in the relative cleavage efficiency indicates that the substrate produced only 50% of the product as the AC1611 control.

[0254] In this experiment, the cleavage percentage (% cleavage) of AC1611 was 20% as quantified by ImageJ. The substrates screened in this experiment showed cleavage of 21%, 39%, 1%, 58%, 24%, 6%, 15%, 1%, 1%, and 25%, where 1% essentially represents below the detection limit and does not represent an exact value. The relative cleavage efficiencies calculated based on the above formula were 0.08, 0.95, -4.34, 1.51, 0.26, -1.76, -0.47, -4.34, -4.34, and 0.32, respectively.

[0255] Conclusion: We determined the relative cleavage efficiency of 10 release segment variants when subjected to uPA compared with AC1611 in the same experiment. Following a similar procedure, we determined the cleavage profiles of 134 release segments, and the results are listed in Table 19, using RSR1517 (AC1611) as a reference control. These release segments cover a wide range of cleavage efficiencies for both individual enzymes and in combination. For example, RSR-1478 had a value of -2.00 for MMP-14, meaning that this substrate only yielded 25% of the product when digested with MMP-14 compared to the reference control RSR-1517. Certain release segments, such as RSR-1951, appear to be better substrates for all seven proteases tested. While these faster release segments may prove clinically useful if systemic toxicity is low / manageable, improvements in efficacy (depending in part on how quickly cleavage occurs to render the bispecific antigen-binding composition in its activated form) are required. [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4] [Table 19-5]

[0256] Example 7: Competitive digestion using RSR-1517 as an internal control This competitive assay is developed to minimize any variation in enzyme concentration or reaction conditions between reactions in different vials within the same experiment. New control plasmids are constructed to address both the control substrate and the RS of interest in the same experiment.

[0257] 1. Molecular cloning of RSR-1517-containing internal control Two internal control plasmids, AC1830 (HD2-V5-AE144-RSR-1517-XTEN288) and AC1840 (HD2-V5-AE144-RSR-1517-XTEN432), were constructed in a similar manner to AC1611 described in Example 6, differing only in the length of the C-terminal XTEN.

[0258] 2. Enzyme digestion A 2x substrate solution is prepared by mixing and diluting purified AC1830 or AC1840 and the desired RS in assay buffer to a final concentration of 6 μM for each substrate. An enzyme master mix is ​​prepared such that, after mixing 1:1 with the 2x substrate solution, the total reaction volume is 20 μL, the final substrate concentration of each component is 3 μM, and the enzyme:substrate ratio is as selected during assay development. The reaction is incubated at 37°C for 2 hours and then stopped as described above.

[0259] 3. Calculation of Relative Cleavage Efficiency The reaction mixture is analyzed using non-reducing 4-12% SDS-PAGE. Because the internal control and the target substrate have different molecular weights, upon cleavage, four bands should be visible in the same sample lane. The percentage of cleavage for both can be calculated, and the relative cleavage efficiency can be derived using the same formula as in Example 6.

number

[0260] The only difference is that now both values ​​are calculated from a reaction mixture in the same vial, whereas previously they were calculated from two reactions sharing the same enzyme mixture.

[0261] Conclusion: We expect this competitive digestion assay using RSR-1517 as an internal control to have less interassay variability compared to the assay described in Example 6. We look forward to adopting this method for further release segment screening.

[0262] Example 9: In vitro caspase 3 / 7 assay of anti-EGFR x anti-CD3 bispecific antigen binding compositions The redirected cytotoxicity of unmasked (proteolytically removed XTEN), masked (with two proteolytically cleavable XTEN and two release segments), and non-cleavable (with two proteolytically insensitive XTEN and a peptide-substituted release segment) anti-EGFR x anti-CD3 bispecific antigen-binding polypeptide compositions was evaluated in an in vitro cell-based assay of caspase 3 / 7 activity in apoptotic cells. Similar to the caspase cytotoxicity assay described in the previous example, PBMCs were mixed with EGFR-positive tumor target cells at a ratio of 1 target cell to 10 effector cells. All anti-EGFR x anti-CD3 bispecific antigen-binding polypeptide compositions were tested using 10-point, 5-fold serial dilutions. Unmasked anti-EGFR x anti-CD3 compositions were evaluated over a final dose range of 0.000012 to 10 nM. Masked and non-cleavable bispecific antigen-binding polypeptide compositions were analyzed over a final dose range of 0.00064 to 250 nM. Suitable EGFR-positive human tumor target cell lines included FaDu (head and neck squamous cell carcinoma, SCCHN), SCC-9 (SCCHN), HCT-116 (KRAS-mutated colorectal tumor), NCI-H1573 (KRAS-mutated colorectal tumor), HT-29 (BRAF-mutated colorectal tumor), and NCI-H1975 (EGFR T790M-mutated). Cell lines representing wild-type EGFR and T790M-mutated, KRAS-mutated, and BRAF-mutated colorectal and SCCHN tumors were selected.

[0263] Upon cell lysis, released caspase 3 / 7 in the culture supernatant was measured by the amount of cleavage of a luminescent caspase 3 / 7 substrate by caspase 3 / 7, generating a "glow-type" luminescent signal (Promega Caspase-Glo 3 / 7, catalog no. G8091). The amount of luminescence is proportional to the amount of caspase activity.

[0264] Results: As shown in Table 20, the EC of the masked anti-EGFR x anti-CD3 bispecific antigen-binding polypeptides when evaluated in the EGFR KRAS mutant HCT-116 cell line was 50 The activity was 3,408 pM. EC 50 is greater than 100,000 pM and the unmasked EC 50 The activity was 0.8 pM.

[0265] EC of masked anti-EGFR × anti-CD3 bispecific antigen-binding polypeptides when assessed in EGFR BRAF-mutant HT-29 cell lines 50 The activity was 10,930 pM. EC of the uncleavable and unmasked compositions 50 The activities were over 100,000 pM and 0.8 pM, respectively.

[0266] In the two EGFR mutant cell lines tested, the masked anti-EGFR x anti-CD3 bispecific antigen-binding polypeptide was approximately 4,000- to 14,000-fold less active than the unmasked anti-EGFR x anti-CD3 bispecific antigen-binding polypeptide. As expected, the activity of the uncleavable variant was the lowest of the three versions evaluated, with EC 50 was over 100,000 pM.

