EGFR antigen-binding fragments and compositions containing the same

Abstract

The present disclosure relates to antigen binding units that specifically bind to EGFR or an epitope thereof. Some embodiments include bispecific anti-EGFR / anti-CD3 constructs with improved expression and / or stability. Related methods are also disclosed.

Description

[Technical Field]

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

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, which 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 drugs are cytotoxic agents that kill both normal and tumor cells. The therapeutic benefits of these cytotoxic agents depend on the fact that tumor cells are more sensitive than normal cells, which makes it possible to achieve a clinical response using doses that do not cause unacceptable side effects. However, all of these nonspecific drugs inherently cause some, if not significant, damage to normal tissue, and often limit their therapeutic applicability.

[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 derived from two monoclonal antibodies into a single composition, enabling combinations of approaches or scopes of application that are 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 a CD3 molecule on a T cell, induces the cytotoxic activity of effector molecules such as TNF-α, IFN-γ, interleukin 2, 4, and 10, perforin, and granzymes by releasing them. Advances in antibody engineering have led to the development of several bispecific antibody forms 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 requiring co-stimulation or conventional 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 concentrations within a therapeutic window long enough to achieve therapeutic effects, or have variable effects, there remains a dual problem in certain patients who experience serious side effects referred to as "cytokine storms" 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), mediated by the release of TNF-α and IFN-γ among other cytokines. Therefore, there is an unmet need in this field for the development of bispecific antibodies effective for use in cancer treatment. [Overview of the project]

[0005] The present invention relates to an anti-epidermal growth factor receptor (EGFR) antigen-binding fragment incorporated into a chimeric fusion protein and a method for using or creating the same. In one embodiment, 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), wherein AF1 a. specifically binds to the epidermal growth factor receptor (EGFR), and b. comprises FR-H1, FR-H2, FR-H3, and FR-H4, wherein FR-H1 has one amino acid sequence from SEQ ID NOs. 14 to 16, FR-H2 has the amino acid sequence of SEQ ID NOs. 18 or 19, FR-H3 has the amino acid sequence of SEQ ID NOs. 20 or 21, and FR-H4 has one amino acid sequence from 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, any one of the amino acid sequences 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, linear antibodies, and single-stranded variable fragments (scFv).

[0006] In another embodiment, AF1 includes a variable weight (VH) amino acid sequence that has or is identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequences of SEQ ID NOs. In a particular embodiment, AF1 includes a variable light (VL) amino acid sequence that has or is identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequences of SEQ ID NOs. 25–27.

[0007] In some embodiments, AF1 further comprises CDR-H3, and CDRH3 has the amino acid sequence of SEQ ID NO: 6. In certain embodiments, AF1 further comprises CDR-H1, CDR-H2, and CDR-H3, each having 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, each having 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, where a. FR-L1 exhibits at least 90% or at least 95% sequence identity with 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 with 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 with or is identical to the amino acid sequences of SEQ ID NOs: 9 to 11; and d. FR-L4 exhibits at least 90% or at least 95% sequence identity with or is identical to the amino acid sequence of SEQ ID NO: 13. In one particular embodiment, FR-L includes 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, FR-L includes 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, FR-L includes 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, FR-H includes 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, and c. FR-H4 having the amino acid sequence of SEQ ID NO: 22 or 23. In another embodiment, FR-H includes 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 a particular embodiment, FR-H includes 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 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 substituted amino acid is 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), where RS1 is a substrate for cleavage by a mammalian protease. In some embodiments, the fusion protein, in its uncleaved state, has a structural configuration of AF1-RS1-XTEN1 or XTEN1-RS1-AF1 from N-terminus to C-terminus.

[0011] In some embodiments, RS1 is a substrate for a protease selected from the group consisting of regmine, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase. 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 with a sequence selected from any one of SEQ ID NOs. 53 to 671. In certain embodiments, RS1 includes 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 listed in Table 5.

[0012] In some embodiments, the polypeptides disclosed herein further comprise a first elongated recombinant polypeptide (XTEN1), wherein 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 to 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 to 675. In another embodiment, XTEN1 includes an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from any one of sequence numbers 676 to 734. In a particular embodiment, XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, 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 listed in Table 7.

[0013] In certain embodiments, AF1 has a higher isoelectric point (pI) than the antigen-binding fragment consisting of the sequence shown in SEQ ID NO: 52. In one embodiment, AF1 is incorporated into a polypeptide to form an anti-EGFR bispecific antibody, the polypeptide exhibiting a higher pI than a control bispecific antibody, the polypeptide comprising AF1 and a reference antigen-binding fragment that binds to the cluster of differentiation 3 T cell receptor (CD3), the control bispecific antigen-binding fragment being identical to the polypeptide except that AF1 is replaced by SEQ ID NO: 52. In another embodiment, AF1 exhibits a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit higher than the pI of the antigen-binding fragment consisting of the sequence shown in SEQ ID NO: 52. In certain embodiments, AF1 exhibits a pI of 5.4 to 6.6 (including boundary values). In other embodiments, AF1 exhibits a pI of approximately 5.4 to 5.6, or approximately 5.5 to 5.7, or approximately 5.6 to 5.8, or approximately 5.7 to 5.9, or approximately 5.8 to 6.0, or approximately 5.9 to 6.1, or approximately 6.0 to 6.2, or approximately 6.1 to 6.3, or approximately 6.2 to 6.4, or approximately 6.3 to 6.5, or approximately 6.4 to 6.6. In yet another embodiment, AF1 exhibits a pI of approximately 5.4, approximately 5.5, or approximately 5.6, or approximately 5.7, or approximately 5.8, or approximately 5.9, or approximately 6.0, or approximately 6.1, or approximately 6.2, or approximately 6.3, or approximately 6.4, or approximately 6.5, or approximately 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 binds to about 0.1 nM to about 100 nM K when determined by an in vitro antigen-binding assay including EGFR or its epitope. d It specifically binds to EGFR.

[0015] In another embodiment, the polypeptide further comprises a second antigen-binding fragment (AF2) that specifically binds to a surface antigen classification 3 T cell receptor (CD3). In a particular embodiment, (1) the AF2 fragment is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, linear antibodies, single-domain antibodies, and single-stranded variable fragments (scFv), or (2) AF1 and AF2 are configured as (Fab')2 or single-stranded diabodies.

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

[0017] In some embodiments, AF2 includes a variable weight (VH) amino acid sequence that has or is identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 766 or SEQ ID NO: 769. In certain embodiments, AF2 includes a variable light (VL) amino acid sequence that has or is identical to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the amino acid sequences of SEQ ID NO: 765, 767, 768, 770, or 771. In other embodiments, AF2 includes an amino acid sequence that has or is identical to at least 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the amino acid sequences of SEQ ID NO: 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 fragments include CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 742, 743, and 744, respectively. In some embodiments, CDR-L includes a. CDR-L1 having the amino acid sequence of SEQ ID NOs. 735 or 736, b. CDR-L2 having the amino acid sequence of SEQ ID NOs. 738 or 739, and c. CDR-L3 having the amino acid sequence of SEQ ID NOs. 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 includes 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 any one of the amino acid sequences 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 any one of the amino acid sequences of SEQ ID NO: 764. In another embodiment, the antigen-binding fragments include 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 includes 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 a particular embodiment, the antigen-binding fragment includes 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 includes 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), where 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 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, where the diabody comprises VL and VH of AF1 and AF2, AF2 specifically binds to CD3, AF1 specifically binds to EGFR, and XTEN1 and XTEN2 have the same or different amino acid lengths or sequences.

[0022] In certain embodiments, RS2 is a substrate for a protease selected from regmine, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase. 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 with a sequence selected from SEQ ID NOs. 53-671. In certain embodiments, RS1 and RS2 are 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 to 6 different amino acids selected from G, A, S, T, E, and P. In certain embodiments, XTEN2 includes an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from sequence numbers 676 to 734. In other embodiments, XTEN2 includes an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, 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 listed in Table 7. In a particular embodiment, XTEN2 includes an amino acid sequence comprising at least three of the amino acid sequences of SEQ ID NOs. 672-675.

[0024] In some embodiments, T of AF2 m The T of the antigen-binding fragment consisting of the sequence of SEQ ID NO: 781 was determined by increasing the melting temperature in an in vitro assay. m At least 2°C higher than, 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 one of the following: CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta-type psilon. In one embodiment, AF2 specifically binds to human or cynomolgus monkey (cyno) CD3. In yet another embodiment, AF2 specifically binds to both human and cynomolgus monkey (cyno) CD3.

[0026] In other embodiments, AF2, when determined by an in vitro antigen-binding assay, has a dissociation constant (K) of approximately 10 nM to approximately 400 nM. d It specifically binds to human or cyno-CD3 at a constant (K). In certain embodiments, AF2, when determined by an in vitro antigen-binding assay, 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. d It specifically binds to human or cyno CD3 at a constant (K). In certain embodiments, AF2 has a dissociation constant (K) weaker than approximately 3 nM, or approximately 10 nM, or approximately 50 nM, or approximately 100 nM, or approximately 150 nM, or approximately 200 nM, or approximately 250 nM, or approximately 300 nM, or approximately 400 nM, as determined by an in vitro antigen-binding assay. d ) specifically binds to human or cyno CD3. In other embodiments, AF2 is used in in vitro antigen binding assays with their respective dissociation constants (K d As determined by ), it specifically binds to human or cyno CD3 with a binding affinity at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times weaker than the 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 times, at least 100 times, or at least 1000 times higher than the binding affinity of AF2 to CD3, as measured by 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 (including boundary values). In other embodiments, AF2 exhibits a pI of approximately 5.5 to 6.6, or approximately 5.6 to 6.4, or approximately 5.8 to 6.2, or approximately 6.0 to 6.2. In some embodiments, AF2 exhibits a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lower than the pI of the reference antigen-binding fragment consisting of the sequence shown in Sequence ID No. 781. In another embodiment, 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 a particular embodiment, AF2 exhibits a pI that is within at least about 0.1 to about 1.5 pH units of the pI of AF1, 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.

[0028] In another aspect, the present disclosure provides a bispecific antigen-binding unit comprising: a. a first antigen-binding fragment (AF1) which specifically binds to EGFR; and b. a second antigen-binding fragment (AF2) which specifically binds to surface antigen classification 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 approximately 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 an in vitro assay. In some embodiments, AF1 includes a variable weight (VH) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with, or is identical to, the amino acid sequences of SEQ ID NOs. 25–27 includes a variable light (VL) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with, or is identical to, the amino acid sequences of SEQ ID NOs. 37–51 includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, and 99% sequence identity with, or is identical to, any one of the amino acid sequences 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 antibodies, single-domain antibodies, and single-stranded variable fragments (scFv), or (2) AF1 and AF2 are configured as (Fab')2 or single-stranded diabodies.

[0029] In some embodiments, the bispecific antigen-binding unit 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 comprises FR-H1, FR-H2, FR-H3, and FR-H4.

[0030] In other embodiments, AF1 further comprises CDR-H3, and CDRH3 has the amino acid sequence of SEQ ID NO: 6. In certain embodiments, AF1 further comprises CDR-H1, CDR-H2, and CDR-H3, each having 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, each having the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively.

[0031] In certain embodiments, FR-H1 has one amino acid sequence from SEQ ID NOs: 14 to 16, FR-H2 has the amino acid sequence of SEQ ID NOs: 18 or 19, FR-H3 has the amino acid sequence of SEQ ID NOs: 20 or 21, and FR-H4 has one amino acid sequence from SEQ ID NOs: 22 to 24. In some embodiments, FR-H includes FR-H1 having the amino acid sequence of SEQ ID NOs: 14, FR-H2 having the amino acid sequence of SEQ ID NOs: 18, FR-H3 having the amino acid sequence of SEQ ID NOs: 20, and FR-H4 having the amino acid sequence of SEQ ID NOs: 22 or 23. In other embodiments, FR-H includes FR-H1 having the amino acid sequence of SEQ ID NOs: 15, FR-H2 having the amino acid sequence of SEQ ID NOs: 19, FR-H3 having the amino acid sequence of SEQ ID NOs: 21, and FR-H4 having the amino acid sequence of SEQ ID NOs: 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 one particular embodiment, FR-L1 exhibits at least 90% or at least 95% sequence identity with, 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 with, 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 with, or is identical to, the amino acid sequences of SEQ ID NOs: 9 to 11; and FR-L4 exhibits at least 90% or at least 95% sequence identity with, or is identical to, the amino acid sequence of SEQ ID NO: 13. In another embodiment, FR-L includes 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, FR-L includes FR-L1 having the amino acid sequence of SEQ ID NO: 7, 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 FR-L4 having the amino acid sequence of SEQ ID NO: 13. In yet another embodiment, FR-L includes 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, the bispecific antigen-binding unit 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, each having the amino acid sequences of SEQ ID NOs. 742, 743, and 744, respectively. In some embodiments, AF2 includes a variable weight (VH) amino acid sequence that has or is identical to the amino acid sequence of SEQ ID NOs. 766 or 769 by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In other embodiments, AF2 includes a variable light (VL) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or is identical to any one of the amino acid sequences of SEQ ID NOs. 765, 767, 768, 770, or 771. In certain embodiments, AF2 includes an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with or is identical to any one of the amino acid sequences 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 includes 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 any one of the amino acid sequences 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 any one of the amino acid sequences of SEQ ID NO: 764. In a particular embodiment, AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen-binding unit includes 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 includes 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 includes 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 a particular embodiment, AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen-binding unit includes 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 mobile peptide linker. In certain embodiments, the mobile 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, each of RS1 and RS2 is a substrate for a protease selected from the group consisting of regmine, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase. In other embodiments, each of RS1 and RS2 includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from any one of SEQ ID NOs: 53-671. In another embodiment, each of RS1 and RS2 includes 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 listed in Table 5.

[0038] In some embodiments, the bispecific antigen-binding unit further comprises a first elongated recombinant polypeptide (XTEN1) and a second elongated recombinant polypeptide, each of XTEN1 and XTEN2 being a. having 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 of the XTEN1 sequence being selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), and c) characterized by at least 4 to 6 different amino acids selected from G, A, S, T, E, and P. In one embodiment, XTEN1 and XTEN2 are identical. In another embodiment, XTEN1 and XTEN2 are different.

[0039] In one particular embodiment, each of XTEN1 and XTEN2 includes an amino acid sequence comprising at least three of the amino acid sequences of SEQ ID NOs. 672-675. In yet another embodiment, each of XTEN1 and XTEN2 includes an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from any one of SEQ ID NOs. 676-734. In other embodiments, each of XTEN1 and XTEN2 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, A The amino acid sequences include sequences selected from the sequences E300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, and having at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, each of which is listed in Table 7.

[0040] In some embodiments, the bispecific antigen-binding unit has the following structural arrangements from the N-terminus to the 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, and the diabody contains the VL and VH of AF1 and AF2.

[0041] In some embodiments, AF1 specifically binds to human or cynomolgus (cyno) EGFR. In other embodiments, AF1 specifically binds to human and cynomolgus (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 (cyno) CD3. In yet another embodiment, AF2 specifically binds to human and cynomolgus (cyno) CD3.

[0042] In one embodiment, AF1 specifically binds to EGFR with a dissociation constant (K d ) of about 0.1 nM to about 100 nM as determined in an in vitro antigen-binding assay that includes EGFR or an epitope thereof.

[0043] In other embodiments, AF1 specifically binds to EGFR with a dissociation constant (K d ) 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. In some embodiments, AF2 has a dissociation constant (K dIt specifically binds to human or cyno CD3 at a constant (K). In certain embodiments, AF2 has a dissociation constant (K) weaker than approximately 3 nM, or approximately 10 nM, or approximately 50 nM, or approximately 100 nM, or approximately 150 nM, or approximately 200 nM, or approximately 250 nM, or approximately 300 nM, or approximately 400 nM, as determined by an in vitro antigen-binding assay. d ) specifically binds to human or cyno CD3. In yet another embodiment, AF2 has a dissociation constant (K) in an in vitro antigen binding assay. d When determined by ), AF2 specifically binds to human or cyno CD3 with a binding affinity at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times weaker than the antibody binding fragment consisting of the amino acid sequence of SEQ ID NO: 781. In some embodiments, AF2 has a dissociation constant (K) in an in vitro antigen binding assay. d When determined by ), it exhibits a binding affinity for CD3 that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or at least 1000 times weaker than the binding affinity for AF1.

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

[0045] In yet another embodiment, the disclosure provides polypeptides disclosed herein for the preparation of agents for treating a disease in subjects requiring treatment of that disease. In some embodiments, the diseases are anaplastic and medullary thyroid cancer, appendiceal cancer, allenoblastoma, biliary tract cancer, bladder cancer, breast cancer, cancers of the bile duct, carcinoid tumors, cervical cancer, cholangiocarcinoma, colorectal cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignant tumors with malignant ascites, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, follicular cancer, gallbladder cancer, gastric cancer. Cancer, gastrointestinal stromal tumors (GIST), GEG-associated 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 cancer, 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, stomach cancer The cancers selected are from the group consisting of cancers such as testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial carcinoma, uterine cancer, serous uterine carcinoma, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0046] In relevant embodiments, the Disclosure provides a method for 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 mice, rats, monkeys, and humans.

[0047] In certain embodiments, the disease is anaplastic and medullary thyroid cancer, appendiceal cancer, allenoblastoma, biliary tract cancer, bladder cancer, breast cancer, bile duct cancer, carcinoid tumor, cervical cancer, cholangiocarcinoma, colorectal 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 tumors (GIST), GEG-associated 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 cancer, 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, stomach cancer The cancers selected are from the group consisting of cancers such as testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial carcinoma, uterine cancer, serous uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor. In other embodiments, the pharmaceutical composition is administered to the subject as 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 a month. In certain embodiments, the pharmaceutical composition is administered to the subject as 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, intra-arterially, intra-abdominately, intraperitoneally, intrathecally, or intramuscularly.

[0048] In certain embodiments, the Disclosure provides isolated nucleic acids, which include (a) polynucleotides encoding polypeptides disclosed herein, or (b) complements of the polynucleotides of (a).

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

[0050] In yet another embodiment, the disclosure provides isolated host cells containing the expression vector disclosed herein. In some embodiments, the host cell is a prokaryote. In certain embodiments, the host cell is E. coli or a mammalian cell.

[0051] Embedding by reference All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. [Brief explanation of the drawing]

[0052] Various features of this disclosure are described in detail in the attached claims. A further understanding of the features and advantages of this disclosure can be obtained by referring to the following embodiments for carrying out the invention and the attached drawings, which describe illustrative embodiments in which the principles of the present invention are utilized. [Figure 1] The individual components of the bispecific antigen-binding fragment composition are shown. Figure 1A shows the antigen-binding fragment with affinity for the target cell marker. Figure 1B shows the antigen-binding fragment with affinity for effector cells. Figures 1C and 1D show XTEN polypeptides of different lengths. Figure 1E shows the cleavable release segment. [Figure 2]Two different forms of polypeptide compositions described herein are shown. Figure 2A shows, on the left, an antigen-binding fragment for effector cells fused with a release segment and XTEN, while the arrow indicates the action of a protease to cleave the release segment, resulting in the release of XTEN from the polypeptide antigen-binding fragment on the right, so that the antigen-binding fragment is no longer shielded by XTEN and thus regains its potential binding affinity (e.g., its maximum binding affinity). Figure 2B shows a bispecific composition having an antigen-binding fragment for effector cells fused with an antigen-binding fragment having a binding affinity for a target cell marker on the left. The release segment and XTEN are also fused with an antigen-binding fragment having affinity for effector cells, while the arrow indicates the action of a protease to cleave the release segment, resulting in the release of XTEN and the fused antigen-binding fragment from the polypeptide on the right, so that they are no longer shielded by XTEN and thus regain their potential binding affinity. [Figure 3] Two different forms of bispecific antigen-binding polypeptides are shown. On the left, a bispecific composition having an antigen-binding fragment for effector cells is fused to an antigen-binding fragment having binding affinity to a target cell marker having a release segment (the scissors indicate sensitivity to protease cleavage), and XTEN is fused to the antigen-binding fragment having binding affinity to effector cells. On the other hand, on the right, a bispecific composition having an antigen-binding fragment for effector cells is fused to an antigen-binding fragment having binding affinity to a target cell marker, and the release segment and XTEN are fused to the antigen-binding fragment having binding affinity to the target cell marker. [Figure 4]Three different forms of bispecific antigen-binding polypeptides are shown. Figure 4A shows a bispecific composition having an scFv antigen-binding fragment fused to an scFv antigen-binding fragment having binding affinity to a target cell marker having a release segment (scissors indicating sensitivity to protease cleavage) for effector cells, and XTEN fused to each antigen-binding fragment. Figures 4B and 4C are variations of Figure 4A, in which the antigen-binding fragment is a diabody configuration, and the release segment (scissors indicating sensitivity to protease cleavage) and XTEN are fused to the antigen-binding fragment for the effector cell marker or target cell marker, respectively. [Figure 5] A schematic diagram shows bispecific antigen-binding polypeptides near tumor tissue (upper part) and normal tissue (lower part). The bispecific antigen-binding polypeptides are preferentially cleaved in tumor tissue, releasing one or more XTEN moieties compared to normal tissue. The cleaved bispecific antigen-binding polypeptides can bind to T cells and tumor cells expressing tumor-specific markers. [Figure 6] The amino acid sequence of the controlled-release segment RSR-1517 (SEQ ID NO: 53) is shown, along with the peptide cleavage sites of the listed proteases. [Modes for carrying out the invention]

[0053] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided only as examples. Without departing from the present invention, those skilled in the art will be able to conceive of numerous variations, modifications, and substitutions. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of those claims and their equivalents are thereby covered.

[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. Methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, but preferred methods and materials are described below. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, the materials, methods, and examples are illustrative and not intended to limit the invention. A number of variations, modifications, and substitutions will be conceivable by those skilled in the art without departing from the invention.

[0055] definition In relation to this application, the following terms have the meanings they represent unless otherwise specified.

[0056] As used herein and in the claims, the terms “a,” “an,” and “the” are used to mean “at least one,” “at least first,” “one or more,” or “more than,” of the components or steps being referenced, unless an upper limit is specifically stated thereafter. Thus, “release segment,” as used herein, means “at least first release segment,” but includes multiple release segments. The operable limits and parameters of any combination, as well as the quantities of any single agent, will be known to those skilled in the art in light of this disclosure.

[0057] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acid groups. These terms also encompass amino acid polymers modified by any other operation, such as disulfide bond formation, glycosylation, lipid formation, acetylation, phosphorylation, or conjugation with labeling components.

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

[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 D and L optical isomers, as well as amino acid analogs and peptide mimes. Amino acids can be designated using standard one- or three-letter codes.

[0060] The term "natural L-amino acids" or "L-amino acids" refers to the L-optical isomer 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 its 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, as long as they exhibit the desired antigen-binding activity or immunological activity. The term “immunoglobulin” (Ig) is used herein interchangeably with “antibody.” Full-length antibodies may be, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies. Antibodies represent a large molecular family including several types of molecules such as IgD, IgG, IgA, IgM, and IgE. The term “immunoglobulin molecule” includes, for example, hybrid antibodies or modified antibodies, as well as fragments thereof. It has been shown that the antigen-binding function of an antibody may be performed by a naturally occurring antibody or a fragment of a monoclonal antibody.

[0062] A “humanized” antibody refers to a chimeric antibody that contains amino acid residues derived from a non-human complementarity-determining region (CDR) and amino acid residues derived from a human framework region (FR). In certain embodiments, the humanized antibody would contain at least one, typically two, substantially all of the variable domains, with all or substantially all of the CDR corresponding to that of a non-human antibody (which may include amino acid substitutions), and all or substantially all of the FR corresponding to that 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 in that population are identical and / or bind to the same epitope, except for possible variant antibodies, such as those containing naturally occurring mutations or those arising during the production of the monoclonal antibody preparation, which are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of antibodies obtained from a substantially homogeneous antibody population and should not be interpreted as requiring antibody production 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 presentation methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are known in the art or described herein.

[0064] As used herein, “antigen-binding fragment” refers to an immunoglobulin molecule and a molecule containing an immunoactive portion of an immunoglobulin molecule, i.e., an antigen-binding site that specifically binds to (and “immunely reacts with) an antigen. Examples include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies (see U.S. Patent No. 5,641,870), single-domain antibodies, single-domain camel 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 having a specific shape that fits, recognizes, and binds to an epitope, in which one or more non-covalent interactions stabilize the complex between the molecular structure and the epitope. An antigen-binding fragment is “specifically binding” to an antigen or “immunoreactive” to an antigen if it binds with greater affinity or binding activity than it would to other reference antigens, including polypeptides or other substances.

[0065] "scFv" or "single-stranded variable fragment" is used interchangeably herein and refers to an antibody fragment form containing two copies of the variable regions of the heavy (VH) and light (VL) chains of an antibody, or the VH chain or the VL chain, which are linked together by a short, mobile peptide linker, thereby enabling the scFv to form a structure desirable for antigen binding. scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin and can be readily expressed in a functional form in E. coli or other host cells.

[0066] A "diabody" refers to a small antibody fragment prepared by constructing an scFv fragment with a short linker (approximately 5-10 residues) between the VH and VL domains. This achieves interchain pairing rather than intrachain pairing of the V domain, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. A bispecific diabody is a heterodimer of two "crossover" scFv fragments, where the VH and VL domains of the two antibodies are located on different polypeptide chains. Diabodies are described more completely, for example, in US7635475.

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

[0068] The terms “antigen,” “target antigen,” and “immunogen” are used interchangeably herein and refer to a structural or binding determinant to which an antibody, antibody fragment, or antibody fragment-based molecule binds or has specificity for it. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound, or a part thereof. An antigen is also a ligand for an antibody or antibody fragment that has binding affinity to the antigen. Non-exclusive exemplary antigens described herein include CD3 and EGFR (and parts 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 to be useful as a diagnostic and / or therapeutic agent when targeting differentiation antigen group 3 (CD3).

[0070] An "EGFR antigen-binding fragment" refers to an antigen-binding fragment that can bind to the epidermal growth factor receptor. EGFR is a member of the ErbB receptor family, which is 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 any tissue or cell possessing the EGFR antigen that is responsible for or a part of a disease condition, such as cancer or an associated condition, but is not limited to these. Affected target tissue or cell sources include organs, tumors, cancer cells or populations of cancer cells, or cells found to form a matrix or in association with a population of cancer cells, bone, skin, and cells that produce cytokines or factors that contribute to the disease condition.

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

[0073] "Affinity" refers to the sum of the 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 the 1:1 interaction between members of a binding pair (e.g., between an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). d ) can be represented by. As used herein, “greater binding affinity” means lower K d It means a value, for example, 1 × 10 -9 M is 1 × 10 -8 It has a higher binding affinity than M. An antibody that binds to a target antigen, such as tumor-associated EGFR antigen, is an antibody that binds to the antigen with sufficient affinity to be useful as a diagnostic and / or therapeutic agent when targeting cells or tissues expressing the antigen, and does not significantly cross-react with other proteins.

[0074] "Dissociation constant" or "K" d The terms "L" and "P" are used interchangeably and refer to the affinity between the ligand "L" and the protein "P," i.e., how strongly the ligand binds to a particular protein. This is represented by formula K. d It can be calculated using =[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 antibody variable domains 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 descriptions are used and included herein; for example, CDR-L1 refers to the first hypervariable CDR region of the light chain, and CDR-H2 refers to the second hypervariable CDR region of the heavy chain. 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" is used interchangeably herein and refers to the pH at which a particular molecule has no net charge or is electrically neutral at a statistical mean. The formal name for isoelectric point is pH, and thus the unit is pH. For example, an antigen-binding fragment with a pI of 6.3 will have a neutral charge in a solution with pH 6.3. Isoelectric point can be determined mathematically, and several algorithms for estimating the isoelectric point of peptides and proteins include, for example, the Henderson-Hasselbalch formula with different pK values. Isoelectric point can also be determined experimentally by in vitro assays such as capillary isoelectric focusing.

[0077] A “framework” or “FR” residue is a variable domain residue in an antigen-binding fragment other than the hypervariable region residues as defined herein, and is generally located between them or adjacent to the CDR. Several FR descriptions are used and encompassed herein, for example, 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 terms “release segment” or “RS” refer to a cleavage sequence within a target composition that can be recognized and cleaved by one or more proteases, influencing the release of antigen-binding fragments and XTEN from the composition. As used herein, “mammalian protease” means a protease normally present in body fluids, cells, and tissues that can be found at higher levels in certain target tissues or cells of mammals, e.g., diseased tissue (e.g., tumors). RS sequences can be manipulated to be cleaved by various mammalian proteases or multiple mammalian proteases that are present in or near the target tissue in the subject, or introduced in an in vitro assay. Other equivalent proteases (endogenous or exogenous) capable of recognizing defined cleavage sites can be utilized. It is particularly intended that RS sequences can be tuned and individualized to the proteases utilized, and that linker amino acids can be incorporated to bind to adjacent polypeptides.

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

[0080] The term “internal” refers to the linking 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, and also includes the insertion of the first polypeptide into the sequence of the second polypeptide. For example, if an RS component is “linked” within a chimeric polypeptide construct, RS may be linked to the N-terminus, the C-terminus, or inserted between any two amino acids of the XTEN polypeptide.

[0081] As applied to the forms of compositions provided herein, “activity” means any action or effect that is generally known in the art for the effector components of a composition, including but not limited to antigen binding, antagonist activity, agonist activity, cellular or physiological response, cell lysis, cell death, or whether measured by in vitro assay, ex vivo assay, or in vivo assay, or by clinical efficacy.

[0082] As used herein, “effector cells” include any eukaryotic cells capable of influencing target cells. For example, effector cells can induce loss of membrane integrity, nuclear condensation, nuclear disintegration, apoptosis, lysis, and / or death of target cells. In another example, effector cells can induce division, growth, differentiation, or alteration of signaling pathways in 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 antigens" refer to molecules expressed by effector cells, including but not limited to cell surface molecules such as proteins, glycoproteins, or lipoproteins. Exemplary effector cell antigens include the CD3 complex or T cell receptor (TCR), the CD4, CD8, CD25, CD38, CD69, CD45RO, CD57, CD95, CD107, and CD154 proteins, as well as effector molecules such as cytokines that associate with, bind to, are expressed within, or are expressed and released by effector cells. Effector cell antigens can function as binding counterparts to the binding domain of a target chimeric polypeptide construct.

[0084] As used herein, “CD3” or “differentiation antigen group 3” means 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 independently. The extracellular domains of CD3 epsilon, gamma, and delta are considered part of the immunoglobulin superfamily because they contain immunoglobulin-like domains. CD3 includes, for example, the 207-amino acid-length human CD3 epsilon protein (NCBI reference sequence number NP_000724) and the 182-amino acid-length human CD3 gamma protein (NCBI reference sequence number NP_000064).

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

[0086] "Host cells" include individual cells or cell cultures that can become recipients of, or are currently recipients of, a target vector into which an exogenous nucleic acid, such as those described herein, has been introduced. Host cells include offspring of a single host cell. Offspring are not necessarily identical to the original parent cell (in terms of total DNA complement morphology or genome) due to spontaneous, accidental, or intentional mutations. Host cells include cells transfected in vivo with the vector of the present invention.

[0087] As used to describe the various polypeptides disclosed herein, “isolated” means a polypeptide identified, isolated, and / or recovered from components of its natural environment or from more complex mixtures (such as those in protein purification). Contaminants in its natural environment are typically materials that would interfere with the diagnostic or therapeutic use of the polypeptide, and these may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As will be apparent to those skilled in the art, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof that do not exist in nature do not require “isolation” to distinguish them from their naturally occurring counterparts. In addition, “concentrated,” “isolated,” or “diluted” polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are distinguishable from their naturally occurring counterparts in that the concentration or number of molecules per unit volume generally exceeds that of their naturally occurring counterparts. Generally, polypeptides produced by recombinant means and expressed in host cells are considered “isolated.”

[0088] An "isolated nucleic acid" is a nucleic acid molecule that is identified and isolated from at least one contaminating nucleic acid molecule that normally associates with the natural source of the nucleic acid that encodes a polypeptide. For example, an isolated polypeptide-encoding nucleic acid molecule is in a form or situation other than that found in nature. Thus, an isolated polypeptide-encoding nucleic acid molecule is distinguished from a nucleic acid molecule that encodes a specific polypeptide because it is present in a native cell. However, isolated polypeptide-encoding nucleic acid molecules include, for example, nucleic acid molecules that encode polypeptides found in cells that normally express polypeptides, where the nucleic acid molecule is located at a chromosomal or extrachromosomal location different from the location in a native cell.

[0089] A "chimeric" protein or polypeptide comprises at least one fusion polypeptide containing at least one region in a sequence different from its naturally occurring position. These regions may normally exist in separate proteins and be combined in the fusion polypeptide, or they may normally exist in the same protein but are positioned in a new configuration within the fusion polypeptide. Chimeric proteins can be created, for example, by chemical synthesis or by constructing and translating polynucleotides in which peptide regions encode in a desired relationship.

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

[0091] "XTENization" is used to refer to a peptide or polypeptide modified by the binding or fusion of one or more XTEN polypeptides (described below) to a peptide or polypeptide, whether by recombinant means or chemical crosslinking means.

[0092] "Operatively linked" means that the linked DNA sequences are in the reading phase or in frame. "In-frame fusion" refers to the joining of two or more open reading frames (ORFs) to form a longer, consecutive ORF, in a manner that maintains the reading frame of the original ORF. For example, a promoter or enhancer is operationally linked to the coding sequence of a polypeptide if it affects the transcription of the polypeptide sequence. Thus, the resulting recombinant fusion protein is a single protein containing two or more segments corresponding to the polypeptide encoded by the original ORF (those segments are not usually linked in this way in nature).

[0093] In relation to polypeptides, a "linear sequence" or "sequence" refers to the order of amino acids in a polypeptide from the amino terminus to the carboxyl terminus (N-terminus to C-terminus), where adjacent residues within that sequence are consecutive in the polypeptide's primary structure. A "partial sequence" is a linear sequence of a polypeptide that is known to contain additional residues in one or both directions.

[0094] "Heterogeneous" means that the organism originates from an organism that is genetically distinct from the other organisms being compared. For example, a glycine-rich sequence that has been removed from a natural coding sequence and operably ligated to a coding sequence other than that natural sequence is a heterogeneous glycine-rich sequence. When applied to polynucleotides and polypeptides, the term "heterogeneous" means that the polynucleotide or polypeptide originates from an organism that is genotypeically distinct from the other organisms being compared.

[0095] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “nucleotide” are used interchangeably. They refer to nucleotides of any length, encompassing both single and multiple nucleic acids, which are either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci defined from ligation 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 may include modified nucleotides such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, where present, may be conferred before or after polymer construction. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeling components.

[0096] The term "complementary polynucleotide" refers to a polynucleotide molecule that has a complementary base sequence and reverse coordination compared to a reference sequence, so that it can hybridize with the reference sequence in perfect fidelity.

[0097] In the context of polynucleotides, "recombination" 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 recombinant proteins within host cells.

[0098] The terms “gene” and “gene fragment” are used interchangeably herein. They refer to polynucleotides containing at least one open reading frame that can code for a particular protein after transcription and translation. A gene or gene fragment can be a genome or cDNA, insofar 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 heterogeneous polynucleotides linked together.

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

[0100] The term "downstream" refers to a nucleotide sequence located 3' to the reference nucleotide sequence. In certain embodiments, a downstream nucleotide sequence refers to a sequence that follows a transcription start site. 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 the reference nucleotide sequence. In certain embodiments, an upstream nucleotide sequence refers to a sequence located 5' to the coding region or at the transcription start site. For example, most promoters are located upstream of the transcription start site.

[0102] "Homologousness" or "homologous" means sequence similarity or compatibility 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 bloom45 or bloom80 may be selected to optimize identity, similarity, or homology scores. Preferably, homologous polynucleotides hybridize under stringent conditions as defined herein and have sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably 97%, more preferably 98%, and even more preferably 99% compared to their respective sequences. Homologous polypeptides preferably have sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95-99% when optimally aligned to sequences of equivalent length.

[0103] In the context of polynucleic acids, "ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments or genes, thereby linking them together. For DNA fragments or genes to ligate together, the ends of the DNA must fit together. In some cases, the ends fit directly after endonuclease digestion. However, sometimes it is necessary to first convert the alternating ends, which are generally produced after endonuclease digestion, into blunt ends to make them fit for ligation.

[0104] The terms “stringent conditions” or “stringent hybridization conditions” refer to conditions under which a polynucleotide hybridizes to its target sequence to a detectably higher degree than other sequences (e.g., at least twice the background). Generally, the stringency of hybridization is expressed in part with respect to the temperature and salt concentration under which the washing step is performed. Typically, stringent conditions are a salt concentration of less than about 1.5 M sodium ion concentration at pH 7.0–8.3, typically about 0.01–1.0 M sodium ion concentration (or other salt), and a temperature of at least about 30°C for short polynucleotides (e.g., 10–50 nucleotides) and at least about 60°C for long polynucleotides (e.g., more than 50 nucleotides). For example, “stringent conditions” could include hybridization in 50% formamide, 1M NaCl, and 1% SDS at 37°C, and three washes (15 minutes each) in 0.1×SSC / 1%SDS at 60°C–65°C. Alternatively, temperatures of approximately 65°C, 60°C, 55°C, or 42°C may be used. The SSC concentration may vary from approximately 0.1–2×SSC, and SDS is present at approximately 0.1%. Such wash temperatures are typically selected to be approximately 5–20°C lower than the thermal melting point of a particular sequence at defined ionic strength and pH. Tm is the temperature at which 50% of the target sequence hybridizes to a probe that perfectly matches (at defined ionic strength and pH). Formulas for calculating the Tm and conditions of 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, blocking reagents are used to block nonspecific hybridization. Such blocking reagents contain, for example, approximately 100–200 μg / ml of sheared and denatured salmon sperm DNA.Organic solvents such as formamide at concentrations of approximately 35-50 v / v% can also be used under specific circumstances, such as RNA:DNA hybridization. The useful variations in these washing conditions will be readily apparent to those skilled in the art.