[0267] Conclusions: The results demonstrate that the anti-EGFR x anti-CD3 bispecific antigen-binding polypeptide is cytotoxically active against EGFR KRAS-mutant and BRAF-mutant cell lines. The masked anti-EGFR x anti-CD3 bispecific antigen-binding polypeptide with two XTENs resulted in potent cytotoxic activity blockade, with cytotoxicity 4,000-14,000-fold lower than that of the unmasked form. [Table 20]

[0268] Example 10: Antitumor properties of anti-EGFR x anti-CD3 bispecific antigen-binding polypeptide compositions in an early-treatment HT-29 in vivo model. In vivo efficacy experiments were conducted to evaluate EGFR-CD3 bispecific antigen-binding polypeptide compositions based on the pJB0169 construct in immunodeficient NOD / SCID mice, characterized by T cell and B cell deficiencies and impaired natural killer cell function. Mice were maintained under sterile, standardized environmental conditions, and the experiments were conducted in accordance with the US Institutional Animal Care Association for Assessment and Use Committee (IACUC) Accreditation of Laboratory Animal Care (AAALAC) guidelines. The efficacy of protease-treated and protease-untreated anti-EGFR x anti-CD3 bispecific antigen-binding polypeptides (e.g., pJB0169) was evaluated using an EGFR BRAF-mutant human HT-29 adenocarcinoma xenograft model. Briefly, on day 0, six NOD / SCID mice were inoculated with 3 x 10 IgG per mouse into the right flank. 6 On the same day, 6 × 10 HT-29 cells per mouse were subcutaneously transplanted into the right flank of cohorts 2 to 7, each consisting of six NOD / SCID mice per group. 6 3 x 10 human PBMCs 6The HT-29 cells were subcutaneously injected with a mixture of HT-29 cells and HT-29 / PBMCs. Treatment began 4 hours after inoculation of the HT-29 or HT-29 / PBMC mixture. Cohorts 1 and 2 were intravenously injected with vehicle (PBS + 0.05% Tween 80). Cohorts 3 and 4 were injected with 0.05 mg / kg intact anti-EGFR x anti-CD3 bispecific construct and 0.5 mg / kg anti-EGFR x anti-CD3 bispecific construct, respectively, that had been treated with protease to remove XTEN from the polypeptide. Cohorts 5 and 6 were injected with 0.143 mg / kg and 1.43 mg / kg intact anti-EGFR x anti-CD3 bispecific construct, respectively. Cohort 7 was injected with 50 mg / kg cetuximab as a positive control. Cohorts 1–6 further received seven booster doses daily on days 1–7 (for a total of eight doses). Cohort 7 received cetuximab twice weekly for 4 weeks for a total of 8 doses.

[0269] Tumors in mice were measured twice weekly over an expected 33-day period using calipers in two perpendicular dimensions, and tumor volumes were calculated as follows: (width) 2 The tumor growth inhibition index (TGI%) was calculated by applying the formula: ((Cohort 2 vehicle control mean tumor volume - test article treated mean tumor volume) / Cohort 2 vehicle control mean tumor volume) × 100. Treatments with a TGI% of 60% or greater were considered therapeutically active.

[0270] Results: On day 33, vehicle-treated Cohort 1 mice bearing tumor cells had a mass of 250±113 mm 3 Cohort 2 mice treated with vehicle in the presence of human effector cells did not inhibit tumor progression, with a mean tumor burden of only 238 ± 228 mm 3The mean tumor burden was 1.02 mg / kg, indicating that human effector cells alone were unable to induce antitumor effects. Treatment with the protease-treated anti-EGFR × anti-CD3 construct at 0.05 mg / kg and 0.5 mg / kg (cohorts 3 and 4, respectively) in the presence of human effector cells exhibited clear tumor growth inhibition with a TGI of 99% in both treatment groups. Importantly, treatment with anti-EGFR × anti-CD3 XPAT at 0.143 mg / kg and 1.43 mg / kg (cohorts 5 and 6, respectively) in the presence of human effector cells also inhibited tumor growth in a dose-dependent manner with a TGI of 70% for the 0.143 mg / kg dose group and 96% for the 1.43 mg / kg cohort. The data suggest that at the 0.143 mg / kg and 1.43 mg / kg dosages, sufficient amounts of the anti-EGFR x anti-CD3 construct were effectively cleaved by proteases in the in vivo tumor environment to form more active, non-XTENed anti-EGFR x anti-CD3 bispecific antigen-binding fragments, resulting in the observed efficacy. Significantly, cohort 7 treated with 50 mg / kg cetuximab did not induce tumor regression, with a TGI% of -20%.

[0271] Conclusion: The results suggest that the anti-EGFR x anti-CD3 bispecific construct can be effectively cleaved in vivo to an active form and is effective in inhibiting tumor progression in the setting of EGFR-BRAF-mutant HT-29 tumors. Additionally, the anti-EGFR x anti-CD3 bispecific construct outperformed the cetuximab control in terms of antitumor activity under these experimental conditions. Notably, no significant weight loss was observed in any of the test-agent-treated groups, indicating that all treatments were well tolerated.

[0272] Example 11: Cell binding assessed by flow cytometry. The bispecific binding of the anti-EGFR x anti-CD3 bispecific antigen binding composition is also assessed by a flow cytometry-based assay utilizing CD3-positive human Jurkat cells and EGFR-positive human cells selected from HT-29, HCT-116, NCI-H1573, NCI-H1975, FaDu, and SCC-9, or a stable CHO cell line expressing EGFR. + Cells and EGFR + Cells are incubated with a dose range of untreated anti-EGFR x anti-CD3 bispecific antigen binding composition (PJB0169, including two XTENs and two RSs), protease-treated PJB0169, and anti-CD3 scFv and anti-EGFR scFv positive controls in a binding buffer containing HBSS with 2% BSA and 5 mM EDTA for 30 minutes at 4 ° C. After washing with binding buffer to remove unbound test material, cells are incubated with FITC-conjugated anti-His tag antibody (Abcam, catalog number ab1206) for 30 minutes at 4 ° C. Unbound FITC-conjugated antibody is washed away with binding buffer, and cells are resuspended in binding buffer and acquired on a FACS Calibur flow cytometer (Becton Dickerson) or equivalent instrument. All flow cytometry data are analyzed using FlowJo software (FlowJo LLC) or equivalent.