[0105] The terms “identity percentage,” “sequence identity percentage,” and “identity %” applied to polynucleotide sequences refer to the percentage of residue matching between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm inserts gaps in a standardized and reproducible manner within the sequences being compared to optimize the alignment between the two sequences, and thus can achieve a more meaningful comparison of the two sequences. The identity percentage may be measured over the length of the entire defined polynucleotide sequence, or over a shorter length, for example, over the length of a fragment taken from a larger defined polynucleotide sequence, e.g., a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210, or at least 450 consecutive residues. Such lengths are illustrative, and it is understood that any fragment length supported by the sequences shown herein in tables, figures, or sequence listings may be used to describe the length over which the identity percentage may be measured. The sequence identity percentage is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of matching positions (resulting in identical residues in both polypeptide sequences), dividing the number of matching 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 considered when calculating sequence identity.

[0106] With respect to polypeptide sequences specified herein, the terms “identity percentage,” “sequence identity percentage,” and “identity %” are defined as the percentage of amino acid residues in a query sequence that are identical to amino acid residues in a second reference polypeptide sequence or a portion thereof of equivalent length, after the sequences have been aligned and gaps introduced as necessary to achieve the maximum sequence identity percentage, and without considering any conservative substitutions as part of the sequence identity. Alignment for determining amino acid sequence identity percentage can be achieved in various ways within the scope 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 algorithm necessary to achieve optimal alignment over the entire length of the sequences being compared. The identity percentage may be measured over the entire length of the defined polypeptide sequence, or over a shorter length, for example, over the length of a fragment taken from a larger defined polypeptide sequence, e.g., a fragment of at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues. Such lengths are illustrative, and it should be understood that any fragment length supported by the sequences shown herein in tables, figures, or sequence listings may be used to describe the length over which the identity percentage may be measured.

[0107] In the context of polynucleotide sequences, "repetition" refers to the degree of internal homology in a sequence, such as the frequency of identical nucleotide sequences of a given length. Repetition 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, such as RNA or polypeptide. This includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or other RNA products, and the translation of mRNA into polypeptides. Expression produces a “gene product.” As used herein, a gene product may be either a nucleic acid, such as messenger RNA produced by the transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids having post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides having post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or protein cleavage.

[0109] A “vector” or “expression vector” is interchangeable and refers to a nucleic acid molecule that self-replicates within a suitable host, preferably transferring the 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 cells, vector replication vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Vectors that provide two or more of the above functions are also included. An “expression vector” is a polynucleotide that, upon introduction into a suitable host cell, can be transcribed and translated into polypeptides. “Expression system” usually implies a suitable host cell containing an expression vector capable of functioning to produce a desired expression product.

[0110] The term "serum degradation resistance" applied to polypeptides typically refers to the polypeptide's ability to withstand degradation in blood or its components, involving proteases in serum or plasma. Serum degradation resistance can typically be measured by combining the protein with human (or, if necessary, mouse, rat, dog, or monkey) serum or plasma at approximately 37°C, typically over a range of days (e.g., 0.25, 0.5, 1, 2, 4, 8, or 16 days). Samples at these time points can be run on a Western blot assay, and the protein is detected using an antibody. The antibody may be against a tag in the protein. If the protein shows a single band on the Western blot (where the size of the protein is identical to the size of the injected protein), no degradation has occurred. In this exemplary method, the point at which 50% of the protein is degraded, as determined by Western blotting 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 "cyclic half-life" are used interchangeably herein and, where used herein, ln(2) / K el This represents the terminal half-life calculated as K. el is the terminal phase elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration against a time curve. Half-life typically refers to the time required for half the amount of an administered substance to be metabolized or removed by normal biological processes in a living organism. When the clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic, having 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 it 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 in relation to polypeptides, molecular weight is calculated based on the amino acid composition, by adding the molecular weights of each type of amino acid in the composition, or by estimating it from a comparison with a molecular weight standard in an SDS electrophoresis gel. The calculated molecular weight of a molecule may differ from its "apparent molecular weight," which generally refers to the molecular weight of a molecule determined by one or more analytical techniques. "Apparent molecular weight coefficient" and "apparent molecular weight" are related terms, and when used in relation to 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 comparing it to a globular protein standard measured in "apparent kD" units using size exclusion chromatography (SEC) or a similar method. The apparent molecular weight coefficient is the ratio between apparent molecular weight and "molecular weight," the latter of which is calculated by adding based on the amino acid composition as described above, or by estimating it from a comparison with a molecular weight standard in an SDS electrophoresis gel. The determination of apparent molecular weight and apparent molecular weight coefficient is described in U.S. Patent No. 8,673,860.

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

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

[0115] Where used herein, “treatment” or “to treat,” “to alleviate,” or “to improve” are interchangeable herein. These terms refer to approaches to obtain beneficial or desired outcomes, including but not limited to therapeutic and / or preventive benefits. A therapeutic benefit means the eradication or improvement of the underlying disease being treated. A therapeutic benefit is achieved by the eradication or improvement of one or more physiological symptoms or improvement of one or more clinical parameters associated with the underlying disease, so that improvement is observed in the subject, even though the subject may still have the underlying disease. In the case of a preventive benefit, the composition may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though the disease has not been diagnosed.

[0116] As used herein, “therapeutic effect” or “therapeutic benefit” refers to a physiological effect, including but not limited to the alleviation, improvement, or prevention of a disease, or the improvement of one or more clinical parameters related to an underlying disease in a subject, or an enhancement of the physical or mental health of a subject resulting from the administration of the polypeptide of the present invention other than the ability of a biologically active protein to induce the production of antibodies against an antigen epitope. In the case of a preventive benefit, the composition may be administered to a subject at risk of developing a particular disease, a recurrence of a previous disease, or a state or symptom of that disease, or to a subject reporting one or more physiological symptoms of a disease despite no diagnosis of the disease being made.

[0117] As used herein, the terms “therapeutic dose” and “therapeutic amount” refer to the amount of a drug or bioactive protein, either alone or as part of a composition, that, when administered to a subject in a single or repeated dose, can produce any detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of any of the symptoms, aspects, parameters, or characteristics of a disease or condition. Such effect does not need to be absolute to be beneficial. Determining the therapeutic dose is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosures provided herein.

[0118] As used herein, the term “therapeutably effective non-toxic dose” refers to a tolerable dose of a composition as defined herein that is sufficiently high to cause depletion of tumor or cancer cells, elimination of tumor, reduction of tumor, or stabilization of disease without causing or essentially causing significant toxicity to the subject. Such therapeutically effective non-toxic doses may be determined by dose-escalation studies as described in the Art and should be below the dose that induces severe adverse side effects.

[0119] As used herein, the term “therapeutic index” refers to the ratio of the blood concentration at which a drug is toxic to the blood concentration at which the drug is effective. One exemplary ratio of the therapeutic index is LD 50 :ED 50 And here, LD 50 This is a dose that results in a 50% mortality rate in the target population, and ED 50 This is the dose that produces efficacy in the target population.

[0120] As used herein, the term “dose regimen” refers to a schedule of multiple doses (i.e., at least two) of a composition administered in succession, 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 any disease or condition in the subject.

[0121] As used herein, “administration” means a method of administering a certain dosage of a compound (e.g., the anti-CD3 antibody of the present invention) or a composition (e.g., a pharmaceutical composition comprising the anti-CD3 antibody of the present invention) to a subject.

[0122] The "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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 "malignant" typically refer to or describe a physiological condition in mammals characterized by uncontrolled cell growth / proliferation. Examples of cancer include carcinoma, Hodgkin lymphoma, non-Hodgkin 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, urogenital cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, serous uterine cancer, endometrial cancer, cervical cancer, colorectal cancer, 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, and stomach cancer. This includes, but is not limited to, 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, sarcomas of any origin, primary hematological malignancies (including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, myeloproliferative neoplasm disorders, or myelodysplastic disorders), myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpasture syndrome.

[0124] As used herein, “tumor” refers to all new cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as used herein.

[0125] As used herein, “tumor-specific marker” refers to an antigen that is present on or within cancer cells that may be found in greater quantities on or within cancer cells compared to normal cells or tissue, but not necessarily.

[0126] I) General Techniques The practice of this invention will, unless otherwise indicated, utilize conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the scope 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 Please refer to Wiley & Sons, Somerset, NJ, 2000, the contents of which are incorporated herein in their entirety by reference.

[0127] Host cells can be cultured in a variety of media. Commercial media such as Ham F10 (Sigma), Minimum Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing eukaryotic cells. In addition, animal cells can be grown in standardized 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. Standardized media that support cell survival maintain viability, morphology, metabolic capacity, and potentially the differentiation capacity of cells, while standardized 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, for example, pH, pO2, temperature, and osmolality. The nutrient requirements of cells are usually provided in standard medium 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. Apart from nutrients necessary to maintain cellular metabolism, most cells also require one or more hormones derived from at least one of the following groups: steroids, prostaglandins, growth factors, pituitary hormones, and peptide hormones, in order to grow in serum-free media (Sato, GH, et al. in “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 from cell type to cell type. Most of these hormones or transport proteins are either exogenously added, or, in rare cases, mutant cell lines have been found that do not require specific factors.Those skilled in the art will know of other factors necessary to maintain cell culture without excessive experimentation.

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

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

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

[0131] The various EGFR-binding antigen-binding fragments of this 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 remains a significant issue in their development potential and is still a major area to focus on in antibody production. Antibody aggregation can be induced by partial unfolding of its domains, leading to monomer-monomer association, followed by nucleation and aggregate growth. While the aggregation tendency of antibodies and antibody-based proteins can be influenced by external experimental conditions, they are strongly dependent on their endogenous antibody properties, determined by their sequence and structure. Although it is well known that proteins are slightly stable in their folded state, it is less understood that in many cases, most proteins are inherently prone to aggregation in their unfolded or partially unfolded state, and that the resulting aggregates are extremely stable and have long-lived potential isomerism. It has also been shown that a reduction in aggregation tendency is accompanied by an increase in expression titer, indicating that reduced protein aggregation is beneficial throughout the development process and may 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 various mechanisms. Controlling aggregation can improve protein stability, manufacturability, abrasion rate, safety, formulation, titer, immunogenicity, and solubility. Intrinsic protein properties such as size, hydrophobicity, electrostatics, and charge distribution play a crucial role in protein solubility. Low solubility of therapeutic proteins due to surface hydrophobicity has been shown to make formulation development more difficult and can lead to poor in vivo distribution, undesirable pharmacokinetic behavior, and immunogenicity. Reducing the overall surface hydrophobicity of candidate monoclonal antibodies can also provide benefits and cost reductions associated with purification and administration regimens. Individual amino acids can be identified as contributing to antibody aggregation ability through structural analysis and can be located in both the CDR and framework regions.In particular, residues may be predicted to pose a high risk of causing hydrophobicity problems in a given antibody. In one embodiment, the disclosure provides an antigen-binding fragment having the ability to specifically bind to a parent antibody or antibody fragment to an EGFR having at least one amino acid substitution of a hydrophobic amino acid within its framework region, the hydrophobic amino acid being 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 within one or more framework regions, the hydrophobic amino acid being selected from isoleucine, leucine, or methionine.

[0132] In relation to a target 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 the antibody fragment, it has a net positive charge. A larger positive charge tends to correlate with increased blood clearance and tissue retention, and generally a shorter half-life. If the pH is above the pI of the antibody fragment, it has a negative charge. A negative charge generally results in decreased tissue uptake and a longer half-life. It is possible to manipulate this charge for framework residues through mutation. These considerations have informed the design of various sequences of antigen-binding fragments in the embodiments described herein, which were made for parental antibodies in which individual amino acid substitutions were used as a starting point. The isoelectric point of a polypeptide can be determined mathematically or experimentally in an in vitro assay. The isoelectric point (pI) is the pH at which a protein has a net charge of zero, and can be calculated using the charge of a specific amino acid in the protein sequence. The estimate of the charge is called the acid dissociation constant or pKa value and is used to calculate the pI. pI can be determined in vitro by methods such as capillary isoelectric focusing (see Datta-Mannan, A., et al. The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies. mAbs 7:1084 (2015), Li, B., et al. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge. mAbs 6, 1255-1264 (2014)) or other methods known in the art.

[0133] In some embodiments of the embodiments disclosed herein, the target 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 its epitope. Additionally or alternatively, the target AF1 of the present disclosure may comprise a CDR-L or CDR-H having at least 60% identity with either of the CDR-L or CDR-H listed in Table 1. In some embodiments, the target 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 higher sequence identity with any of the sequence numbers listed in Table 1. Furthermore, the target 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 embodiments, the subject AF1 of the Disclosure may be obtained by comprising an FR-L or FR-H exhibiting at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more sequence identity with any of the sequence numbers listed in Table 2. In one embodiment, any of the subject composition embodiments of the Disclosure described herein is a chimeric or humanized antigen-binding fragment. In one embodiment, any of the subject composition embodiments of the Disclosure described herein is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, linear antibodies, and single-chain variable fragments (scFv). AF1 having CDR-H and CDR-L may be configured with an N-terminus to C-terminus (CDR-H)-(CDR-L) orientation.

[0134] In one embodiment, the disclosure provides a polypeptide comprising AF1, wherein AF1 comprises CDR-L and CDR-H, and a heavy chain framework region (FR-H), and AF1 (a) specifically bound to EGFR, and (b) comprising FR-H1, FR-H2, FR-H3, and FR-H4, wherein FR-H1 has one amino acid sequence from SEQ ID NOs. 14 to 16, FR-H2 has the amino acid sequence of SEQ ID NOs. 18 or 19, FR-H3 has the amino acid sequence of SEQ ID NOs. 20 or 21, and FR-H4 has one amino acid sequence from SEQ ID NOs. 22 to 24. In another embodiment, the polypeptide of the subject composition embodiment described herein comprises AF1, wherein AF1 comprises CDR-H3, and CDR-H3 has the amino acid sequence of SEQ ID NOs. 6. In yet another embodiment, AF1 comprises CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 4, 5, and 6, respectively.

[0135] In another embodiment, the disclosure provides a polypeptide comprising AF1, wherein AF1 has an isoelectric point (pI) higher than that of an antigen-binding fragment consisting of the sequence shown in SEQ ID NO: 52, as demonstrated by an in vitro assay. In one embodiment, AF1 is incorporated into a polypeptide to form an anti-EGFR bispecific antibody, the polypeptide exhibiting a higher pI than a control bispecific antibody, the polypeptide comprising AF1 and a reference antigen-binding fragment that binds to a surface antigen classification 3 T cell receptor (CD3), the control bispecific antigen-binding fragment being identical to the polypeptide except that AF1 is replaced by SEQ ID NO: 52. In the above embodiment, AF1 exhibits a pI 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 that of an antigen-binding fragment consisting of the sequence shown in SEQ ID NO: 52. In the embodiments described above, the in vitro assay for determining pI may be capillary isoelectric focusing or other assays known in the art.

[0136] In another embodiment, any polypeptide among the subject composition embodiments described herein comprises AF1, which comprises CDR-L, CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H), and AF1 comprises (a) CDR-H1, CDR-H2, and CDR-H3 configured to specifically bind to EGFR, (b) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs. 4, 5, and 6, respectively, and (c) FR-H1 , comprising FR-H2, FR-H3, and FR-H4, each exhibiting or being identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequences 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 comprising FR-L, where FR-L (a) has the amino acid sequence of SEQ ID NO. 7 (b) FR-L1 exhibits or is identical to the amino acid sequence of SEQ ID NO: 8 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, or is identical to the amino acid sequence of SEQ ID NO: 9, (c) FR-L2 exhibits or is identical to the amino acid sequence of SEQ ID NO: 9 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, or is identical to the amino acid sequence of SEQ ID NO: 9 (d) FR-L3 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of (d) SEQ ID NO. 13 and includes FR-L4 exhibiting or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO. 13.

[0137] In another embodiment, any polypeptide among the subject composition embodiments described herein comprises AF1, AF1 comprising CDR-L, CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H), AF1 comprising (a) configured to specifically bind to EGFR, (b) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs. 4, 5, and 6, respectively, and (c) FR-H1, FR-H 2) comprising FR-H3 and FR-H4, each exhibiting or being identical to, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with, respectively, the amino acid sequences 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, (d) further comprising FR-L, where FR-L is (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 with the amino acid sequence of SEQ ID NO. 8, or being identical thereto; (ii) FR-L2 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. 8, or being identical thereto; (iii) SEQ ID NO. 1 (iv) FR-L3 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of (0), or is identical thereto, and (iv) FR-L4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of (iv) SEQ ID NO: 13, or is identical thereto.

[0138] In another embodiment, any polypeptide among the subject composition embodiments described herein comprises AF1, AF1 comprising CDR-L, CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H), AF1 comprising (a) configured to specifically bind to EGFR, (b) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs. 4, 5, and 6, respectively, and (c) FR-H1, F (d) comprising R-H2, FR-H3, and FR-H4, each exhibiting or being identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequences 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 comprising FR-L, where FR-L is (i) minimally identical to the amino acid sequence of SEQ ID NO. 7. (ii) FR-L1 exhibits or is identical to 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: 8, or (iii) FR-L2 exhibits or is identical to 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: 11 (iv) It includes FR-L3 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of (iv) Sequence ID No. 13 which exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with or is identical to the amino acid sequence of Sequence ID No. 13.

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

[0140] In another embodiment, any polypeptide among the subject composition embodiments described herein comprises AF1, AF1 comprising CDR-L, CDR-H, a light chain framework region (FR-L), and a heavy chain framework region (FR-H), AF1 comprising (a) configured to specifically bind to EGFR, (b) CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs. 4, 5, and 6, respectively, and (c) FR-L1 (d) comprising FR-L2, FR-L3, and FR-L4, each exhibiting or being identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequences of SEQ ID NOs. 7, 8, 9-11, and 13, respectively, (d) comprising FR-H1, FR-H2, FR-H3, and FR-H4, where FR-H1 is the amino acid of SEQ ID NO. 15 FR-H1 has a sequence that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity, or is identical thereto, and FR-H2 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity, or is identical thereto, and FR-H 3 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 21, and FR-H4 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 24.

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

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

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

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

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

[0146] It will be understood that the use of the term “antigen-binding fragment” in relation to the composition embodiments disclosed herein is not limiting and is intended to include a portion or fragment of an antibody that retains the ability to bind to an antigen that is a ligand for the corresponding intact antibody. In such embodiments, the antigen-binding fragment may be, but is not limited to, other polypeptides known in the art, including CDRs and intervening framework regions, variable or hypervariable regions (VL, VH) of the antibody's light and / or heavy chains, variable fragments (Fv), Fab' fragments, F(ab')2 fragments, Fab fragments, single-chain antibodies (scAb), VHH camelid antibodies, single-chain variable fragments (scFv), linear antibodies, single-domain antibodies, complementarity-determining regions (CDRs), domain antibodies (dAb), BHH-type or BNAR-type single-domain heavy-chain immunoglobulins, single-domain light-chain immunoglobulins, or fragments of antibodies capable of binding to an antigen. The VL and VH of the two antigen-binding fragments can also be configured as single-stranded diabody structures; that is, the VL and VH of AF1 and AF2 can be composed of linkers of appropriate length, enabling their arrangement as diabody structures.

[0147] In certain embodiments, the antigen-binding fragments VL and VH are fused together by a relatively long linker consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids, which have mobility properties. 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 sequences 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, any AF1 from the subject composition embodiments described herein specifically binds to human or cynomolgus monkey (cyno)EGFR. In yet another embodiment, any AF1 from the subject composition embodiments described herein specifically binds to both human and cynomolgus monkey (cyno)EGFR.

[0149] In another embodiment, the disclosure provides AF1 having a specific binding affinity to EGFR for incorporation into a target composition in which one or more individual amino acids of the framework region are modified to increase the pI of AF1 to a parent antigen-binding fragment in order to enhance the stability of the bispecific polypeptide into which it is incorporated. In one embodiment, any polypeptide among the target composition embodiments described herein comprises AF1, and 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 an in vitro assay. In another embodiment, any polypeptide among the target composition embodiments described herein comprises AF1, and AF1 exhibits a pI of 5.4 to 6.6 (including boundary values), as demonstrated by an in vitro assay. In another embodiment, any polypeptide among the subject composition embodiments described herein comprises AF1, which exhibits a pI of about 5.4–6.6, or about 5.6–6.4, or about 5.8–6.2, or about 6.0–6.2, or about 6.1–6.3, or about 6.2–6.4, or about 6.3–6.5, or about 6.4–6.6, as calculated or demonstrated by an in vitro assay.

[0150] In another embodiment, the Disclosure provides AF1 having a specific binding affinity to EGFR for incorporation into a subject composition in which the binding affinity to the EGFR antigen is within a specified range. In one embodiment, any polypeptide among the subject composition embodiments described herein comprises AF1, wherein AF1 has a binding affinity of about 0.1 nM to about 100 nM when determined by an in vitro antigen-binding assay containing the EGFR antigen. d It specifically binds to EGFR. In another embodiment, AF1 has a binding affinity 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 (K in in vitro binding assay). d It specifically binds to EGFR (as determined by...). In another embodiment, any polypeptide among the subject composition embodiments described herein comprises AF1 and AF2, 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 by an in vitro antigen binding assay. d It will be understood that a binding affinity of, for example, 1 nM, is greater than a binding affinity of 10 nM. The binding affinity of a target composition to a target ligand can be assayed using a binding assay or competitive binding assay, such as a Biacore assay using a chip-binding receptor or binding protein, or an ELISA assay as described in U.S. Patent No. 5,534,617, the assay described in the examples herein, a radioreceptor assay, or any other assay known in the art. The binding affinity constant can then be determined using a standard method such as Scatchard analysis as described by van Zoelen, et al., Trends Pharmacol Sciences (1998)19)12):487, or any other method known in the art.

[0151] In another embodiment, the Disclosure provides AF1 having a specific binding affinity to EGFR for incorporation into a target composition in which one or more individual amino acids of the framework region are modified to reduce the hydrophobicity of the antigen-binding framework with respect to a parent antigen-binding fragment in order to enhance the stability of the bispecific polypeptide into which it is incorporated. In one embodiment, any polypeptide among the target composition embodiments described herein comprises AF1, which specifically binds to EGFR, and AF1 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 substituted amino acid is selected from arginine, threonine, or glutamine. In another embodiment, AF1 has at least two amino acid substitutions of one or more hydrophobic amino acids 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 substituted 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 disclosure relates to release segment (RS) peptides suitable for incorporation into the subject compositions described herein, which are substrates of one or more mammalian proteases associated with or produced by cells found in or near affected tissue. Such proteases include, but are not limited to, classes of proteases such as metalloproteases, cysteine ​​proteases, aspartate proteases, and serine proteases. RS is particularly useful for conferring a prodrug form to subject compositions that can be activated by cleavage of the RS by a mammalian protease. As described herein, the RS is incorporated into the subject composition embodiments described herein, with the incorporated antigen-binding fragment linked to XTEN (whose composition is described more fully below), thereby releasing the antigen-binding fragment and XTEN from the composition upon cleavage of the RS by the action of one or more proteases of which the RS is a substrate, and the antigen-binding fragment, no longer shielded by XTEN, increases their binding potential to their respective ligands. In specific features, the RS functions as a substrate for proteases that are found in close association with or co-localize with affected tissues or cells, including but not limited to tumors, cancer cells, and inflammatory tissues. Upon cleavage of the RS, antigen-binding fragments are released from the composition that are otherwise shielded by XTEN in the composition (and therefore have lower binding affinity to their respective ligands), restoring the increased ability to bind to target and / or effector cell ligands. In another embodiment, the RS of the polypeptide composition comprises an amino acid sequence that is a substrate for a cellular protease located within the target cell. In another specific feature of the composition described herein, the RS, which is a substrate for two or three classes of proteases, is designed using a sequence that allows different proteases to cleave the RS sequence at different positions, a representative example of which is shown in Figure 6.Therefore, RS, which is a substrate for two, three, or more classes of proteases, has two, three, or more distinct cleavage sites in its RS sequence; nevertheless, cleavage by a single protease results in the release of antigen-binding fragments and XTEN from the RS-containing composition.

[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, and RS1 is a substrate for a protease selected from the group consisting of regmine, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase. In other cases, any polypeptide among the subject composition embodiments described herein comprises a first release segment (RS1) sequence, where RS1 is meprin, neprilysin (CD10), PSMA, BMP-1, A disintegrin and metalloproteinase (ADAM), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM having a thrombospongin motif (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3 (MMP-3, stromelysin 1), matrix metalloprotein Matrix metalloproteinase-7 (MMP-7, matricylsin 1), matrix metalloproteinase-8 (MMP-8, collagenase 2), matrix metalloproteinase-9 (MMP-9, gelatinase B), matrix metalloproteinase-10 (MMP-10, stromelysin 2), matrix metalloproteinase-11 (MMP-11, stromelysin 3), matrix metalloproteinase-12 (MMP- 12. Macrophage elastase), Matrix metalloproteinase-13 (MMP-13, collagenase 3), Matrix metalloproteinase-14 (MMP-14, MT1-MMP), Matrix metalloproteinase-15 (MMP-15, MT2-MMP), Matrix metalloproteinase-19 (MMP-19), Matrix metalloproteinase-23 (MMP-23, CA-MMP),Matrix metalloproteinase-24 (MMP-24, MT5-MMP), matrix metalloproteinase-26 (MMP-26, matrilysin 2), matrix metalloproteinase-27 (MMP-27, CMMP), regmine, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X, cathepsin D, cathepsin E, secretase, urokinase (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, prostate-specific antigen (PSA, KLK3), human neutrophil elastin It is 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), matryptase, matryptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast-activating protein (FAP), kallikrein-related peptidases (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14.

[0154] In another embodiment, the Disclosure provides a polypeptide comprising a first release segment (RS1) sequence for incorporation into a target 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 with a sequence selected from SEQ ID NOs. 53-671. In another embodiment, RS1 includes 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 listed in Table 5. As described in more detail in the following description of the composition and properties of the target polypeptide composition, the release segment is fused between the antigen-binding fragment and the XTEN polypeptide such that XTEN is released from the composition upon cleavage of the release segment.

[0155] In other embodiments, the Disclosure provides a polypeptide comprising a first release segment (RS1) sequence and a second release segment (RS2) for incorporation into a target polypeptide composition described herein, wherein RS1 and RS2 are identical. In yet another embodiment, the Disclosure provides a polypeptide comprising a first release segment (RS1) sequence and a second release segment (RS2) for incorporation into a target polypeptide composition, wherein RS1 and RS2 are different. In some of the embodiments described above, RS1 and RS2 are each substrates for cleavage by a mammalian protease selected from the group consisting of regmine, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase. In another embodiment, the Disclosure provides a polypeptide comprising RS1 and RS2 sequences for incorporation into a target polypeptide composition described herein, wherein RS1 and RS2 are each substrates for cleavage by one or more mammalian proteases, and each 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 with a sequence selected from SEQ ID NOs. 53 to 671. In another embodiment, RS1 and RS2 each contain amino acid sequences 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 listed in Table 5. As described in more detail in the paragraphs relating to the composition and properties of the target polypeptide composition below, the release segments are fused between the antigen-binding fragment and the XTEN polypeptide such that adjacent XTEN is released from the composition upon cleavage of each release segment. [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 embodiment, release segments (either RS1 and / or RS2) for incorporation into any of the target composition embodiments described herein may be designed to be selectively sensitive to a variety of proteases that are substrates, having different cleavage rates and different cleavage efficiencies. Because a given protease may be found at different concentrations in affected tissue, including but not limited to tumors, hematological malignancies, or inflammatory tissue or sites, compared to healthy tissue or circulating blood, the Disclosure provides RSs that ensure the polypeptide is preferentially converted from prodrug form to active form (i.e., by separation and release of antigen-binding fragments and XTEN from the polypeptide after cleavage of the release segment) when near target cells or tissue and their co-localized proteases, by manipulating the individual amino acid sequences to have higher or lower cleavage efficiencies for a given protease compared to the cleavage rate of the release segment in healthy tissue or circulating blood, so that the released antigen-binding fragment has a greater ability to bind to ligands in affected tissue compared to the prodrug form remaining in circulating blood. Such selective design improves the therapeutic index of the resulting composition and reduces 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 ratio of the percentage of the test substrate containing cleaved release segments when subjected to the protease enzyme in the biochemical assay in which the reaction is carried out (further detailed in the Examples), to the percentage of the cleaved control substrate RSR-1517 (AC1611), where the initial substrate concentration is 6 μM, the reaction is stopped after incubation at 37°C for 2 hours (e.g., by adding EDTA), and the amount of digested 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] Therefore, a cleavage efficiency of -1 means that the amount of the test substrate cleaved was 50% compared to the amount of the control substrate, and a cleavage efficiency of +1 means that the amount of the test substrate cleaved was 200% compared to the amount of the control substrate. A higher cleavage rate by the test protease compared to the control results in higher cleavage efficiency, and a slower cleavage rate by the test protease compared to the control results in lower cleavage efficiency. As detailed in the Examples, when the cleavage rate by individual proteases was tested in an in vitro biochemical assay, the control RS sequence AC1611 (RSR-1517) having the amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO: 53) was established as having appropriate baseline cleavage efficiencies by the proteases regmine, MMP-2, MMP-7, MMP-9, MMP-14, uPA, and matryptase. A library of RS peptides was constructed by selectively substituting amino acids at individual positions within the RS peptide, evaluated against a panel of seven proteases (more fully detailed in the Examples), and profiles were obtained. These profiles were used to establish guidelines for appropriate amino acid substitutions to achieve RS peptides with desired cleavage efficiency. While substitutions using hydrophilic amino acids A, E, G, P, S, and T are preferred when constructing RS peptides with desired cleavage efficiency, 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 sensitivity to protease cleavage.

[0159] IV) XTEN polypeptide In other embodiments, the Disclosure relates to polypeptides comprising at least a first elongated recombinant polypeptide (XTEN) incorporated into the subject composition embodiments described herein, thereby serving to both 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 intact and not cleaved, as fully described below. In some embodiments, the Disclosure provides polypeptides comprising a single XTEN fused to the terminus of an RS located between the antigen-binding fragment and the XTEN. In other embodiments, the Disclosure provides polypeptides comprising a first XTEN and a second XTEN (XTEN1 and XTEN2) fused to the N-terminus and C-terminus of RS1 and RS2, respectively, located between each antigen-binding fragment and the XTEN.

[0160] Without being constrained by theory, the incorporation of XTEN may be incorporated into the design of a target composition to confer to the following properties: 1) if the polypeptide composition is in its intact prodrug form, the property of providing the composition with XTEN that shields antigen-binding fragments and reduces their binding affinity to target cell markers and effector cell antigens; 2) the property of providing the polypeptide composition with XTEN that provides an enhanced half-life when administered to a subject; 3) the property of contributing to the solubility and stability of the intact composition, thereby enhancing the pharmaceutical properties of the target composition; and 4) the property of providing the polypeptide composition with XTEN that reduces extravasation in normal tissues and organs, but allows some extravasation in affected tissues (e.g., tumors) with larger pore sizes due to vascular structures, and nevertheless may be released from the tissue by the action of certain mammalian proteases, thereby allowing the antigen-binding fragments of the composition to penetrate more easily into affected tissues, e.g., tumors, and to bind to target cell markers on effector cells and tumor cells and link them together. To meet these needs, the Disclosure provides compositions comprising one or more XTENs, wherein the XTENs provide the resulting composition with increased mass and hydrodynamic radius. The XTEN polypeptides of the embodiments not only provide the composition with increased mass and hydrodynamic radius, but their unstructured mobility may also provide a shielding effect on antigen-binding fragments of the composition, thereby providing a particular advantage in the design of the composition in that it reduces binding to antigens in normal tissue or vascular structures of normal tissue that do not express target cell markers and / or effector cell antigens or express them at reduced levels. In addition, the incorporation of XTENs into the composition can enhance the solubility and proper folding of single-chain antibody-binding fragments during expression and recovery.

[0161] XTEN is a polypeptide having substantially non-repeating sequences that do not exist in nature and have, to some extent, a secondary or tertiary structure under physiological conditions, or not having them, as well as one or more additional properties as 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 amino acids. In one embodiment, the disclosure provides a polypeptide comprising XTEN1, characterized in that XTEN1 has at least about 36 amino acid residues, wherein 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), glutamic acid (E), and proline (P), and it has at least 4 to 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 instances, 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, wherein 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues are selected from 4 to 6 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, characterized in that XTEN1 has at least about 36 to about 1000 amino acid residues, wherein 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 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 from any of the embodiments described herein, comprising XTEN1, characterized in that XTEN1 has at least about 36 to about 1000 amino acid residues, or at least about 100 to about 900 amino acid residues, or at least 144 to about 868 amino acid residues, wherein 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 of 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, such that any length of at least 36 amino acids (e.g., 36, 48, 60, 72, 84, 96 amino acids, etc.) can be achieved in 12-amino acid increments. In other cases, any polypeptide among the subject composition embodiments described herein may include XTEN1, characterized in that XTEN1 has at least about 36 to about 1000 amino acid residues, or at least about 100 to about 900 amino acid residues, 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 to 734. In another embodiment, any of the XTENs in the subject composition embodiments described herein may have an affinity tag of HHHHHH (SEQ ID NO: 794), HHHHHHHH (SEQ ID NO: 795), or sequence EPEA (SEQ ID NO: 796) attached to the N-terminus or C-terminus of the XTEN of the composition to facilitate the purification 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, such as IMAC chromatography or C-tagXL chromatography, or by the methods described in the following examples.

[0163] In another embodiment, the disclosure provides a polypeptide comprising XTEN1, wherein XTEN1 comprises AE36 (a sequence selected from any three sequences of sequence numbers 672-675), or AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_ The amino acid sequences include sequences selected from the sequences of 1, AE288_2, AE288_3, AE292, AE293, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is listed in Table 7.

[0164] In some aspects of the embodiments disclosed herein, the target polypeptide comprises XTEN1 and XTEN2. The composition of the polypeptide comprising XTEN1 and XTEN2, among other components, is described below herein. In one embodiment, the disclosure provides a polypeptide comprising XTEN1 and XTEN2, wherein XTEN2 has 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 of the XTEN2 sequence are selected from at least three of the sequences of SEQ ID NOs. 672 to 675. In another embodiment, the disclosure provides polypeptides comprising XTEN1 and XTEN2, each characterized by having at least about 36 to about 1000 amino acid residues, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN2 sequence are selected from the sequences of SEQ ID NOs. 676 to 734. In another embodiment, any polypeptide among the subject composition embodiments described herein may include XTEN1 and XTEN2, each of which is AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_ The amino acid sequences include sequences selected from the sequences of 1, AE288_2, AE288_3, AE292, AE293, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is listed in Table 7. In some cases of the embodiments described above in this paragraph, XTEN1 and XTEN2 are identical. In other cases of the embodiments described above in this paragraph, XTEN1 and XTEN2 have different amino acid sequences.In some cases, XTEN1 in either 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 in either 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 in either 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] This disclosure envisions any of the embodiments described herein, including XTENs of intermediate length to those in Table 7, and XTENs of longer length than those in Table 7, for example, those in which the 12 amino acid motifs in Table 6 are added to the N-terminus or C-terminus of the XTENs in Table 7.

[0166] In another embodiment, the Disclosure envisions a polypeptide composition among the embodiments described herein, comprising XTEN1 and XTEN2, each further comprising a His tag of sequence EPEA (SEQ ID NO: 796) at the N-terminus of the polypeptide composition, to facilitate the 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 matrices, and other such methods (including, but not limited to, those described in the embodiments below).

[0167] Additional examples of XTEN sequences that can be used in accordance with this disclosure are U.S. Patent Publications 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1, or This is 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, this disclosure relates to antigen-binding fragments (AF2) having specific binding affinity to effector cell antigens, which can be incorporated into any of the subject composition embodiments described herein. In some cases, the effector cell antigen is expressed on the surface of effector cells 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 antigen-binding fragments that have a binding affinity to the EGFR antigen associated with the affected cell or tissue in a compositional form that leads to cell death of the affected cell or tissue. Binding specificity can be determined by a complementarity-determining region or by a CDR such as a light-chain CDR or a heavy-chain CDR. 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 to the effector cell antigen compared to other reference antigens. A bispecific composition resulting from linking a first antigen-binding fragment (AF1) to EGFR with a second antigen-binding fragment (AF2) that has binding specificity to the effector cell antigen via a short, mobile peptide linker is bispecific in that each antigen-binding fragment has a specific binding affinity to its respective ligand. In such compositions, it will be understood that AF1 directed towards the EGFR of the affected tissue is used in combination with AF2 directed towards an effector cell marker to bring effector cells close to the cells of the affected tissue and induce cytolysis of the affected tissue cells. Furthermore, AF1 and AF2 are incorporated into a specifically designed polypeptide containing a cleavable release segment and XTEN to confer prodrug properties to the composition, which is activated by the release of fused AF1 and AF2 upon cleavage of the release segment when near affected tissue having 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 composition in question has a binding affinity to effector cell antigens expressed on the surface of T cells. In another embodiment, AF2 of the composition in question has a binding affinity to CD3. In yet another embodiment, AF2 of the composition in question has a binding affinity to 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 independently combined forms. In yet another embodiment, AF2 has a binding affinity to CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, or CD3 beta.