[0273] While the anti-EGFR scFv is not expected to bind to Jurkat cells, the anti-CD3 scFv, untreated PJB0169, and protease-treated PJB0169 are all expected to bind to Jurkat cells, as indicated by increased fluorescence intensity compared to Jurkat cells incubated with FITC-conjugated anti-His tag antibody alone. Similarly, the anti-EGFR scFv, protease-treated, and untreated PJB0169 are all expected to bind to EGFR-positive cells, while the anti-CD3 scFv is not expected to bind to EGFR-positive cells. It is expected that these data reflect the bispecific binding capacity of the anti-EGFR x anti-CD3 bispecific antigen binding composition, which recognizes both the CD3 antigen and the EGFR antigen expressed on a panel of Jurkat cell lines and EGFR-expressing human cell lines, respectively. Furthermore, due to some interference with surface binding by the XTEN polymer, it is expected that the untreated anti-EGFR x anti-CD3 bispecific antigen binding composition will bind to both the CD3 and EpCAM antigens with lower affinity than the protease-treated bispecific antigen binding composition.

[0274] Example 12: Cytolysis assessed by flow cytometry. Cytolysis by the anti-EGFR x anti-CD3 bispecific antigen binding composition is assessed by flow cytometry using human PBMCs and EGFR-positive cell lines. EGFR-positive HCT-116 target cells (or target cells selected from HT-29, NCI-H1573, NCI-H1975, FaDu, and SCC-9, or a stable CHO cell line expressing EGFR) are labeled with the fluorescent membrane dye CellVue Maroon dye (Affymetrix / eBioscience, catalog number 88-0870-16) according to the manufacturer's instructions. Alternatively, PKH26 (Sigma, catalog numbers MINI26 and PKH26GL) can also be used. Briefly, HCT-116 cells are washed twice with PBS and then diluted at 2 x 10 in 0.1 mL of diluent C provided in the CellVue Maroon labeling kit. 6In a separate tube, 2 μL of CellVue Maroon dye is mixed with 0.5 mL of Diluent C, and then 0.1 mL is added to the HCT-116 cell suspension. The cell suspension and CellVue Maroon dye are mixed and incubated at room temperature for 2 minutes. The labeling reaction is then quenched by adding 0.2 mL of fetal calf serum (FCS). The labeled cells are washed twice with complete cell culture medium (RPMI-1640 containing 10% FCS), and the total via...

Claims

1. A polypeptide comprising an antibody-binding fragment (AF1), wherein the AF1 comprises a light chain complementarity determining region (CDR-L), a heavy chain complementarity determining region (CDR-H), a light chain framework region (FR-L), and a heavy chain framework region (FR-H), wherein the AF1 comprises: a. specifically binds to the epidermal growth factor receptor (EGFR); b. A polypeptide comprising FR-H1, FR-H2, FR-H3, and FR-H4, wherein FR-H1 has the amino acid sequence of any one of SEQ ID NOs: 14 to 16, FR-H2 has the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19, FR-H3 has the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, and FR-H4 has the amino acid sequence of any one of SEQ ID NOs: 22 to 24.

2. The polypeptide of claim 1, wherein the AF1 further comprises a CDR-H3, the CDRH3 having the amino acid sequence of SEQ ID NO:

6.

3. The polypeptide of claim 1 or 2, wherein the AF1 further comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

4. The polypeptide according to any one of claims 1 to 3, wherein the AF1 has a higher isoelectric point (pI) than the isoelectric point (pI) of an antigen-binding fragment consisting of the sequence set forth in SEQ ID NO:

52.

5. 5. The polypeptide of any one of claims 1 to 4, wherein the AF1 is incorporated into the polypeptide to form an anti-EGFR bispecific antibody, the polypeptide exhibiting a higher pI relative to a control bispecific antibody, the polypeptide comprising the AF1 and a reference antigen-binding fragment that binds to cluster of differentiation 3 T-cell receptor (CD3), the control bispecific antigen-binding fragment being identical to the polypeptide except for the AF1 being replaced with SEQ ID NO:

52.

6. 6. The polypeptide of claim 4 or 5, wherein AF1 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units higher than the pI of the antigen-binding fragment consisting of the sequence set forth in SEQ ID NO:

52.

7. the AF1 further comprises FR-L1, FR-L2, FR-L3, and FR-L4; a. FR-L1 exhibits at least 90%, or at least 95%, sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 7; b. FR-L2 exhibits at least 90%, or at least 95%, sequence identity to or is identical to the amino acid sequence of SEQ ID NO:8; c. FR-L3 exhibits at least 90%, or at least 95%, sequence identity to, or is identical to, the amino acid sequence of SEQ ID NOs: 9-11; d. A polypeptide according to any one of the preceding claims, wherein FR-L4 exhibits at least 90%, or at least 95% sequence identity to, or is identical to, the amino acid sequence of SEQ ID NO:

13.

8. 10. The polypeptide of any one of the preceding claims, wherein the AF1 CDR-L comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively.

9. The FR-L is a. FR-L1 having the amino acid sequence of SEQ ID NO: 7; b. FR-L2 having the amino acid sequence of SEQ ID NO: 8; c. FR-L3 having the amino acid sequence of SEQ ID NO: 9; d. A polypeptide according to any one of the preceding claims, comprising FR-L4 having the amino acid sequence of SEQ ID NO:

13.

10. The FR-L is a. FR-L1 having the amino acid sequence of SEQ ID NO: 7; b. FR-L2 having the amino acid sequence of SEQ ID NO: 8; c. FR-L3 having the amino acid sequence of SEQ ID NO: 10; d. The polypeptide of any one of claims 1 to 8, comprising FR-L4 having the amino acid sequence of SEQ ID NO:

13.

11. The FR-L is a. FR-L1 having the amino acid sequence of SEQ ID NO: 7; b. FR-L2 having the amino acid sequence of SEQ ID NO: 8; c. FR-L3 having the amino acid sequence of SEQ ID NO: 11, and d. The polypeptide of any one of claims 1 to 8, comprising FR-L4 having the amino acid sequence of SEQ ID NO:

13.

12. The FR-H is a. FR-H1 having the amino acid sequence of SEQ ID NO: 14; b. FR-H2 having the amino acid sequence of SEQ ID NO: 18; c. FR-H3 having the amino acid sequence of SEQ ID NO: 20; d. The polypeptide of any one of claims 1 to 11, comprising FR-H4 having the amino acid sequence of SEQ ID NO: 22 or 23.

13. The FR-H is a. FR-H1 having the amino acid sequence of SEQ ID NO: 15; b. FR-H2 having the amino acid sequence of SEQ ID NO: 19; c. FR-H3 having the amino acid sequence of SEQ ID NO: 21; d. The polypeptide of any one of claims 1 to 11, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

24.