[0171] The antigen-binding fragments envisioned by this disclosure may originate from naturally occurring antibodies or fragments thereof, non-naturally occurring antibodies or fragments thereof, humanized antibodies or fragments thereof, synthetic antibodies or fragments thereof, hybrid antibodies or fragments thereof, or engineered antibodies or fragments thereof. Methods for generating antibodies against a given target marker are well known in the art. For example, monoclonal antibodies may be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by the recombinant DNA method (U.S. Patent No. 4,816,567). The structures of antibodies and their fragments, the variable regions of the heavy and light chains of antibodies (VH and VL), the single-stranded variable region (scFv), the complementarity-determining region (CDR), and domain antibodies (dAb) are well understood. Methods for generating polypeptides having a desired antigen-binding fragment with binding affinity to a given antigen are known in the art.

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

[0173] The various CD3-binding AF2s of this disclosure are specifically modified to enhance their stability in the polypeptide embodiments described herein. Antibody protein aggregation remains a significant issue in their developmental potential and remains a major area of ​​focus in antibody production. Antibody aggregation can be induced by partial unfolding of its domains, leading to monomer-monomer association, followed by nucleation and aggregate growth. While the aggregation tendency of antibodies and antibody-based proteins can be influenced by external experimental conditions, they are strongly dependent on their endogenous antibody properties, determined by their sequence and structure. Although it is well known that proteins are slightly stable in their folded state, it is less understood that in many cases, most proteins are inherently prone to aggregation in their unfolded or partially unfolded state, and that the resulting aggregates are extremely stable and have long-lived potential isomerism. It has also been shown that a reduction in aggregation tendency is accompanied by an increase in expression titer, indicating that reduced protein aggregation is beneficial throughout the development process and may 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 various mechanisms. Controlling aggregation can improve protein stability, manufacturability, abrasion rate, safety, formulation, titer, immunogenicity, and solubility. Intrinsic protein properties such as size, hydrophobicity, electrostatics, and charge distribution play a crucial role in protein solubility. Low solubility of therapeutic proteins due to surface hydrophobicity has been shown to make formulation development more difficult and can lead to poor in vivo distribution, undesirable pharmacokinetic behavior, and immunogenicity. Reducing the overall surface hydrophobicity of candidate monoclonal antibodies can also provide benefits and cost reductions associated with purification and administration regimens. Individual amino acids can be identified by structural analysis as contributing to antibody aggregation and can be located in both the CDR and framework regions. In particular, residues may be predicted to have a high risk of causing hydrophobic problems in a given antibody.In one embodiment, the disclosure provides an AF2 having the ability to specifically bind to a parent antibody or antibody fragment to a CD3 having at least one amino acid substitution of a hydrophobic amino acid within its framework region, the hydrophobic amino acid being selected from isoleucine, leucine, or methionine. In another embodiment, the CD3 AF2 has at least two amino acid substitutions of a hydrophobic amino acid within one or more framework regions, the hydrophobic amino acid being 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 the antibody or antibody fragment, it has a net positive charge. A larger positive charge tends to correlate with increased blood clearance and tissue retention, and generally a shorter half-life. If the pH is above the pI of the antibody or antibody fragment, it has a negative charge. A negative charge generally results in decreased tissue uptake and a longer half-life. It is possible to manipulate this charge for framework residues through mutation. These considerations informed the design of the AF2 sequence in the embodiments described herein, which was performed on the parent antibody from which individual amino acid substitutions were used as a starting point. The isoelectric point of a polypeptide can be determined mathematically (e.g., computationally) or experimentally in an in vitro assay. The isoelectric point (pI) is the pH at which a protein has a net charge of zero, and can be calculated using the charge of a particular amino acid in the protein sequence. The estimate of the charge is called the acid dissociation constant or pKa value and is used to calculate the pI. pI can be determined in vitro by methods such as capillary isoelectric focusing (see Datta-Mannan, A., et al. The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies. mAbs 7:1084 (2015), Li, B., et al. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge. mAbs 6, 1255-1264 (2014)) or other methods known in the art. In some embodiments, the isoelectric points of AF1 and AF2 are designed to be within a specific range of each other, thereby promoting stability.

[0175] In one embodiment, the Disclosure provides AF2 for use in any of the polypeptide embodiments described herein, including CDR-L and CDR-H, wherein AF2 comprises (a) 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, including CDR-L and CDR-H, AF2 comprising (a) CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 742, 743, and 744, respectively, and (c) CDR-L, which comprises CDR-L1 having the amino acid sequence of SEQ ID NOs. 735 or 736, CDR-L2 having the amino acid sequence of SEQ ID NOs. 738 or 739, and CDR-L3 having the amino acid sequence of SEQ ID NOs. 740.In another embodiment, the AF2 embodiment described above in this paragraph further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 comprises FR-L1 which exhibits or is identical to, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with, or is identical to, the amino acid sequence of SEQ ID NO: 746, and at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, FR-L2 exhibits or is identical to 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 748-751, and FR-L3 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO. 754, and FR-L2 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, and 99% sequence identity with the amino acid sequence of SEQ ID NO. 754. FR-L4 exhibits 7%, 98%, and 99% sequence identity, or is identical thereto, and FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity, or is identical thereto, to the amino acid sequence of SEQ ID NO: 755 or SEQ ID NO: 756, and FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity, to the amino acid sequence of SEQ ID NO: 759. It includes FR-H2 which exhibits or is identical to the above, FR-H3 which exhibits or is identical to the amino acid sequence of SEQ ID NO: 760 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and FR-H4 which exhibits or is identical to the amino acid sequence of SEQ ID NO: 764 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and is identical to the above.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 has FR-L1 which exhibits or is identical to, 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: 746, and at least 86%, 87%, 88%, 8% sequence identity with, or identical to, the amino acid sequence of SEQ ID NO: 747. FR-L2 exhibits or is identical to 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 748, or exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 754, or exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and 95% sequence identity with the amino acid sequence of SEQ ID NO: 754 FR-L4 exhibits or is identical to FR-L4, which has sequence identity of %, 96%, 97%, 98%, and 99%, and FR-H1 exhibits or is identical to FR-H1, which exhibits or is identical to FR-L4, which has sequence identity of at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% with the amino acid sequence of FR-H1, which exhibits or is identical to FR-H1, which has sequence identity of at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% with the amino acid sequence of. The product comprises FR-H2 which is or is identical thereto, FR-H3 which exhibits or is identical to the amino acid sequence of SEQ ID NO: 760 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and FR-H4 which exhibits or is identical to the amino acid sequence of SEQ ID NO: 764 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and is identical thereto.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 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with or is identical to FR-L1 of the amino acid sequence of SEQ ID NO: 746, and sequence FR-L2 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with, or is identical to, the amino acid sequence of sequence number 747, or FR-L2 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with, or is identical to, the amino acid sequence of sequence number 749. L3 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 754, or is identical to FR-L4, which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 755, or is identical to FR-L4. FR-H1 is identical to the amino acid sequence of SEQ ID NO: 759, exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity, or FR-H2 is identical to it, exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 760. It comprises FR-H3 which is or is identical thereto, and FR-H4 which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or is identical to the amino acid sequence of SEQ ID NO: 764.In another embodiment, AF2 of the target polypeptide embodiment described herein comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 has FR-L1 which exhibits or is identical to, 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: 746, and at least 86%, 87%, 88%, 89%, 90%, 91%, 99% sequence identity with, or identical to, the amino acid sequence of SEQ ID NO: 747. FR-L2 exhibits or is identical to 2%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 750, and FR-L3 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 754, and FR-L2 exhibits or is identical to 2%, 93%, 94%, 95%, 96%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 754, and FR-L3 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, and 97% sequence identity with the amino acid sequence of SEQ ID NO: 754. FR-L4 exhibits or is identical to FR-L4 exhibiting %, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 755, or exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 759, or exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 759, It includes FR-H2 which is or is identical thereto, FR-H3 which exhibits or is identical to the amino acid sequence of SEQ ID NO: 760 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and FR-H4 which exhibits or is identical to the amino acid sequence of SEQ ID NO: 764 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and is identical thereto.In another embodiment, AF2 of the target polypeptide embodiment described herein comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF2 has FR-L1 which exhibits or is identical to, 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: 746, and at least 86%, 87%, 88%, 89%, 90%, 91%, 99% sequence identity with, or identical to, the amino acid sequence of SEQ ID NO: 747. FR-L2 exhibits or is identical to 2%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 751, and FR-L3 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 754, and FR-L2 exhibits or is identical to 2%, 93%, 94%, 95%, 96%, and 99% sequence identity with the amino acid sequence of SEQ ID NO: 754, and FR-L3 exhibits or is identical to at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, and 97% sequence identity with the amino acid sequence of SEQ ID NO: 754. FR-L4 exhibits or is identical to FR-L4 exhibiting %, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 756, or exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 759, or exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 759, It includes FR-H2 which is or is identical thereto, FR-H3 which exhibits or is identical to the amino acid sequence of SEQ ID NO: 760 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and FR-H4 which exhibits or is identical to the amino acid sequence of SEQ ID NO: 764 with at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity, and is identical thereto.

[0176] In another embodiment, the Disclosure provides AF2 for use in any of the polypeptide embodiments described herein, wherein AF2 comprises a variable weight (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or being 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 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or being 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, AF2 comprising a variable weight (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or being 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 with or being 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%, or 99% sequence identity with or identical to any one of the amino acid sequences of SEQ ID NOs. 776-780.

[0178] In another embodiment, the Disclosure provides AF2 antigen-binding fragments that bind to CD3 protein complexes and have enhanced stability compared to CD3-binding antibodies or antigen-binding fragments known in the Art. In addition, the CD3 antigen-binding fragments of the Disclosure are designed to confer higher stability to the chimeric bispecific antigen-binding fragment compositions into which they are incorporated, thereby resulting in improved expression and recovery of the fusion protein, increased shelf life, and enhanced stability when administered to a subject. In one approach, the CD3 AF2 of the Disclosure is designed to have higher thermal stability compared to certain CD3-binding antibodies and antigen-binding fragments known in the Art. As a result, CD3 AF2 used as a component of the chimeric bispecific antigen-binding fragment composition into which they are incorporated exhibit desirable pharmaceutical properties, including high thermal stability and low aggregation tendency, resulting in improved expression and recovery during manufacturing and storage, and promoting a longer serum half-life. Biophysical properties such as thermal stability are often limited by antibody variable domains, and their intrinsic properties vary considerably. High thermal stability is often associated with high expression levels and other desired 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 defined as the "melting temperature" (T), which is the temperature at which half of the molecule denatures. m The melting temperature of each heterodimer is determined by measuring the temperature. The melting temperature of each heterodimer indicates its thermal stability. The method includes the method described in the following examples. mIn vitro assays for determining the melting point of the heterodimer are known in the art. The melting point of the heterodimer can be measured using techniques such as differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60, Ghirlando et al (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of the heterodimer can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9) or as described in the following examples.

[0179] The thermal denaturation curves of the CD3 binding fragment of the present disclosure and the anti-CD3 bispecific antibody comprising the anti-CD3 binding fragment and a reference binding fragment demonstrate that the constructs of the present disclosure are more resistant to thermal denaturation than the antigen binding fragment comprising the sequence shown in SEQ ID NO: 781 or a control bispecific antibody, the control bispecific antigen binding fragment comprising SEQ ID NO: 781 and a reference antigen binding fragment bound to the EGFR embodiment described herein. In one embodiment, any polypeptide of the subject composition embodiments described herein comprises the anti-CD3 AF2 of the embodiment described herein, and the T m The T of the antigen-binding fragment consisting of the sequence of SEQ ID NO: 781 was determined by increasing the melting temperature in an in vitro assay. m At least 2°C higher than, 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, any polypeptide among 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 about 300 nM, as determined by an in vitro antigen-binding assay including a human or cyno CD3 antigen. dThe polypeptide comprises AF2 that specifically binds to human or cyno CD3 at a constant of (K). In another embodiment, any polypeptide among the subject composition embodiments described herein has 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 by an in vitro antigen binding assay. d ) contains AF2 that specifically binds to human or cyno-CD3. For clarity, 400K d The antigen-binding fragment having 10 nM K d It binds to its ligand more weakly than those having a higher dissociation constant (K) in an in vitro antigen-binding assay. In another embodiment, any polypeptide among the subject composition embodiments described herein has a dissociation constant (K) in an in vitro antigen-binding assay. d The present disclosure includes AF2 which binds specifically to human or cyno CD3 with a binding affinity at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times weaker than the antibody binding fragment consisting of the amino acid sequence of SEQ ID NO: 781, as determined by ). In another embodiment, the present disclosure includes AF2 which binds specifically to human or cyno CD3 with a respective dissociation constant (K) in an in vitro antigen binding assay. dThe present invention provides a bispecific polypeptide comprising AF2 that exhibits a binding affinity to CD3 that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or at least 1000 times weaker than the binding affinity of the AF1 EGFR embodiment incorporated into the target polypeptide, as determined by the method described herein. The binding affinity of the target composition to the target ligand can be assayed using a binding assay or a competitive binding assay, such as a Biacore assay using a chip-binding receptor or binding protein, or an ELISA assay as described in U.S. Patent No. 5,534,617, the assay described in the examples herein, a radioreceptor assay, or any other assay known in the art. The binding affinity constant can then be determined using a standard method such as the Scatchard analysis described by van Zoelen, et al., Trends Pharmacol Sciences (1998) 19) 12): 487, or any other method known in the art.

[0181] In a related embodiment, the Disclosure provides AF2 incorporated into a chimeric bispecific polypeptide composition that binds to CD3 and has an isoelectric point (pI) that imparts enhanced stability to the composition of the Disclosure compared to a corresponding composition comprising a CD3-binding antibody or antigen-binding fragment known in the Art. In one embodiment, any polypeptide among the subject composition embodiments described herein comprises a CD3-binding AF2, wherein the AF2 exhibits a pI of 6.0 to 6.6 (including boundary values). In another embodiment, any polypeptide among the subject composition embodiments described herein comprises a CD3-binding AF2, wherein the AF2 exhibits a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lower than the pI of a reference antigen-binding fragment consisting of the sequence shown in SEQ ID NO: 781. In another embodiment, any polypeptide among the subject composition embodiments described herein comprises AF2 bound to CD3 fused to AF1 bound to the EGFR antigen, wherein AF2 exhibits a pI 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 bound to the EGFR antigen or its epitope. In another embodiment, any polypeptide among the subject composition embodiments described herein comprises AF2 bound to CD3 fused to AF1 bound to the EGFR antigen, wherein AF2 exhibits a pI within 0.1 to 1.5 pH units of the pI of AF1, or at least 0.3 to 1.2, or at least 0.5 to 1.0, or at least 0.7 to 0.9 pH units. The design, with the pI of these two antigen-binding fragments within a certain range, is specifically intended to result in the resulting fused antigen-binding fragments conferring greater stability to the chimeric bispecific antigen-binding fragment composition into which they are incorporated, leading to 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.To put it separately, having AF2 and AF1 within a relatively narrow pI range allows for the selection of buffers or other solutions in which both AF2 and AF1 are stable, thereby promoting the overall stability of the composition.

[0182] In certain embodiments, the antigen-binding fragments VL and VH are fused together by a relatively long linker consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids, which have mobility properties. In one embodiment, 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 sequences 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 sequences SGGGGS (SEQ ID NO: 797), GGGGS (SEQ ID NO: 798), GGSGGS (SEQ ID NO: 799), GGS, or GSP. In another embodiment, the disclosure provides a composition comprising a single-stranded diabody in which, after folding, a first domain (VL or VH) pairs with a 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 as well as the third and last domains are fused together by one of the aforementioned short linkers, and the second and third variable domains are fused 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 mispairing of adjacent variable domains, thereby facilitating the formation of a single-stranded diabody configuration comprising the VL and VH of the first and second antigen-binding fragments. [Table 8] [Table 9] [Table 10] [Table 11-1] [Table 11-2]

[0183] VI) Bispecific antigen-binding compositions - composition and functional properties In another aspect, the present disclosure relates to novel chimeric bispecific antigen-binding compositions that bind to antigens or epitopes of the CD3 protein complex of effector cells (e.g., T cells) and EGFR antigens associated with affected cells or tissues. They may therefore be referred to as T-cell engagers. As described in more detail below, the bispecific antigen-binding compositions that confer superior 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 upon administration to a subject and increased half-life in circulating blood, the ability to be activated at the intended site of therapy but not in normal healthy tissue, and exhibiting binding affinity to target cells and effector cells at least comparable to that of corresponding conventional bispecific IgG antibodies when activated by proteolytic cleavage of the release segment and release of fused AF1 and AF2. When fused AF1 and AF2 cells bind to effector and target cells, an immunological synapse is formed that leads to activation of the effector cells and promotes the subsequent destruction of target cells through apoptosis or cell lysis.

[0184] The various bispecific antigen-binding compositions of this disclosure described herein are specifically designed to be in prodrug form, in that the XTEN component shields the antigen-binding fragment, reducing its ability to bind to their ligands until it is released from the composition by protease cleavage of one of the protease cleavage sites located within the release segment. Proteases known to be associated with affected cells or tissues include, but are not limited to, the specific proteases described herein, including, but are not limited to, serine proteases, cysteine ​​proteases, aspartate proteases, and metalloproteases. This prodrug property of the bispecific antigen-binding compositions improves the specificity of the compositions to affected 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 highly expressed target cells or affected tissues where the EGFR antigen and protease can cleave the release segment, the bispecific antigen-binding fragment and its construct XTEN are released upon cleavage of the release segment. The fused AF1 and AF2 cross-link cytotoxic effector cells with cells expressing the EGFR antigen in a highly specific manner, thereby directing the cytotoxic properties of T cells toward the target cells. After protease cleavage, the fused AF1 and AF2 are no longer shielded and effectively regain their ability to bind to target cells and effector cells such as cytotoxic T cells possessing the EGFR antigen by binding to the CD3 antigen. This leads to the formation of a T cell receptor complex and triggers T cell activation that mediates the subsequent lysis of target cells expressing a specific EGFR antigen. Thus, the bispecific antigen-binding composition is intended to present potent, specific, and efficient target cell death. In such cases, cells are selectively removed, thereby reducing the potential for toxic side effects.

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

[0186] In one embodiment, the Disclosure provides a bispecific antigen-binding composition having two antigen-binding fragments, AF1 and AF2, which are either of the antigen-binding fragment embodiments described herein, wherein AF2 is fused to AF1 by a mobile peptide linker. In one embodiment, the bispecific antigen-binding fragment composition comprises a first antigen-binding fragment (AF1) in which AF1 specifically binds to EGFR or its epitope, and a second antigen-binding fragment (AF2) in which AF2 specifically binds to a surface antigen classification 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 in the range of 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, when determined computationally or by an in vitro assay. In one embodiment of the bispecific antigen-binding composition, AF1 is determined by an in vitro antigen-binding assay including EGFR or its epitope to be approximately 0.1 nM to approximately 100 nM, or approximately 0.5 nm to approximately 50 nM, or approximately 1 nm to approximately 20 nM, or approximately 2 nM to approximately 10 nM of K. d And it specifically binds to EGFR. In another embodiment of the bispecific antigen-binding composition, AF2 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 by an in vitro antigen-binding assay. d ) ) specifically binds to human or cyno CD3. In another embodiment of the bispecific antigen-binding composition, AF2 has a dissociation constant (K) of about 10 nM to about 400 nM when determined by an in vitro antigen-binding assay. d It specifically binds to human or cyno-CD3 at a constant (K). In yet another embodiment of the bispecific antigen-binding composition, 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 by an in vitro antigen-binding assay. d) ) specifically binds to human or cyno CD3. In another embodiment of the bispecific antigen-binding composition, 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 by an in vitro antigen-binding assay. d ) specifically binds to human or cyno CD3. In another embodiment of the bispecific antigen-binding composition, AF2 has the respective dissociation constant (K) in an in vitro antigen-binding assay. d When determined by ), it specifically binds to human or cyno CD3 with a binding affinity at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times weaker than the antibody-binding fragment consisting of the amino acid sequence of SEQ ID NO: 781. In another embodiment of the bispecific antigen-binding composition, AF2 has a respective dissociation constant (K) in an in vitro antigen-binding assay. d When determined by ), it exhibits a binding affinity for CD3 that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or at least 1000 times weaker than the binding affinity for AF1. For clarity, 400K d The antigen-binding fragment having 10 nM K d It binds to its ligand more weakly than those that possess it.

[0187] In another embodiment of any of the bispecific antigen-binding compositions among the applicable embodiments described herein, having two antigen-binding fragments (AF1 and AF2), a single RS, and a single XTEN, the polypeptide, in its uncleaved state, may have the structural configuration 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 embodiment, when the RS of the bispecific antigen-binding composition is cleaved by a mammalian protease in the environment of the target cell and converted from a prodrug form to an activated or apolipoprotein form, the fused AF1 and AF2 bind to effector cells targeted by AF2 (e.g., T cells having CD3) and affected cells having the EGFR antigen of the target cell targeted by AF1 (e.g., tumor or cancer cells), thereby ligating them together and activating the effector cells, which is a characteristic of any of the various designed compositions in any of the embodiments described herein. In one embodiment, the cleavage of the RS of the bispecific antigen-binding composition, the release of the antigen-binding fragment, and the subsequent co-binding to the effector cell and target cell result in at least 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, or 1000-fold activation of the effector cell, which is assessed by cytokine production, cytolytic protein production, or target cell lysis, as evaluated by an in vitro cell-based assay. In another embodiment, the simultaneous binding of the released antigen-binding fragment to T cells possessing the CD3 antigen and target cells possessing the EGFR antigen forms an immunological synapse, which leads to the release of T cell-derived effector molecules that can lyse the affected cells.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, ELISAs, radiometric 51Cr release assays, fluorescence-based europium release assays, Calcein AM release assays, photometric MTT assays, XTT assays, WST-1 assays, Alamar blue assays, radiometric 3H-Thd incorporation assays, cloning assays for measuring cell division activity, fluorescence-based Rhodamine 123 assays for measuring mitochondrial transmembrane gradients, FACS-based apoptosis assays monitored by phosphatidylserine exposure, ELISA-based TUNEL test assays, caspase activity assays, and cell morphology assays, or other assays known in the art for cytokines, cell-lysing proteins, or cell lysis, or methods described in the following examples.

[0189] In other embodiments, the Disclosure provides bispecific antigen-binding compositions having any two antigen-binding fragments from the embodiments described herein, any two RSs from the embodiments described herein, and any two XTENs from the embodiments described herein. The design of these compositions was driven by consideration of further reducing the binding affinity of the uncleaved composition to the respective ligands of the AF1 and AF2 antibody fragments by adding a second XTEN, thereby further reducing unintended binding of the composition to healthy tissue or cells when administered to a subject, and thereby further improving the therapeutic index of the subject composition compared to a composition having only one RS and one XTEN. The addition of a second RS and a second XTEN resulted in a remarkable reduction in the binding affinity of the uncleaved intact polypeptide to the respective ligands of the AF1 and AF2 antibody fragments when assayed in vitro, compared to a composition having a single RS and XTEN, and also resulted in reduced toxicity in animal models of the disease when administered as a therapeutically effective dose, as described in the following examples. In embodiments of a composition having two antigen-binding fragments, two RSs, and two XTENs, the composition may, in its uncleaved state, have the following structural configurations from N-terminus to C-terminus: 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 (diabodies containing VL and VH of AF1 and AF2), or XTEN1-RS1-diabody-RS2-XTEN2 (diabodies containing VL and VH of AF1 and AF2).

[0190] Without being constrained by any particular theory, it is conceivable that, by using the aforementioned bispecific antigen-binding composition format, upon cleavage of RS, the released fused AF1 and AF2 can kill target cells by recruiting cytotoxic effector cells without requiring pre-stimulation and / or co-stimulation. Furthermore, the independence of effector cells from pre-stimulation and / or co-stimulation may substantially contribute to the extremely high cytotoxicity mediated by the released fused AF1 and AF2 antigen-binding fragments. In some embodiments, the released AF1 and AF2, with AF1 remaining fused to AF2 by a linker peptide, are designed with binding specificity such that AF1 has the ability to bind to pre-selected EGFR antigens 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 affected tissue) and link them together in close proximity, thereby resulting in immunological synapses and selective, targeted, and local effects of the released cytokines and effector molecules on target disease or cancer cells, resulting in damage or destruction of disease or cancer cells and providing therapeutic benefits to the target. The released AF2, which binds to effector cell antigens, can modulate one or more functions of the effector cells and target tumor The effector cell antigens induce or contribute to a cytolytic effect on tumor cells. Effector cell antigens may be expressed by effector cells or other cells. In one embodiment, the effector cell antigens are expressed on the cell surface of effector cells. Non-limiting examples of effector cell antigens are CD3, CD4, CD8, CD16, CD25, CD38, CD45RO, CD56, CD57, CD69, CD95, CD107, and CD154. Thus, those skilled in the art will understand that the composition of the composition is intended to selectively or disproportionately deliver the active form of the composition to target tumor tissue or cancer cells compared to healthy tissue or healthy cells in the subject to which the composition is administered, thereby resulting in a therapeutic benefit. As is evident from the foregoing, this disclosure provides a large family of polypeptides in configurations designed to produce desired properties.

[0191] The object of this disclosure is to design a target bispecific antigen-binding composition in which a shielding effect is conferred by the intact circulating composition XTEN, and simultaneously a reduction in the likelihood of binding to effector cells and target tissue, resulting in a reduction in the production of Th1 T cell-related 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 not linked to a shielding portion such as XTEN. As important components of cellular immunity, the production of IL-2, TNF-alpha, and IFN-gamma is a prominent feature of the Th1 response, 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) (Romagnani ST-cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol. 2000. 85(1):9-18). IL-4, IL-6, and IL-10 are also important pro-inflammatory cytokines 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, the uncleaved, intact bispecific antigen-binding compositions of the embodiments described herein may exhibit at least a 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold, or at least 7-fold, or at least 8-fold, or at least 9-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 1000-fold, reduction in the potential to produce Th1 and / or pro-inflammatory cytokines compared to Th1 and / or pro-inflammatory 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, for example, at equivalent molar concentrations, when the uncleaved, intact polypeptide is in contact with effector and target cells in an in vitro cell-based cytokine stimulation assay. In one embodiment described herein, Th1 cytokine production is assayed in an in vitro assay involving effector cells such as PBMCs or CD3+ T cells, and target cells having the EGFR antigen disclosed herein. In another embodiment, cytokines can be evaluated from blood, body fluid, or tissue samples taken from subjects to which the polypeptide composition has been administered. In the embodiments described herein, subjects may be mice, rats, monkeys, and humans. However, an advantage of the target bispecific antigen-binding compositions of the embodiments described herein is that the cytolytic properties of the composition do not require prior stimulation by cytokines, and the formation of immunological synapses of effector cells bound to target cells by antigen-binding fragments is sufficient to influence cytolysis or apoptosis in the target cells. Nevertheless, the production of pro-inflammatory cytokines is a useful marker for evaluating the potency or effect of the target polypeptide composition, whether by in vitro assays or by monitoring the treatment of subjects with tumors.

[0192] In relation to the use of the bispecific antigen-binding fragment composition in the subject, the subject bispecific antigen-binding composition is designed to take advantage of the difference in pore size of vascular structures in tumor or inflammatory tissue compared to healthy vascular structures with the addition of XTEN, so that the extravasation of the intact bispecific antigen-binding composition in normal tissue is reduced, but in the leakage environment of tumor vascular structures or other inflammatory areas, the intact construct may leak proteases in the affected cell environment, thereby being activated and releasing the antigen-binding fragment to effector and target cells (see, for example, Figure 5). In the case of the RS of the bispecific antigen-binding composition, this design takes advantage of the situation in which the bispecific antigen-binding composition, when near affected tissue that produces one or more proteases, e.g., a tumor, may have an RS sequence sensitive to one or more proteases expressed by the tumor that can be cleaved by the proteases (as fully described above). The action of the 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 will be understood, the reduction in molecular weight and hydrodynamic radius of the composition also confers properties that result in the released fusion AF1 and AF2 moving more freely in solution, traveling through smaller pore spaces in tissues and tumors, more readily leaking out of larger pores in tumor vascular structures, more readily penetrating into tumors, and having an increased ability to bind to effector cells and tumor cells and link them together. Such properties can be measured by different assays. Thus, those skilled in the art will understand that, in relation to the treatment of the target using the composition in question, the bispecific antigen-binding composition exists in a prodrug form and is converted to a more active form when it enters a particular cellular environment by the action of a protease co-localized with the affected tissue or cells. Upon release from the composition by the action of proteases in the target tissue, AF2, which has binding specificity to effector cell antigens, and the linked AF1, which has binding specificity to target cell antigens, fully regain their ability to bind to effector cells and target cells and link them together, forming immunological synapses.Immunological synapse formation activates effector cells, which in turn activate new gene transcription via various signaling pathways, leading to the release of effector molecular contents from their vesicles via exocytosis. Depending on the type of effector cell, different cytokines and lymphokines are released. For example, type 1 helper T cells (Th1) release cytokines such as IFN-gamma, IL-2, and TNF-alpha, while 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 (collectively known as "effector molecules") such as perforin and granzymes that kill their targets. When the bispecific antigen-binding fragments of the bispecific antigen-binding composition bind to effector cells and target tumor cells simultaneously, linking them together, it is particularly intended that, at a very low effector:target (E:T) ratio, tumor cells are acted upon by effector molecules released into intercellular immunological synapses by effector cells, resulting in tumor cell damage, perforin-mediated lysis, granzyme B-induced cell death, and / or apoptosis. Therefore, in another embodiment, although not constrained by theory, when an activatable bispecific antigen-binding fragment composition is administered to a subject with a tumor, the prodrug form remains in the circulatory system in normal tissue, but the prodrug form of the construct can be activated by a protease co-localized with the tumor, and the released antigen-binding fragment can leak into the more permeable vascular structure of the tumor so that it binds to effector cells (e.g., T cells) and tumor cells expressing the EGFR antigen targeted by AF1 of the composition, linking them together, thereby activating the effector cells and resulting in the lysis of tumor cells, which is a characteristic feature of the designed composition.In other words, in some cases, the more permeable vascular structure in tumor tissue allows bispecific antigen-binding polypeptides to leak into that tissue, where tumor-associated proteases act on the release segment (RS), cleaving it and releasing the binding site, which can then bind to effector cells and tumor-associated cells, linking them together. In normal tissue, extravasation may be blocked by a tighter vascular barrier, or if some bispecific antigen-binding polypeptides leak, they may persist mainly in the "pro" form, as there may be insufficient proteases in the healthy tissue to release the binding site, resulting in a net effect of no immunological synapse formation. In some cases, fused AF1 and AF2 released in the target tumor bound to both tumor cells and effector cells exhibit at least 10-fold, or at least 30-fold, or at least 100-fold, or at least 200-fold, or at least 300-fold, or at least 400-fold, or at least 500-fold, or at least 1000-fold increased ability to activate effector cells compared to the corresponding uncleaved, intact bispecific antigen-binding composition. In other cases, fused AF1 and AF2 released in the target tumor bound to both tumor cells and effector cells exhibit at least 10-fold, or at least 30-fold, or at least 100-fold, or at least 200-fold, or at least 300-fold, or at least 400-fold, or at least 500-fold, or at least 1000-fold increased ability to lyse tumor cells compared to the corresponding uncleaved, intact bispecific antigen-binding composition in the tumor. In the embodiments described above, effector cell activation and / or cytotoxicity can be assayed by conventional methods known in the art, such as cell counting of activated effector cells, cytokine assays, tumor size measurement, or histopathology. In the embodiments described above, the subjects may be mice, rats, dogs, monkeys, and humans.Specifically, the composition is designed so that, when administered to subjects with a disease to which AF2 can bind an EGFR antigen, the bispecific antigen-binding composition exhibits enhanced therapeutic indicators and reduced incidence of side effects compared to conventional bispecific antibodies known in the art, achieved by a combination of the shielding effect of XTEN on the binding affinity to the antigen-binding fragment in prodrug form and steric hindrance, but particularly intended to allow the RS to release bispecific AF1 and AF2 near or within the target tissue (e.g., tumor) that produces the substrate protease (achieved by inclusion of the cleavage sequence into the RS).

[0193] VII) Methods and Uses of Bispecific Antigen-Conjugating Compositions In another aspect, the Disclosure provides activatable bispecific antigen-binding compositions and pharmaceutical compositions comprising bispecific antigen-binding compositions particularly useful in medical conditions, e.g., for the prevention, treatment, and / or improvement of certain cancers, tumors, or inflammatory diseases. For use in the treatment of a disease, the bispecific antigen-binding compositions of the present invention would be formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to healthcare professionals.

[0194] In particular, several therapeutic strategies, including the modulation of T cell responses by targeting TcR signaling using the VL and VH portions of anti-human CD3 monoclonal antibodies widely used clinically in immunosuppressive regimens, are being used to design polypeptide compositions for use in treating subjects with cancerous diseases. CD3-specific monoclonal OKT3 was the first such monoclonal approved for use in humans (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 the CD3 complex in the membrane of T cells, blocking its function, and the CD3 complex is associated with the antigen-recognizing structure of T cells (TCRs), which is 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 suppressor T cell induction. In cancer, attempts have been made to use cytotoxic T cells to lyse cancer cells. Without being constrained by theory, it is thought 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 associate with the appropriate antigen on the target cell. This creates immunological synapses, which then initiate a signaling cascade within cytotoxic T cells, leading to T cell activation and the production of various cytotoxic cytokines and effector molecules.Perforin and granzyme are highly toxic molecules stored in preformed granules present in activated cytotoxic T cells. After recognition of the target cell, the cytoplasmic granules of the associated cytotoxic T cells move towards and ultimately fuse with the cytotoxic T cell membrane, releasing their contents in a directed manner within the immunological synapse to form pores in the membrane of the target cell and disrupt the tumor cell plasma membrane. The pores created act as an entry for granzyme, a family of serine proteases that induce apoptosis of the tumor cells.

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

[0196] This disclosure envisions a method of using a bispecific antigen-binding composition engineered to target a broad range of malignant cells, including tumors, in addition to effector cells, in order to yield beneficial therapeutic outcomes. The bispecific antigen-binding composition may be designed to initiate target cell lysis, with one antigen-binding fragment binding to and associating with CD3 to activate cytotoxic T cells, while a second antigen-binding fragment targets an EGFR marker characteristic of a specific malignant tumor, thereby creating an immunological synapse. A particular advantage of this design is that the physical binding of cytotoxic effector cells and EGFR-possessing cells eliminates the need for antigen processing, MHCI / β2-microglobulin, and costimulatory molecules. Due to the range of EGFR-possessing cells, the resulting composition will be understood to have utility against a variety of cancers, including solid and hematological malignancies. In one embodiment, this disclosure provides a method for treating a subject with a tumor. The tumors to be treated may include tumor cells resulting 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, mammary tissue cells, prostate cells, kidney cells, brain cells, colon cells, ovarian cells, uterine cells, bladder cells, skin cells, gastric cells, urogenital tract cells, cervical cells, uterine cells, small intestinal cells, hepatocytes, 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 cytotoxic effector cells are not consumed during the damage / destruction of cross-linked target cancer cells; therefore, after causing the lysis of one target cell, the activated effector cells can be released, migrate through local tissue toward other target cancer cells, bind to the EGFR antigen, and initiate additional cell lysis. In addition, under localized environments such as solid tumors, the release of effector cell molecules such as perforin and granzyme is intended to cause damage to tumor cells adjacent to a given molecule of the bispecific binding domain but not bound by it, thereby leading to stagnation of tumor growth or regression.

[0197] Therefore, after administering a therapeutically effective dose of a pharmaceutical composition comprising the bispecific antigen-binding composition described herein to a subject having a cancer or tumor having an 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, whereby an immunological synapse can be created by the linkage of EGFR-bearing cells and effector cells, and as a result, effector molecules derived from effector cells capable of lysing target cells are released into the synapse, and the usefulness of the present disclosure that apoptosis, cytolysis, or death of the target cancer or tumor cells will be brought about will be understood. Furthermore, those skilled in the art will understand that when an immunological synapse is formed by the binding of the released binding domain to effector cells and target cancer cells, the use of the bispecific antigen-binding composition can result in a beneficial therapeutic effect that is systemic rather than "single-shot killing" and is more lasting.

[0198] In one aspect, the present disclosure relates to a method of treating a disease in a subject, such as a subject having cancer. In some embodiments, the present disclosure provides a method of treating a disease in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising any of the bispecific antigen-binding compositions of the embodiments described herein to a subject who needs it. The therapeutically effective amount of the pharmaceutical composition can vary depending on factors such as the medical condition, age, gender, and weight of the individual, as well as the ability of the antibody or antibody portion to elicit the desired response in the individual. A therapeutically effective amount is also an amount at which the therapeutically beneficial effect outweighs any toxic or detrimental effects of the subject composition. A prophylactically effective amount refers to the amount of the pharmaceutical composition required over a period necessary to achieve the desired prophylactic result.