14. The FR-H is a. FR-H1 having the amino acid sequence of SEQ ID NO: 16; b. FR-H2 having the amino acid sequence of SEQ ID NO: 19; c. FR-H3 having the amino acid sequence of SEQ ID NO: 20; d. The polypeptide of any one of claims 1 to 11, comprising FR-H4 having the amino acid sequence of SEQ ID NO: 22 or 23.

15. 10. The polypeptide of any one of the preceding claims, wherein said AF1 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NOs: 28-32.

16. 10. The polypeptide of any one of the preceding claims, wherein the AF1 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NOs: 25-27.

17. 10. The polypeptide of any one of the preceding claims, wherein AF1 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of any one of SEQ ID NOs: 37-51.

18. 2. The polypeptide of any one of the preceding claims, wherein said AF1 specifically binds to human or cynomolgus monkey (cyno) EGFR.

19. 2. The polypeptide of any one of the preceding claims, wherein said AF1 specifically binds to human and cynomolgus monkey (cyno) EGFR.

20. 10. The polypeptide of any one of the preceding claims, wherein said AF1 exhibits a pI of from about 5.4 to about 5.6, or from about 5.5 to about 5.7, or from about 5.6 to about 5.8, or from about 5.7 to about 5.9, or from about 5.8 to about 6.0, or from about 5.9 to about 6.1, or from about 6.0 to about 6.2, or from about 6.1 to about 6.3, or from about 6.2 to about 6.4, or from about 6.3 to about 6.5, or from about 6.4 to about 6.

6.

21. 10. The polypeptide of any one of the preceding claims, wherein the AF1 exhibits a pI of about 5.4, or about 5.5, or about 5.6, or about 5.7, or about 5.8, or about 5.9, or about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.

6.

22. 10. The polypeptide of any one of the preceding claims, wherein said AF1 exhibits a pI of 5.4 to 6.6, inclusive.

23. The AF1 has a K of about 0.1 nM to about 100 nM as determined in an in vitro antigen binding assay involving EGFR or an epitope thereof. d 10. A polypeptide according to any one of the preceding claims which specifically binds to EGFR at

24. 5. The polypeptide of claim 1, wherein the AF1 has at least one amino acid substitution of a hydrophobic amino acid in a framework region relative to the amino acid sequence of SEQ ID NO: 52, wherein the hydrophobic amino acid is selected from isoleucine, leucine, or methionine, and the substituted amino acid is selected from arginine, threonine, or glutamine.

25. 25. The polypeptide of claim 24, wherein AF1 has at least two amino acid substitutions of hydrophobic amino acids in one or more framework regions relative to the amino acid sequence of SEQ ID NO: 52, wherein the hydrophobic amino acids are selected from isoleucine, leucine, or methionine, and the substituted amino acids are selected from arginine, threonine, or glutamine.

26. 10. The polypeptide of any one of the preceding claims, further comprising a first release segment peptide (RS1), said RS1 being a substrate for cleavage by a mammalian protease.

27. 10. The polypeptide of any one of the preceding claims, wherein the RS1 is a substrate for a protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase.

28. 10. The polypeptide of any one of the preceding claims, wherein the RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 53-671.

29. 2. The polypeptide of any one of the preceding claims, wherein the RS1 comprises an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which is set forth in Table 5.

30. and further comprising a first extended recombinant polypeptide (XTEN1), said XTEN1 comprising: a. it has at least about 36 amino acids; b. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P); c. A polypeptide according to any one of the preceding claims, characterized in that it has at least 4 to 6 different amino acids selected from G, A, S, T, E and P.

31. 10. The polypeptide of any one of the preceding claims, wherein the XTEN1 comprises an amino acid sequence comprising at least three of the amino acid sequences of SEQ ID NOs: 672-675.

32. 10. The polypeptide of any one of the preceding claims, wherein the XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOs: 676-734.

33. The XTEN1 is AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE86 4, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7.

34. 2. The polypeptide of any one of the preceding claims, wherein the AF1 is a chimeric or humanized antigen-binding fragment.

35. 10. The polypeptide of any one of the preceding claims, wherein said AF1 is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, linear antibody, and single chain variable fragment (scFv).

36. 10. The polypeptide of any one of the preceding claims, expressed as a fusion protein, said fusion protein having, in its uncleaved state, the structural arrangement of AF1-RS1-XTEN1 or XTEN1-RS1-AF1 from N-terminus to C-terminus.

37. 10. The polypeptide of any one of the preceding claims, further comprising a second antigen-binding fragment (AF2) that specifically binds to the cluster of differentiation 3 T-cell receptor (CD3).

38. 38. The polypeptide of claim 37, wherein the AF2 binds to a CD3 complex subunit selected from any one of CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta epsilon.

39. 39. The polypeptide of claim 37 or 38, wherein the AF2 specifically binds to human or cynomolgus monkey (cyno) CD3.

40. 39. The polypeptide of claim 37 or 38, wherein the AF2 specifically binds to human and cynomolgus monkey (cyno) CD3.

41. The polypeptide of any one of claims 37 to 40, wherein the AF2 comprises a light chain complementarity determining region (CDR-L) and a heavy chain complementarity determining region (CDR-H), and the AF2 comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 742, 743, and 744, respectively.

42. the CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 735 or 736; b. a CDR-L2 having the amino acid sequence of SEQ ID NO: 738 or 739, and c) The polypeptide of claim 41, comprising a CDR-L3 having the amino acid sequence of SEQ ID NO:

740.

43. The AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the AF2 a. FR-L1 having the amino acid sequence of SEQ ID NO: 746; b. FR-L2 having the amino acid sequence of SEQ ID NO: 747; c. FR-L3 having the amino acid sequence of any one of SEQ ID NOs: 748-751; d. FR-L4 having the amino acid sequence of SEQ ID NO: 754; e. FR-H1 having the amino acid sequence of SEQ ID NO: 755 or SEQ ID NO: 756; f. FR-H2 having the amino acid sequence of SEQ ID NO: 759; g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h) The polypeptide of claim 41 or 42, comprising FR-H4 having the amino acid sequence of any one of SEQ ID NO:

764.

44. the AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen-binding fragment a. FR-L1 having the amino acid sequence of SEQ ID NO: 746; b. FR-L2 having the amino acid sequence of SEQ ID NO: 747; c. FR-L3 having the amino acid sequence of SEQ ID NO: 748; d. FR-L4 having the amino acid sequence of SEQ ID NO: 754; e. FR-H1 having the amino acid sequence of SEQ ID NO: 755; f. FR-H2 having the amino acid sequence of SEQ ID NO: 759; g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h) The polypeptide of any one of claims 41 to 43, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

764.