[0199] The therapeutically effective dose of the bispecific antigen-binding composition described herein generally provides a therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the bispecific antigen-binding composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal experiments are used to determine the LD50 (Lethal dose for 50% of the population) and ED 50 (The effective therapeutic dose in 50% of the population) can be determined. The dose ratio between toxic effects and therapeutic effects is the LD 50 / ED 50 This is a therapeutic index that can be expressed as a ratio. A bispecific antigen-binding composition exhibiting a large therapeutic index is preferred. In one embodiment, the bispecific antigen-binding molecule according to the present invention exhibits a high therapeutic index. Using data obtained from cell culture assays and animal observations, a range of doses suitable for human use can be formulated. The dosage is determined to have little to no toxicity in the ED. 50 It is preferable that the blood concentration is within the range including [specific component]. The dosage may vary within this range depending on various factors, such as the dosage form used, the route of administration utilized, and the condition of the subject. The exact formulation, route of administration, and dosage may be selected by the individual physician, taking into account 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, bispecific antigen-binding compositions may only provide partial benefit rather than a cure. In some embodiments, any physiological change that provides some benefit is also considered therapeutically beneficial. Therefore, in some embodiments, the amount of bispecific antigen-binding composition that provides a physiological change is considered an "effective dose" or "therapeutic effective dose." The subject, patient, or individual requiring 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 in the embodiments described herein may be administered concurrently with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent administered to treat symptoms or diseases in an individual requiring such treatment. Such additional therapeutic agents may include any active ingredients suitable for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, an immunotumor antibody, a cell proliferation inhibitor, 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 embodiments, the additional therapeutic agent is an anticancer agent, such as a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis activator, or an anti-angiogenic agent.

[0201] In one embodiment of a method for treating diseases in a subject, the diseases to be treated include anaplastic and medullary thyroid carcinoma, appendiceal cancer, allenoblastoma, biliary tract cancer, bladder cancer, breast cancer, bile duct cancer, carcinoid tumors, cervical cancer, cholangiocarcinoma, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignant tumors with malignant ascites, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, follicular cancer, gallbladder cancer, and gastric cancer. Cancer, gastrointestinal stromal tumors (GIST), GEG-associated 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 cancer, 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, stomach cancer It may be cancer, testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial carcinoma, uterine cancer, serous uterine carcinoma, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0202] A therapeutically effective dose can produce beneficial effects in assisting the treatment (e.g., cure or reduction of severity) or prevention (e.g., reduction of the likelihood of recurrence) of cancer or tumors. In another embodiment of the method for treating the disease in a subject, the pharmaceutical composition is administered to the subject as two or more therapeutically effective doses, administered twice weekly, once weekly, 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 the administration of 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 a group consisting of at least approximately 0.005 mg / kg, at least approximately 0.01 mg / kg, at least approximately 0.02 mg / kg, at least approximately 0.04 mg / kg, at least approximately 0.08 mg / kg, and at least approximately 0.1 mg / kg, and one or more subsequent maintenance doses administered are at least approximately 0.02 mg / kg, at least approximately 0.05 mg / kg, at least approximately 0.1 mg / kg, at least approximately 0.16 mg / kg, at least approximately 0.18 mg / kg, at least approximately 0.20 mg / kg, and at least approximately 0.22 mg / kg. The group is selected from the following: 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, intra-arterially, 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 over 5 minutes to 96 hours, insofar as it is tolerable to the 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 over 5 minutes to 96 hours, and this dose is 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 The group is selected from the following: 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 a subject as one or more therapeutically effective bolus doses or by infusion over a period of 5 minutes to 96 hours, and the administration to the subject results in a Cmax plasma concentration of at least about 0.1 ng / mL to at least about 2 μg / mL of the uncleaved, intact bispecific antigen-binding composition in the subject, which 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.The therapeutically effective dose is at least approximately 0.005 mg / kg, at least approximately 0.01 mg / kg, at least approximately 0.02 mg / kg, at least approximately 0.04 mg / kg, at least approximately 0.08 mg / kg, at least approximately 0.1 mg / kg, at least approximately 0.12 mg / kg, at least approximately 0.14 mg / kg, at least approximately 0.16 mg / kg, at least approximately 0.18 mg / kg, at least approximately 0.20 mg / kg, at least approximately 0.22 mg / kg, at least approximately 0.2 The amounts are 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 the subsequent doses are 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, and at least about 0.2 The group is selected from 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 embodiments described above, administration to the subject results in a plasma polypeptide concentration of at least approximately 0.1 ng / mL to at least approximately 2 ng / mL or higher in the subject for at least approximately 3 days, at least approximately 7 days, at least approximately 10 days, at least approximately 14 days, or at least approximately 21 days. In the embodiments described above of this method, the subject may be a mouse, rat, monkey, or human.

[0203] (vIII) Nucleic acid sequences In some embodiments, the present invention provides isolated polynucleotide sequences encoding any AF1 sequence, or AF2 sequence, or release segment sequences (RS1 and RS2), or XTEN sequence, or any combination of any of the constituent embodiments described herein, or complements thereof to such polynucleotide sequences. In one embodiment, the present invention provides isolated polynucleotide sequences encoding any polypeptide or bispecific antigen-binding composition, or complements thereof to such polynucleotide sequences. In one embodiment, the present invention provides isolated polynucleotide sequences encoding polypeptides or bispecific antigen-binding compositions, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polynucleotide sequences listed in Table 12.

[0204] In another aspect, the Disclosure relates to methods for generating polynucleotide sequences encoding a polypeptide or bispecific antigen-binding composition of any of the embodiments described herein, or sequences complementary to a polynucleotide sequence (including homologous variants thereof), and methods for expressing a protein expressed by a polynucleotide sequence. Generally, the Method comprises generating a polynucleotide sequence encoding a proteinogenic polypeptide or bispecific antigen-binding composition of any of the embodiments described herein, and incorporating the encoding gene into an expression vector suitable for a host cell. For generating a polypeptide or bispecific antigen-binding composition encoded in any of the embodiments described herein, the Method comprises transforming a suitable host cell with an expression vector, and culturing the host cell under conditions that cause or enable the expression of the polypeptide or bispecific antigen-binding composition resulting from any of the embodiments described herein in the transformed host cell, thereby generating the polypeptide or bispecific antigen-binding composition, which is recovered by the Method described herein or by standard protein purification methods known in the Art. Standard recombination techniques in molecular biology are used to construct the polynucleotides and expression vectors of the Disclosure.

[0205] According to this disclosure, a nucleic acid sequence (or its complement) encoding any of the polypeptides or bispecific antigen-binding compositions described herein is used to generate recombinant DNA molecules directed towards expression in appropriate host cells. Several cloning strategies are suitable for carrying out this disclosure, many of which are used to generate constructs or their complements containing genes encoding the compositions of this disclosure. In one embodiment, a cloning strategy is used to produce a gene encoding a construct containing nucleotides encoding a polypeptide or bispecific antigen-binding composition used to transform host cells to express the composition. In the embodiments described above in this paragraph, the gene may comprise, in the configuration disclosed herein, an antigen-binding fragment, a release segment, and a nucleotide encoding XTEN.

[0206] In one approach, a construct containing a DNA sequence encoding a polypeptide or a bispecific antigen-binding composition construct is first prepared. An exemplary method for preparing such a construct is described in the Examples. This construct is then used to create an expression vector suitable for transforming host cells, such as prokaryotic or eukaryotic host (e.g., mammalian) cells, 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. An exemplary method for preparing the expression vector, transforming host cells, and expressing and recovering XTEN is described in the Examples.

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

[0208] Subsequently, polynucleotides resulting from encoding polypeptides or bispecific antigen-binding composition sequences can be individually cloned into expression vectors. Nucleic acid sequences are inserted into vectors by various procedures. Generally, DNA is inserted into appropriate restriction endonuclease sites using techniques known in the art. Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Constructing a suitable vector containing one or more of these components is done using standard ligation techniques known to those skilled in the art. Such techniques are well known in the art and are adequately described in the scientific and patent literature. Various vectors are publicly available. Vectors can be in the form of plasmids, cosmids, viral particles, or phages that are conveniently suited to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is introduced. Thus, vectors can be self-replicating vectors, i.e., vectors that exist as extrachromosomal entities and whose replication does not depend on chromosomal replication, such as plasmids. Alternatively, the vector may be one that, upon introduction into a host cell, is integrated into the host cell genome and replicates together with the chromosome into which it is integrated. Upon introduction 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 a light chain CDR or heavy chain CDR transcription mRNA, an antigen-binding fragment, or a 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 techniques (see, for example, U.S. Patents No. 5,405,783, 5,412,087, and 5,445,934) using probes complementary to any region of the antigen-binding unit polynucleotide.

[0209] This disclosure provides the use of plasmid expression vectors comprising a replication sequence and a regulatory sequence that are compatible with and recognized by host cells and operably ligated to a gene encoding a polypeptide for controlled expression of the polypeptide. The vectors typically carry a replication site and a protein-coding sequence that can provide phenotypic selection in transformed cells. Such vector sequences are well known in various bacteria, yeasts, and viruses. Useful expression vectors that can be used include, for example, chromosomal segments, non-chromosomal segments, and synthetic DNA sequences. “Expression vector” means a DNA construct comprising a DNA sequence operably ligated to a suitable regulatory sequence that can result in the expression of DNA encoding a polypeptide in a suitable host. It is required that the vector is replicable and viable in selected host cells. Low-copy-number or high-copy-number vectors may be used as desired.

[0210] Suitable vectors include, but are not limited to, SV40 and derivatives of pcDNA and known bacterial plasmids, e.g., col EI, pCRl, pBR322, pMal-C2, pET, pGEX (described in Smith, et al., Gene 57:31-40 (1988)), pMB9, and their derivatives; plasmids, e.g., RP4; phage DNA, e.g., numerous derivatives of phage I such as NM989; and other phage DNAs, e.g., M13 and filamentous single-stranded phage DNA; yeast plasmids, e.g., 2-micron plasmids or derivatives of 2-micron plasmids; and centromere and integrated yeast shuttle vectors; vectors useful in eukaryotic cells, e.g., vectors useful in insect or mammalian cells; and vectors derived from combinations of plasmids and phage DNA, e.g., plasmids modified to use phage DNA or expression regulatory sequences. Similarly, yeast expression systems that may be used in this disclosure include, but are not limited to, non-fusion pYES2 vectors (Invitrogen), fusion pYESHisA, B, C (Invitrogen), and pRS vectors. The regulatory sequence of a vector includes a promoter that induces transcription, an optional operator sequence that controls such transcription, a sequence that codes for a suitable mRNA-ribosome binding site, and sequences that control the termination of transcription and translation. The promoter may be any DNA sequence that exhibits transcriptional activity in a selected host cell and may originate from a gene that codes for a protein that is either homologous or heterologous to the host cell.Suitable promoters for use in expression vectors using prokaryotic hosts include, for example, β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978), Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase and tryptophan (trp) promoter systems [Goeddel, Nucleic Acids Res., 8:4057 (1980), EP36, 776], and hybrid promoters, such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)], all of which are operably ligated to the DNA encoding the XTEN polypeptide. Promoters for use in bacterial systems may also include Shine-Dalgano (SD) sequences operably ligated to the DNA encoding the polypeptide polypeptide.

[0211] Vector expression can also be determined by examining the components of the antigen-binding fragment or the expressed bispecific antigen-binding composition. Various techniques for protein analysis are available in the art. These include, but are not limited to, radioimmunoassays, ELISA (enzyme-linked immunoradioassay), "sandwich" immunoassays, immunoradioassays, in situ immunoassays (e.g., using colloidal gold, enzymes, or radioisotope labeling), Western blot analysis, immunoprecipitation assays, immunofluorescence assays, and SDS-PAGE.

[0212] IX) Method for preparing polypeptides and bispecific antigen-binding compositions In another embodiment, the Disclosure provides a method for producing a composition of interest. In one embodiment, the method comprises culturing a host cell containing a nucleic acid construct encoding a polypeptide or bispecific antigen-binding composition from any of the embodiments described herein under conditions that promote the expression of the polypeptide or bispecific antigen-binding composition, and then recovering the polypeptide or bispecific antigen-binding composition using a standard purification method (e.g., column chromatography, HPLC, etc.) in which the composition is recovered, 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 properly folded. In another embodiment of the method for production, the expressed polypeptide or bispecific antigen-binding composition is recovered, where 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 monomer-soluble form.

[0213] In another embodiment, the disclosure provides an expression vector encoding constructs useful for methods of producing polypeptides and bispecific antigen-binding compositions of functional proteins at high fermentation expression levels using E. coli or mammalian host cells, and for generating polypeptide construct compositions that are cytotoxically active at high expression levels. In one embodiment, the method comprises 1) preparing a polynucleotide encoding a polypeptide from any of the embodiments disclosed herein; 2) cloning the polynucleotide into an expression vector, which may be a plasmid or other vector under the control of transcriptional and translational sequences suitable for high levels of protein expression in a biological system; 3) transforming a suitable host cell with the expression vector; and 4) culturing the host cell in a conventional nutrient medium under conditions suitable for expression of the polypeptide composition. Preferably, the host cell is E. coli. By this method, polypeptide expression results in a fermentation titer of at least 0.05 g / L, or at least 0.1 g / L, or at least 0.2 g / L, or at least 0.3 g / L, or at least 0.5 g / L, or at least 0.6 g / L, or at least 0.7 g / L, or at least 0.8 g / L, or at least 0.9 g / L, or at least 1 g / L of the host cell expression product, and at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the expressed protein is properly folded. As used herein, the term “properly folded” means that the antigen-binding fragment components of the composition have the ability to specifically bind to their target ligand.In another embodiment, the Disclosure provides a method for producing a polypeptide or a bispecific antigen-binding composition, comprising culturing host cells containing a vector encoding the polypeptide or bispecific antigen-binding composition under conditions effective for expressing the polypeptide product, at a dry weight host cell concentration of more than about 10 milligrams / 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, when the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, wherein the antigen-binding fragment of the expressed protein is properly folded. In another embodiment, the Disclosure provides a method for producing a polypeptide or a bispecific antigen-binding composition, comprising culturing host cells containing a vector encoding the composition under conditions effective for expressing the polypeptide product, at a dry weight host cell concentration of more than about 10 milligrams / 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, 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 evaluations of compositions of this disclosure. It will be understood that various other embodiments can be implemented considering the overview provided above. [Examples]

[0215] Example 1: Construction of a bispecific antigen-binding polypeptide having two release segments. To generate a plasmid that can remove individual scFvs by restriction digestion, pCW1700 encoding an anti-EpCAM-anti-CD3 (UCHT1) bispecific tandem scFv was digested with SacII and BstXI together with the release segment RSR2486, AE866 XTEN, and a 6X His tag affinity tag (SEQ ID NO: 794), removing the 3’ end of the anti-EpCAM binding domain, the linker between the anti-EpCAM domain and the anti-CD3 domain, and the 5’ end of the anti-CD3 domain. A DNA fragment encoding the same region was synthesized by 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. Then, pJB0035 was 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 anneal to the NheI and BsaI overhangs. These oligonucleotides were annealed together and ligated into the digested pJB0035 to obtain pCW1880 encoding an 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 with 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 with restriction sites at the 5’ and 3’ ends to function as DNA overlaps for Gibson DNA Assembly. The synthetic DNA fragment was cloned into the digested backbone using a Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to construct pJB0205.

[0217] To generate a bispecific antigen-binding polypeptide having both N-terminal and C-terminal XTENs, AE292 XTEN was PCR-amplified from a plasmid using primers containing a 17-21 bp 5' homologous region to the N-terminal backbone DNA and the C-terminal uncleavable release segment (RSR3058, amino acid sequence TTGEAGEAAGATSAGATGP (SEQ ID NO: 111)). A second PCR product encoding the light chain and a portion of its heavy chain of the anti-EpCAM antibody 4D5MOCB was amplified using primers containing a 16-21 bp 5' homologous region to the N-terminal RSR3058 and the C-terminal 4D5MOCB heavy chain. These PCR fragments were cloned into a BsiWI-SacII digested skeletal vector encoding the remainder of the 4D5MOCB heavy chain / anti-CD3 tandem scFv, a second copy of the uncleavable release segment RSR3058, and 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 reader, whose components are (N-terminus to C-terminus) AE292 XTEN, the uncleavable release segment RSR3058, and an anti-EpCAM-anti-CD3 bispecific tandem scFv, with RSR3058 fused to AE867 XTEN with a 6xHIS affinity tag (SEQ ID NO: 794). The resulting construct is pJB0084, having the DNA sequence and encoded amino acid sequence provided in Table 12.

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

[0219] To introduce novel CD3 scFv with altered isoelectric point 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, containing 40 bp homology to the digested vector at both the 5' and 3' ends, were synthesized to facilitate Gibson DNA assembly. Plasmids pJB0358–pJB0372 were constructed, each containing structures of 15 anti-EGFR scFv variants paired with 6xHIS affinity tag (SEQ ID NO: 794), AE292 XTEN, RSR2295, and, individually, anti-CD3 scFv, RSR2295, and AE868 XTEN with C-tag affinity tag (DNA and protein sequences in Table 12).