45. the AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen-binding fragment a. FR-L1 having the amino acid sequence of SEQ ID NO: 746; b. FR-L2 having the amino acid sequence of SEQ ID NO: 747; c. FR-L3 having the amino acid sequence of SEQ ID NO: 749; d. FR-L4 having the amino acid sequence of SEQ ID NO: 754; e. FR-H1 having the amino acid sequence of SEQ ID NO: 756; f. FR-H2 having the amino acid sequence of SEQ ID NO: 759; g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h) The polypeptide of any one of claims 41 to 43, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

764.

46. the AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen-binding fragment a. FR-L1 having the amino acid sequence of SEQ ID NO: 746; b. FR-L2 having the amino acid sequence of SEQ ID NO: 747; c. FR-L3 having the amino acid sequence of SEQ ID NO: 750; d. FR-L4 having the amino acid sequence of SEQ ID NO: 754; e. FR-H1 having the amino acid sequence of SEQ ID NO: 756; f. FR-H2 having the amino acid sequence of SEQ ID NO: 759; g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h) The polypeptide of any one of claims 41 to 43, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

764.

47. the AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen-binding fragment a. FR-L1 having the amino acid sequence of SEQ ID NO: 746; b. FR-L2 having the amino acid sequence of SEQ ID NO: 747; c. FR-L3 having the amino acid sequence of SEQ ID NO: 751; d. FR-L4 having the amino acid sequence of SEQ ID NO: 754; e. FR-H1 having the amino acid sequence of SEQ ID NO: 756; f. FR-H2 having the amino acid sequence of SEQ ID NO: 759; g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h) The polypeptide of any one of claims 41 to 43, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

764.

48. 48. The polypeptide of any one of claims 37-47, wherein the AF2 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NO:766 or SEQ ID NO:

769.

49. 49. The polypeptide of any one of claims 37-48, wherein the AF2 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of any one of SEQ ID NOs: 765, 767, 768, 770, or 771.

50. 50. The polypeptide of any one of claims 37 to 49, wherein the AF2 comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of any one of SEQ ID NOs: 776-780.

51. T of the AF2 m is determined by an increase in melting temperature in an in vitro assay, the T of the antigen-binding fragment consisting of the sequence of SEQ ID NO: 781 m 51. The polypeptide of any one of claims 37 to 50, wherein the temperature is at least 2°C higher, or at least 3°C ​​higher, or at least 4°C higher, or at least 5°C higher, 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 than

52. The AF2 has a dissociation constant (K) of about 10 nM to about 400 nM as determined in an in vitro antigen binding assay. d 52. The polypeptide of any one of claims 37 to 51, which specifically binds to human or cyno CD3 at a constant

53. The AF2 has a dissociation constant (K) of about 10 nM to about 400 nM, or about 50 nM to about 350 nM, or about 100 nM to 300 nM, as determined in an in vitro antigen binding assay. d 53. The polypeptide of any one of claims 37 to 52, which specifically binds to human or cyno CD3 at a constant

54. The AF2 has a dissociation constant (K) weaker than about 3 nM, or 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 400 nM, as determined in an in vitro antigen binding assay. d 53. The polypeptide of any one of claims 37 to 52, which specifically binds to human or cyno CD3 at the C3 domain.

55. The AF2 has a dissociation constant (K d 55. The polypeptide of any one of claims 37-54, which specifically binds to human or cyno CD3 with a binding affinity 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 than an antibody binding fragment consisting of the amino acid sequence of SEQ ID NO: 781, as determined by a method similar to that of any one of claims 37-54.

56. 56. The polypeptide of any one of claims 37 to 55, wherein the AF2 is fused to the AF1 by a flexible peptide linker.

57. 57. The polypeptide of claim 56, wherein the flexible linker comprises two or three types of amino acids selected from the group consisting of glycine, serine, and proline.

58. 58. The polypeptide of any one of claims 37 to 57, wherein (1) the AF2 fragment is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, linear antibodies, single domain antibodies, and single chain variable fragments (scFv), or (2) the AF1 and AF2 are configured as (Fab')2 or a single chain diabody.

59. 59. The polypeptide of any one of claims 37 to 58, wherein the AF2 exhibits an isoelectric point (pI) of 6.6 or less.

60. 60. The polypeptide of any one of claims 37 to 59, wherein said AF2 exhibits a pI of 5.5 to 6.6, inclusive.

61. 61. The polypeptide of any one of claims 37 to 60, wherein the AF2 exhibits a pI of about 5.5 to 6.6, or about 5.6 to about 6.4, or about 5.8 to about 6.2, or about 6.0 to about 6.

2.

62. 62. The polypeptide of any one of claims 37-61, wherein the AF2 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the pI of a reference antigen-binding fragment consisting of the sequence set forth in SEQ ID NO:

781.

63. 63. The polypeptide of any one of claims 37-62, wherein AF2 is within at least about 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 about 1.5 pH units of the pI of AF1.

64. 64. The polypeptide of any one of claims 37-63, wherein AF2 exhibits a pI that is within at least about 0.1 to about 1.5, or at least about 0.3 to about 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 to about 0.9 pH units of the pI of AF1.

65. 65. The polypeptide of any one of claims 37 to 64, further comprising a second release segment (RS2), wherein said RS2 is a substrate for cleavage by a mammalian protease.

66. 66. The polypeptide of claim 65, wherein the RS2 is a substrate for a protease selected from legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase.

67. 67. The polypeptide of claim 65 or 66, wherein the RS2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 53-671.

68. 68. The polypeptide of any one of claims 65 to 67, wherein the sequences of RS1 and RS2 are identical.

69. 68. The polypeptide of any one of claims 65 to 67, wherein the sequences of RS1 and RS2 are not identical.

70. 70. The polypeptide of any one of claims 65 to 69, wherein RS1 and RS2 are each substrates for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence.

71. and further comprising a second extended recombinant polypeptide (XTEN2), said XTEN2 comprising: a. it has at least about 36 amino acids; b. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P); c) A polypeptide according to any one of claims 65 to 70, characterized in that it has at least 4 to 6 different amino acids selected from G, A, S, T, E, and P.

72. 72. The polypeptide of claim 71, wherein the XTEN2 comprises an amino acid sequence comprising at least three of the amino acid sequences of SEQ ID NOs: 672-675.

73. 73. The polypeptide of claims 71 or 72, wherein the XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs:676-734.

74. The XTEN2 is AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, A E144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE864 74. The polypeptide of any one of claims 71 to 73, comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the sequences of AE864_1, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7.

75. The polypeptide comprises, from N-terminus to C-terminus, one of the following: XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-diabody-RS1-XTEN1, or XTEN 75. The polypeptide of any one of claims 71-74, having a structural configuration of 1-RS1-diabody-RS2-XTEN2, wherein the diabody comprises the VL and VH of the AF1 and AF2, wherein the AF2 specifically binds CD3 and the AF1 specifically binds EGFR, and wherein XTEN1 and XTEN2 are of the same or different amino acid length or sequence.

76. 76. The polypeptide of any one of claims 71 to 75, wherein the binding affinity of AF1 to EGFR is at least 10 times higher, or at least 100 times higher, or at least 1000 times higher than the binding affinity of AF2 to CD3, as measured in an in vitro antigen binding assay.

77. 10. A pharmaceutical composition comprising a polypeptide according to any one of the preceding claims and one or more pharmaceutically suitable excipients.

78. 78. The pharmaceutical composition of claim 77, wherein the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intraperitoneal, intrathecal, or intramuscular administration.

79. 79. The pharmaceutical composition of claim 78, wherein the pharmaceutical composition is in liquid form.

80. 80. The pharmaceutical composition of any one of claims 77 to 79, wherein the pharmaceutical composition is in a pre-filled syringe for single injection.

81. 78. The pharmaceutical composition of claim 77, wherein the pharmaceutical composition is formulated as a lyophilized powder that is reconstituted prior to administration.

82. 77. Use of a polypeptide according to any one of claims 1 to 76 in the preparation of a medicament for the treatment of a disease in a subject.

83. The diseases include anaplastic and medullary thyroid cancer, appendix cancer, allenoblastoma, biliary tract cancer, bladder cancer, breast cancer, bile duct cancer, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignant tumor with malignant ascites, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, follicular cancer, gallbladder cancer, gastric cancer, cancer), gastrointestinal stromal tumor (GIST), GE junction cancer, genitourinary cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocellular carcinoma, HR+ and HER2+ breast cancer, Hürthle cell carcinoma, inflammatory breast cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroendocrine carcinoma, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary carcinoma, parathyroid cancer, peritoneal dissemination, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, gastric cancer (stomach cancer) 83. The use of claim 82, wherein the cancer is selected from the group of cancers consisting of: testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial cancer, uterine cancer, uterine serous cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

84. 82. A method of treating a disease in a subject, comprising administering to said subject in need thereof one or more therapeutically effective doses of the pharmaceutical composition of any one of claims 77 to 81.

85. The diseases include anaplastic and medullary thyroid cancer, appendix cancer, allenoblastoma, biliary tract cancer, bladder cancer, breast cancer, bile duct cancer, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignant tumor with malignant ascites, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, follicular cancer, gallbladder cancer, gastric cancer, cancer), gastrointestinal stromal tumor (GIST), GE junction cancer, genitourinary cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocellular carcinoma, HR+ and HER2+ breast cancer, Hürthle cell carcinoma, inflammatory breast cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroendocrine carcinoma, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary carcinoma, parathyroid cancer, peritoneal dissemination, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, gastric cancer (stomach cancer) 85. The method of claim 84, wherein the cancer is selected from the group of cancers consisting of: breast cancer, testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial cancer, uterine cancer, uterine serous cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

86. 86. The method of claim 84 or 85, wherein the pharmaceutical composition is administered to the subject as one or more therapeutically effective doses administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, or once a month.

87. 87. The method of any one of claims 84-86, wherein the pharmaceutical composition is administered to the subject in one or more therapeutically effective doses over a period of at least 2 weeks, or at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months.

88. 88. The method of any one of claims 84-87, wherein the dose is administered intradermally, subcutaneously, intravenously, intraarterially, intraperitoneally, intraperitoneally, intrathecally, or intramuscularly.

89. 89. The method of any one of claims 84 to 88, wherein the subject is selected from the group consisting of a mouse, a rat, a monkey, and a human.

90. 100. An isolated nucleic acid comprising: (a) a polynucleotide encoding a polypeptide of any one of claims 1 to 76; or (b) the complement of the polynucleotide of (a).

91. 91. An expression vector comprising the polynucleotide sequence of claim 90 and a recombinant regulatory sequence operably linked to the polynucleotide sequence.

92. 92. An isolated host cell comprising the expression vector of claim 91.

93. 93. The host cell of claim 92, wherein the host cell is a prokaryote.

94. 94. The host cell of claim 92 or 93, wherein the host cell is E. coli.

95. A bispecific antigen-binding unit, comprising: a. a first antigen-binding fragment (AF1), wherein the AF1 specifically binds to EGFR; and b. a second antigen-binding fragment (AF2), wherein the AF2 specifically binds to cluster of differentiation 3 T-cell receptor (CD3); A bispecific antigen-binding unit, wherein the difference between the isoelectric point (pI) of the second antigen-binding fragment and the pI of the first antigen-binding fragment is 0 to about 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, as determined by in vitro assay.

96. 96. The bispecific antigen-binding unit of claim 95, wherein the AF1 comprises a light chain complementarity-determining region (CDR-L), a heavy chain complementarity-determining region (CDR-H), a light chain framework region (FR-L), and a heavy chain framework region (FR-H), and the AF1 comprises FR-H1, FR-H2, FR-H3, and FR-H4, wherein FR-H1 has the amino acid sequence of any one of SEQ ID NOs: 14 to 16, FR-H2 has the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19, FR-H3 has the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, and FR-H4 has the amino acid sequence of any one of SEQ ID NOs: 22 to 24.

97. 97. The bispecific antigen binding unit of claim 96, wherein the AF1 further comprises a CDR-H3, wherein the CDRH3 has the amino acid sequence of SEQ ID NO:

6.

98. The bispecific antigen-binding unit of claim 96 or 97, wherein the AF1 further comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

99. the AF1 further comprises FR-L1, FR-L2, FR-L3, and FR-L4; a. FR-L1 exhibits at least 90%, or at least 95%, sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 7; b. FR-L2 exhibits at least 90%, or at least 95%, sequence identity to or is identical to the amino acid sequence of SEQ ID NO:8; c. FR-L3 exhibits at least 90%, or at least 95%, sequence identity to, or is identical to, the amino acid sequence of SEQ ID NOs: 9-11; d. The bispecific antigen-binding unit of any one of claims 95 to 98, wherein FR-L4 exhibits at least 90%, or at least 95%, sequence identity to or is identical to the amino acid sequence of SEQ ID NO:

13.

100. 100. The bispecific antigen-binding unit of any one of claims 95 to 99, wherein the AF1 CDR-L comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively.

101. The FR-L is a. FR-L1 having the amino acid sequence of SEQ ID NO: 7; b. FR-L2 having the amino acid sequence of SEQ ID NO: 8; c. FR-L3 having the amino acid sequence of SEQ ID NO: 9; d. The bispecific antigen-binding unit of any one of claims 95 to 100, comprising FR-L4 having the amino acid sequence of SEQ ID NO:

13.

102. The FR-L is a. FR-L1 having the amino acid sequence of SEQ ID NO: 7; b. FR-L2 having the amino acid sequence of SEQ ID NO: 8; c. FR-L3 having the amino acid sequence of SEQ ID NO: 10; d. The bispecific antigen-binding unit of any one of claims 95 to 100, comprising FR-L4 having the amino acid sequence of SEQ ID NO:

13.

103. The FR-L is a. FR-L1 having the amino acid sequence of SEQ ID NO: 7; b. FR-L2 having the amino acid sequence of SEQ ID NO: 8; c. FR-L3 having the amino acid sequence of SEQ ID NO: 11, and d. The bispecific antigen-binding unit of claims 95 to 100, comprising FR-L4 having the amino acid sequence of SEQ ID NO:

13.

104. The FR-H is a. FR-H1 having the amino acid sequence of SEQ ID NO: 14; b. FR-H2 having the amino acid sequence of SEQ ID NO: 18; c. FR-H3 having the amino acid sequence of SEQ ID NO: 20; d. The bispecific antigen-binding unit of any one of claims 95 to 100, comprising FR-H4 having the amino acid sequence of SEQ ID NO: 22 or 23.

105. The FR-H is a. FR-H1 having the amino acid sequence of SEQ ID NO: 15; b. FR-H2 having the amino acid sequence of SEQ ID NO: 19; c. FR-H3 having the amino acid sequence of SEQ ID NO: 21; d. The bispecific antigen-binding unit of any one of claims 95 to 100, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

24.

106. The FR-H is a. FR-H1 having the amino acid sequence of SEQ ID NO: 16; b. FR-H2 having the amino acid sequence of SEQ ID NO: 19; c. FR-H3 having the amino acid sequence of SEQ ID NO: 20; d. The bispecific antigen-binding unit of any one of claims 95 to 100, comprising FR-H4 having the amino acid sequence of SEQ ID NO: 22 or 23.

107. 107. The bispecific antigen-binding unit of any one of claims 95 to 106, wherein AF1 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NOs: 28 to 32.

108. 108. The bispecific antigen-binding unit of any one of claims 95 to 107, wherein AF1 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NOs: 25 to 27.

109. The bispecific antigen-binding unit of any one of claims 95 to 108, wherein AF1 comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of any one of SEQ ID NOs: 37 to 51.

110. The bispecific antigen-binding unit of any one of claims 95 to 109, wherein the AF1 specifically binds to human or cynomolgus monkey (cyno) EGFR.

111. The bispecific antigen-binding unit of any one of claims 95 to 109, wherein the AF1 specifically binds to human and cynomolgus monkey (cyno) EGFR.

112. 112. The bispecific antigen-binding unit of any one of claims 95 to 111, wherein AF2 binds to a CD3 complex subunit selected from any one of CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta epsilon.

113. The bispecific antigen-binding unit of any one of claims 95 to 112, wherein the AF2 specifically binds to human or cynomolgus monkey (cyno) CD3.

114. The bispecific antigen-binding unit of any one of claims 95 to 112, wherein the AF2 specifically binds to human and cynomolgus monkey (cyno) CD3.

115. The bispecific antigen-binding unit of any one of claims 95 to 114, wherein the AF2 comprises a light chain complementarity determining region (CDR-L) and a heavy chain complementarity determining region (CDR-H), and the antigen-binding unit comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 742, 743, and 744, respectively.

116. the CDR-L of AF2 is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 735 or 736; b. a CDR-L2 having the amino acid sequence of SEQ ID NO: 738 or 739, and c) The bispecific antigen-binding unit of claim 115, comprising a CDR-L3 having the amino acid sequence of SEQ ID NO:

740.

117. The AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the AF2 a. FR-L1 having the amino acid sequence of SEQ ID NO: 746; b. FR-L2 having the amino acid sequence of SEQ ID NO: 747; c. FR-L3 having the amino acid sequence of any one of SEQ ID NOs: 748-751; d. FR-L4 having the amino acid sequence of SEQ ID NO: 754; e. FR-H1 having the amino acid sequence of SEQ ID NO: 755 or SEQ ID NO: 756; f. FR-H2 having the amino acid sequence of SEQ ID NO: 759; g. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and h) The bispecific antigen-binding unit of claim 115 or 116, comprising FR-H4 having the amino acid sequence of any one of SEQ ID NO:

764.

118. The AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen binding unit comprises: i. FR-L1 having the amino acid sequence of SEQ ID NO: 746; j. FR-L2 having the amino acid sequence of SEQ ID NO: 747; k. FR-L3 having the amino acid sequence of SEQ ID NO: 748; 1. FR-L4 having the amino acid sequence of SEQ ID NO: 754; m. FR-H1 having the amino acid sequence of SEQ ID NO: 755; n. FR-H2 having the amino acid sequence of SEQ ID NO: 759; o. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and p. The bispecific antigen-binding unit of any one of claims 115 to 117, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

764.

119. The AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen binding unit comprises: i. FR-L1 having the amino acid sequence of SEQ ID NO: 746; j. FR-L2 having the amino acid sequence of SEQ ID NO: 747; k. FR-L3 having the amino acid sequence of SEQ ID NO: 749; 1. FR-L4 having the amino acid sequence of SEQ ID NO: 754; m. FR-H1 having the amino acid sequence of SEQ ID NO: 756; n. FR-H2 having the amino acid sequence of SEQ ID NO: 759; o. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and p. The bispecific antigen-binding unit of any one of claims 115 to 117, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

764.

120. The AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen binding unit comprises: i. FR-L1 having the amino acid sequence of SEQ ID NO: 746; j. FR-L2 having the amino acid sequence of SEQ ID NO: 747; k. FR-L3 having the amino acid sequence of SEQ ID NO: 750; 1. FR-L4 having the amino acid sequence of SEQ ID NO: 754; m. FR-H1 having the amino acid sequence of SEQ ID NO: 756; n. FR-H2 having the amino acid sequence of SEQ ID NO: 759; o. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and p. The bispecific antigen-binding unit of any one of claims 115 to 117, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

764.

121. The AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen binding unit comprises: i. FR-L1 having the amino acid sequence of SEQ ID NO: 746; j. FR-L2 having the amino acid sequence of SEQ ID NO: 747; k. FR-L3 having the amino acid sequence of SEQ ID NO: 751; 1. FR-L4 having the amino acid sequence of SEQ ID NO: 754; m. FR-H1 having the amino acid sequence of SEQ ID NO: 756; n. FR-H2 having the amino acid sequence of SEQ ID NO: 759; o. FR-H3 having the amino acid sequence of SEQ ID NO: 760, and p. The bispecific antigen-binding unit of any one of claims 115 to 117, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

764.

122. 122. The bispecific antigen-binding unit of claims 112 to 121, wherein AF2 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of SEQ ID NO: 766 or SEQ ID NO:

769.

123. 123. The bispecific antigen-binding unit of any one of claims 112 to 122, wherein AF2 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or identical to, the amino acid sequence of any one of SEQ ID NOs: 765, 767, 768, 770, or 771.

124. The bispecific antigen-binding unit of any one of claims 112 to 123, wherein AF2 comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% sequence identity to, or is identical to, the amino acid sequence of any one of SEQ ID NOs: 776 to 780.

125. The AF1 has a K of about 0.1 nM to about 100 nM as determined in an in vitro antigen binding assay involving EGFR or an epitope thereof. d The bispecific antigen-binding unit of any one of claims 115 to 124, which specifically binds to EGFR at

126. The AF1 has a dissociation constant (K) of about 0.1 nM to about 100 nM, or about 0.5 nM to about 50 nM, or about 1.0 nM to 20 nM, or about 2.0 nM to about 10 nM, as determined in an in vitro antigen binding assay. d 126. The bispecific antigen-binding unit of any one of claims 112 to 125, which specifically binds to EGFR with a constant

127. The AF2 has a dissociation constant (K) of about 10 nM to about 400 nM as determined in an in vitro antigen binding assay. d 127. The bispecific antigen-binding unit of any one of claims 112 to 126, which specifically binds to human or cyno CD3 with a constant

128. The AF2 has a dissociation constant (K) of about 10 nM to about 400 nM, or about 50 nM to about 350 nM, or about 100 nM to 300 nM, as determined in an in vitro antigen binding assay. d 128. The bispecific antigen-binding unit of any one of claims 112 to 127, which specifically binds to human or cyno CD3 with a constant

129. The AF2 has a dissociation constant (K) weaker than about 3 nM, or 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 400 nM, as determined in an in vitro antigen binding assay. d The bispecific antigen-binding unit of any one of claims 112 to 128, which specifically binds to human or cyno CD3 at

130. The AF2 has a dissociation constant (K d 130. The bispecific antigen-binding unit of any one of claims 112 to 129, which specifically binds to human or cyno CD3 with a binding affinity 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 than an antibody-binding fragment consisting of the amino acid sequence of SEQ ID NO: 781, as determined by a method similar to that of any one of claims 112 to 129.

131. The AF2 has a dissociation constant (K d 131. The bispecific antigen-binding unit of any one of claims 112 to 130, wherein the bispecific antigen-binding unit exhibits a binding affinity for CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold weaker than the binding affinity of said AF1, as determined by

132. The bispecific antigen-binding unit of any one of claims 112 to 131, wherein the AF2 is fused to the AF1 by a flexible peptide linker.

133. 133. The bispecific antigen-binding unit of claim 132, wherein the flexible linker comprises two or three types of amino acids selected from the group consisting of glycine, serine, and proline.

134. The bispecific antigen-binding unit of any one of claims 112 to 133, wherein (1) each of the AF1 and AF2 fragments is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, linear antibodies, single-domain antibodies, and single-chain variable fragments (scFv), or (2) the AF1 and AF2 are configured as (Fab')2 or single-chain diabodies.

135. 135. The bispecific antigen-binding unit of any one of claims 112 to 134, further comprising a first release segment peptide (RS1) and a second release segment peptide (RS2), each of said RS1 and RS2 being a substrate for cleavage by a mammalian protease.

136. The bispecific antigen-binding unit of claim 135, wherein each of RS1 and RS2 is a substrate for a protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase.

137. The bispecific antigen-binding unit of claim 135 or 136, wherein each of RS1 and 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 selected from any one of SEQ ID NOs: 53-671.

138. Each of RS1 and RS2 is RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RS 138. The bispecific antigen-binding unit of any one of claims 135 to 137, comprising an amino acid sequence selected from the sequences of N-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which is set forth in Table 5.

139. The bispecific antigen-binding unit of any one of claims 135 to 138, wherein RS1 and RS2 are identical.

140. The bispecific antigen-binding unit of any one of claims 135 to 138, wherein RS1 and RS2 are different.

141. and a second extended recombinant polypeptide (XTEN2), each of which comprises: a. it has at least about 36 amino acids; b. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P); c) The bispecific antigen-binding unit of any one of claims 135 to 140, characterized in that it has at least 4 to 6 different amino acids selected from G, A, S, T, E, and P.

142. 142. The bispecific antigen binding unit of claim 141, wherein each of the XTEN1 and XTEN2 comprises an amino acid sequence comprising at least three of the amino acid sequences of SEQ ID NOs: 672-675.

143. 143. The bispecific antigen-binding unit of claim 141 or 142, wherein each of the XTEN1 and 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 selected from any one of SEQ ID NOs: 676-734.

144. Each of the XTEN1 and XTEN2 is selected from the group consisting of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE86 144. The bispecific antigen-binding unit of any one of claims 141 to 143, comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the sequences of AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7.

145. 145. The bispecific antigen-binding unit of any one of claims 141 to 144, wherein the XTEN1 and XTEN2 are identical.

146. 146. The bispecific antigen-binding unit of any one of claims 141 to 145, wherein the XTEN1 and XTEN2 are different.

147. 147. The bispecific antigen binding unit of any one of claims 141-146, wherein the bispecific antigen binding unit has the following structural configuration from N-terminus to C-terminus: XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-diabody-RS1-XTEN1, or XTEN1-RS1-diabody-RS2-XTEN2, wherein the diabody comprises the VL and VH of the AF1 and AF2.

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