[0220] pAH0025 and pAH0026 were constructed by first digesting pJB0368 and pJB0373 with BtsI to remove anti-CD3 scFv. A 40 bp homologous region to the digested backbone and adjacent DNA fragments encoding anti-CD3.32 scFv were ordered. These fragments were introduced into pJB0368 and pJB0373 by Gibson Assembly to construct plasmids encoding AE868 XTEN with a 6xHIS affinity tag (SEQ ID NO: 794), RSR2295, anti-EGFR-anti-CD3 bispecific tandem scFv, RSR2295, and C-tag affinity tag, constructed with two different anti-EGFR binding domains, EGFR.23 and EGFR.2, resulting in the pAH0025 and pAH0026 constructs (DNA and protein sequences in Table 12). Using a similar methodology, constructs having 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 scFv in any combination or orientation (i.e., AF1-AF2 or AF2-AF1 from the N-terminus to the C-terminus) are prepared and their sequences 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 12-100

Table 12-113

Table 12-119

Table 12-120

Table 12-130

Table 12-139

Table 12-148

Table 12-170

Table 12-180

Table 12-200

Table 12-231

Table 12-234

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

[0222] 1. Melting temperature (T m ) decision The thermal stability of each scFv variant was determined by measuring its melting temperature. Briefly, a homogeneous amount of scFv in 200 μL of 1% BSA-PBST was dispensed into PCR tubes. The tubes were incubated for 1 hour at several different temperatures (50°C, 51.4°C, 53.7°C, 57.3°C, 61.7°C, 65.5°C, and 68°C). 50 μL of each sample was used to test CD3 The sample was added to an ELISA plate coated with TIFF0007847433000350.tif78μ target antigen (Creative Biomart) or BSA (reference to address stickiness). The wells of the ELISA plate were pre-filled with 1% BSA-PBST (50 μl / well). The plate was incubated at room temperature for 1 hour. The plate was washed three times with water and 0.05% TWEEN to remove unbound scFv. Binding scFv was detected by adding an anti-YOL antibody (Thermo Scientific, no. MA180189) (1:500 dilution (0.05%) in 1% BSA-PBST) that detects the porcine alpha-tubulin motif in the linker between the heavy and light chains. The sample was incubated at room temperature for 1 hour. The plate was washed three times with water and 0.05% TWEEN to remove unbound scFv. Anti-YOL antibody, anti-rat-HRP antibody (Thermo Scientific, No. 31470) (1:7500 dilution (0.05%) in 1% BSA-PBST) [100 Detection was performed by adding TIFF0007847433000351.tif79°L / well) and incubating at room temperature for 1 hour. Unbound antibodies were removed by washing the plates three times with water and 0.05% TWEEN. The 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 at 450 nM. Absorbance at each temperature was graphed. The melting temperature was determined to be the EC50 of each sample, which is 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 scFv 3.23 and 3.24 had a Tm 5°C higher than the parent CD3.9, while CD3.25 and CD3.26 (sequences shown in Table 14) scFv had a Tm equivalent to that of the parent CD3.9. m This indicates that it possessed. [Table 14]

[0224] 2. Determination of binding affinity to CD3 The binding affinity of each scFv was measured using a ForteBio BLItz instrument. Dilution series of each scFv were prepared in PBS (300 μL / tube) using a 1:1 dilution step: for CD3.24-26, starting at 1000 nM and going down to 62.5 nM; for CD3.23, using a 1:1 dilution step: from 400 nM to 25 nM. Biotinylated CD3 TIFF0007847433000353.tif78μ antigen (Creative Biomart) was diluted in PBS to a final concentration of 30 ug / ml. Streptavidin biosensor (ForteBio) was activated in PBS for 10 minutes. The streptavidin biosensor was applied to the BLitz instrument for measurement. A tube containing 300 μL of PBS was transferred to the BLitz instrument over 30 seconds. Biotinylated CD3 A tube containing TIFF0007847433000354.tif78μ (30ug / ml, 300μL / tube) was transferred to a BLitz instrument, and antigen capture to the sensor was measured over 120 seconds. A tube containing 300μL of PBS was transferred to a BLitz instrument over 30 seconds, and the baseline signal was measured. A tube containing test scFv (30ug / ml, 300μL / tube) was transferred to a BLitz instrument, and the association of scFv to the antigen loading biosensor was measured over 120 seconds. A tube containing 300μL of PBS was transferred to a BLitz instrument over 120 seconds, and the dissociation of scFv from the antigen loading biosensor was measured. This protocol was repeated for each scFv dilution. K for each antibody D This was determined using BLI software (ForteBio). The results are shown 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] Conclusion: Two novel anti-CD3 scFvs with improved thermal stability have been identified. Each of the novel scFvs has 8-9 mutations against CD3.9, primarily present in the CDR. While these mutations result in reduced affinity of the scFvs to their target (CD3) compared to the parental CD3.9, the bispecific T cell engager utilizing CD3.23 remains effective in cell death assays and in vivo.

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

[0228] Construct ID pJB0169 is a molecule having eight distinct domains. From the N-terminus to the C-terminus, the molecule 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 four C-terminal residues: glutamate, proline, glutamate, alanine (C-tag) (XTEN_AE868).

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

[0230] Purification: The frozen cell pellet of pJB0169 was resuspended three times in a pH 4.5 lysis buffer (60 mM acetic acid, 350 mM NaCl) and the cells were lysed by homogenization. The homogenate was agglutinated overnight at pH 4.5 and 2-8°C. The agglutinated homogenate was centrifuged and the supernatant was retained. The supernatant was diluted approximately three times 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 with a 0.22 μm filter. The filtrate was adjusted to pH 7.0 with disodium phosphate.

[0231] Purification: The molecule pJB0169 was captured from the first purified lysate and purified by protein L chromatography (TOYOPEARL AF-rProtein L-650F). Subsequently, the N-terminal His6 tag (SEQ ID NO: 794) was selected using IMAC chromatography (GE IMAC Sepharose 6 FF), and then the C-terminal EPEA tag (SEQ ID NO: 796) was selected using C-tag affinity chromatography (CaptureSelect C-tagXL Affinity Matrix). HMWC was removed using anion exchange chromatography (BIA CIMmultus QA monolith), and the molecule was polished to final purity.

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

[0233] Results: Table 16 shows a summary of aggregation (SEC-HPLC monomer %) of construct pJB0169 after each unit operation. A quality threshold of 95% or more monomer recovery at final polishing was used to determine if the molecule was stable or processable. [Table 16]

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

[0235] Stability Improvement and Evaluation: Novel 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 (fused by short peptide linkers) scFv molecules 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, and SEC-HPLC results were monitored through purification to evaluate relative stability compared to pJB0169 or other constructs (e.g., αEGFR.2-αCD3.23). The new design pairs may be more stable than the corresponding αEGFR.2-αCD3.23 (e.g., a molecule consisting of 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 four C-terminal residues glutamate, proline, glutamate, alanine (C-tag) (XTEN_AE868) from the N-terminus to the C-terminus). The pAH0025 and pAH0026 constructs may also be expected to show a simultaneous improvement in monomer content percentage measured by SEC-HPLC after the unit operations (or subsets thereof) shown in the table below (Table 17). Any construct that meets the monomer purity target of 95% or higher will be considered stable or processable. [Table 17]

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

[0237] The binding constant of anti-EpCAM × anti-CD3 bispecific antigen-binding polypeptides that bind to EpCAM-expressing cells and CD3-expressing cells was measured by competitive binding with fluorescently labeled protease-treated bispecific antigen-binding polypeptides. Fluorescently labeled protease-treated bispecific antigen-binding polypeptides were prepared by conjugation of Alexa Fluor 647 C2 maleimide (Thermo Fisher, catalog number A20347) to a cysteine-containing protease-treated bispecific antigen-binding polypeptide variant (MMP-9 treated pCW1645). Binding experiments were performed on 10,000 cells in 100 μL of binding buffer (2% FCS, 5 mM EDTA, HBSS) at 4°C for 1 hour. The cells were washed once with cold binding buffer, then resuspended in 1% formaldehyde in phosphate-buffered saline, and immediately analyzed using a Millipore Guava easyCyte flow cytometer. Binding of fluorescently labeled protease-treated pCW1645 resulted in apparent K2 levels of 1 nM in hEp-CHO 4-12B cells and 4 nM in CD3+ Jurkat cells. d It was found that it has a value.

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

[0239] Competitive binding experiments were performed on 10,000 Jurkat cells at 4°C for 1 hour using 10 nM fluorescently labeled protease-treated pCW1645 in 100 μL total volume binding buffer (2% FCS, 5 mM EDTA, HBSS). The cells were washed once with cold binding buffer, then resuspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed using a Millipore Guava easyCyte flow cytometer. Competitive binding of fluorescently labeled protease-treated pCW1645 to Jurkat cells using cleaved bispecific antigen-binding polypeptides (pJB0189 hEp.2-hCD3.9 or AC1984 hEp.2-hCD3.23) 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 four times weaker than the affinity 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 × anti-CD3 bispecific antigen-binding polypeptide composition. The binding affinity of anti-EGFR × anti-CD3 bispecific antigen-binding polypeptide constructs to human EGFR and human CD3 is measured using flow cytometry with EGFR-positive human cells selected from HT-29, HCT-116, NCI-H1573, NCI-H1975, and CD3 Jurkat cells.

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

[0243] Competitive coupling experiments, K from the aforementioned coupling experiments d 10,000 hEGFR cells were treated with fluorescently labeled protease-treated pJB0297 at a concentration close to the direct binding constant of fluorescently labeled pJB0297 in 100 μL of binding buffer (2% FCS, 5 mM EDTA, HBSS) at 4°C for 1 hour. The cells were washed once with cold binding buffer, then resuspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed using a Millipore Guava easyCyte flow cytometer. The 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 that of fluorescently labeled pJB0297.

[0244] Competitive binding experiments were conducted at approximately 300 nM (or K from the aforementioned binding experiments). d 10,000 Jurkat cells were treated with fluorescently labeled protease-treated pJB0297 (at a concentration close to 100 μL) in a total volume of binding buffer (2% FCS, 5 mM EDTA, HBSS) for 1 hour at 4°C. The cells were washed once with cold binding buffer, then resuspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed using a Millipore Guava easyCyte flow cytometer. The 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 concentration range.

[0245] Example 6: Enzyme activation, storage, and digestion of XTEN AC1611 (RSR-1517) containing 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, matryptase, regmine, MMP-2, MMP-7, MMP-9, and MMP-14. The amino acid sequence of AC1611 is shown 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 matryptase were provided as activating enzymes and stored at -80°C until use. Recombinant mouse MMP-2, recombinant human MMP-7, and recombinant mouse MMP-9 were provided as enzyme precursors and required activation with 4-aminophenylacetate mercuric (APMA). APMA was first dissolved in 0.1 M NaOH to a final concentration of 10 mM, and then the pH was readjusted to neutral using 0.1 M HCl. Further dilution of the APMA stock to 2.5 mM was performed in 50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM CaCl2. To activate pro-MMP, 1 mM APMA and 100 μg / mL pro-MMP were incubated in 50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM CaCl2 for 1 hour (MMP-2, MMP-7) or 24 hours (MMP-9) at 37°C. To activate MMP-14, 0.86 μg / mL recombinant human fluline and 40 μg / mL pro-MMP-14 were incubated in 50 mM Tris (pH 9), 1 mM CaCl2 for 1.5 hours at 37°C. To activate regmine, 100 μg / mL pro-regmine was incubated in 50 mM sodium acetate (pH 4), 100 mM NaCl for 2 hours at 37°C. 100% ultra-high purity glycerol was added to all activated enzymes (including uPA and MTSP1) to a final concentration of 50% glycerol, and then stored at -20°C for several weeks.

[0247] 2. Enzyme digestion An enzyme panel was tested to determine the cleavage efficiency of each enzyme in AC1611. 6 μM of substrate was incubated with each enzyme in the following enzyme:substrate molar ratios and conditions: in 20 μL of reaction product, uPA (1:25 in 50 mM Tris (pH 8.5)), matryptase (1:25 in 50 mM Tris (pH 9) and 50 mM NaCl), regmine (1:20 in 50 mM MES (pH 5) and 250 mM NaCl), MMP-2 (1:1200 in 50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM CaCl2), MMP-7 (1:1200 in 50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM CaCl2), MMP-9 (50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM The reaction was stopped by adding 20 mM EDTA in CaCl2 (1:2000) and MMP-14 (50 mM Tris (pH 8.5), 3 mM CaCl2, 1 μM ZnCl2, 1:30). After incubating the reaction mixture at 37°C for 2 hours, the reaction was stopped by adding 20 mM EDTA in the case of the MMP reaction, by heating at 85°C for 15 minutes in the case of the uPA reaction and the matryptase reaction, and by adjusting the pH to 8.5 in the case of the legmaine reaction.

[0248] 3. Analysis of cutting 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 an SDS-PAGE and the sample was analyzed by staining with Stains-All (Sigma Aldrich) to determine the percentage of cleavage products. The corresponding band intensities were analyzed using ImageJ software to determine the cleavage percentage. During cleavage by various proteases in the release segment, the substrate RSR-1517-containing XTEN yielded two fragments. The larger fragment was used for the cleavage percentage calculation (amount of reaction product divided by the 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, matryptase, legmine, 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 for all seven enzymes, defined by the cleavage percentage under current standard experimental conditions. This release segment exhibits moderate cleavage efficiency for all enzymes, allowing faster or slower variants to cleave within the assay window during screening, thus enabling accurate ranking. [Table 18]

[0250] Example 7: Screening of release segments using RSR-1517 (AC1611) as a control. In this example, uPA is selected to illustrate how release segment screening was performed. The same procedure was applied to all seven tumor-associated proteases to define their relative cleavage profiles for each substrate, which are a number of arrays illustrating how well each enzyme can cleave it compared to the control substrate RSR-1517. All polypeptides in Table 19 had the amino acid sequence of AC1611, but with the substitution of the release segment peptide of the indicated construct, which was swapped with the EAGRSANHEPLGLVAT sequence (SEQ ID NO: 53) of AC1611. For example, BSRS-4 has the release segment sequence LAGRSDNHSPLGLAGS (SEQ ID NO: 945), but otherwise has complete sequence identity with AC1611.

[0251] 1. Enzyme digestion All release-segment-containing XTEN variants and control AC1611 were diluted to 12 μM in 50 mM Tris (pH 7.5), 150 mM NaCl, and 10 mM CaCl2 in individual Eppendorf tubes. The uPA master mix was mixed with each substrate in a 1:1 ratio, and then prepared so that the total reaction volume was 20 μL, the initial substrate concentration was 6 μM, and the enzyme:substrate ratio varied between 1:20 and 1:3000 depending on the enzyme, to obtain uncleaved substrates that could be visualized in the reaction products and endpoints. All reactants were incubated at 37°C for 2 hours, 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. AC1611 digests were always included as a staining control for each gel to normalize the staining between different gels.

[0252] 2. Calculation of relative cutting efficiency The cleavage percentage of each substrate was analyzed using ImageJ software and calculated as described above. For each variant, the relative cleavage efficiency was calculated as follows.

number

[0253] Under the experimental conditions identified above, a value of +1 in relative cleavage efficiency indicates that the substrate produced twice as much product as the AC1611 control, while a value of -1 in relative cleavage efficiency indicates that the substrate produced only 50% of the product compared to 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 efficiencies of 10 release segment variants when subjected to uPA compared to 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, both for individual enzymes and combinations. For example, RSR-1478 has a value of -2.00 against MMP-14, meaning that this substrate produced only 25% of the product compared to the reference control RSR-1517 when digested with MMP-14. 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 (partly depending on how quickly cleavage occurs to activate the bispecific antigen-binding composition) are needed. [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. A novel control plasmid is constructed to resolve both the control substrate and the target RS in the same example.

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

[0258] 2. Enzyme digestion Prepare the 2× substrate solution by mixing and diluting purified AC1830 or AC1840 and the target RS in assay buffer so that the final concentration of each substrate is 6 μM. Prepare the enzyme master mix by mixing it 1:1 with the 2× substrate solution so that 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. Incubate the reaction mixture at 37°C for 2 hours, then stop it using the procedure described above.

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

number

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

[0261] Conclusion: We anticipate that this competitive digestion assay using RSR-1517 as an internal control will have fewer assay intervals compared to the assay described in Example 6. We expect to adopt this method for further release segment screening.

[0262] Example 9: In vitro caspase 3 / 7 assay of anti-EGFR × anti-CD3 bispecific antigen-binding composition The redirected cytotoxicity of unmasked (XTEN removed by proteolysis), masked (having two XTENs and two release segments cleavable by proteolysis), and uncleavable (having two XTENs not sensitive to proteolysis and a release segment replaced by a peptide) anti-EGFR × anti-CD3 bispecific antigen-binding polypeptide compositions was evaluated using an in vitro cell-based assay of caspase 3 / 7 activity in apoptotic cells. Similar to the caspase cytotoxicity assay described in the above examples, PBMCs were mixed with EGFR-positive tumor target cells at a ratio of 10 effector cells per target cell. All anti-EGFR × anti-CD3 bispecific antigen-binding polypeptide compositions were tested using 10-point 5-fold serial dilution dose concentrations. Unmasked anti-EGFR × anti-CD3 compositions were evaluated in the final dose range of 0.000012 to 10 nM. Masked and uncleavable bispecific antigen-binding polypeptide compositions were analyzed in the final dose range of 0.00064 to 250 nM. Appropriate EGFR-positive human tumor target cell lines included FaDu (head and neck squamous cell carcinoma, SCCHN), SCC-9 (SCCHN), HCT-116 (colorectal tumor with KRAS mutation), NCI-H1573 (colorectal tumor with KRAS mutation), HT-29 (colorectal tumor with BRAF mutation), and NCI-H1975 (EGFR T790M mutation). Cell lines representing wild-type EGFR and colorectal tumors and SCCHN tumors with T790M mutation, KRAS mutation, and BRAF mutation were selected.

[0263] During cell lysis, the amount of caspase 3 / 7 released into the culture supernatant was measured by the amount of luminescent caspase 3 / 7 substrate cleavage, generating a "glow-type" luminescence signal (Promega Caspase-Glo 3 / 7, catalog number G8091). The amount of luminescence was proportional to the amount of caspase activity.

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

[0265] When evaluated using EGFR BRAF mutant HT-29 cell lines, the EC of masked anti-EGFR × anti-CD3 bispecific antigen-binding polypeptides was observed. 50 The activity was 10,930 pM. EC of the non-cleavable composition and the unmasked composition. 50 The activity levels were over 100,000 pM and 0.8 pM, respectively.

[0266] In the two EGFR mutant cell lines tested, the masked anti-EGFR × anti-CD3 bispecific antigen-binding polypeptide exhibited approximately 4,000 to 14,000 times lower activity than the unmasked anti-EGFR × anti-CD3 bispecific antigen-binding polypeptide. As expected, the activity of the uncleavable variant was the lowest among the three versions evaluated, and EC 50 It was over 100,000 pM.

[0267] Conclusion: The results showed that the anti-EGFR × anti-CD3 bispecific antigen-binding polypeptide was cytotoxically active against EGFR KRAS mutant and BRAF mutant cell lines. Masked anti-EGFR × anti-CD3 bispecific antigen-binding polypeptide with two XTENs resulted in potent cytotoxic blockade, which was 4,000 to 14,000 times lower in cytotoxicity compared to the unmasked form. [Table 20]

[0268] Example 10: Antitumor properties of an anti-EGFR × anti-CD3 bispecific antigen-binding polypeptide composition 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 natural killer cell dysfunction. Mice were maintained under sterile, standardized environmental conditions, and the experiment was 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 × 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, 3 × 10⁶ cells per mouse were applied to the right flank of six NOD / SCID mice. 6 One HT-29 cell was subcutaneously transplanted (Cohort 1). On the same day, 6 × 10¹⁶ HT-29 cells were transplanted into the right flank of Cohorts 2-7, each consisting of 6 NOD / SCID mice. 6 Individual human PBMCs and 3 x 10 6A mixture with HT-29 cells was subcutaneously injected. Treatment was initiated 4 hours after inoculation of HT-29 or HT-29 / PBMC mixture. Cohorts 1 and 2 were intravenously injected with a vehicle (PBS + 0.05% Tween 80), cohorts 3 and 4 were injected with 0.05 mg / kg of intact anti-EGFR × anti-CD3 bispecific constructs and 0.5 mg / kg of anti-EGFR × anti-CD3 bispecific constructs, respectively, which 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 of intact anti-EGFR × anti-CD3 bispecific constructs, and cohort 7 was injected with 50 mg / kg of cetuximab as a positive control. Cohorts 1–6 also received seven additional doses daily from day 1 to day 7 (a total of eight doses). Cohort 7 received cetuximab twice a week for a total of eight doses over four weeks.

[0269] The tumors in mice were measured twice a week over a predicted 33-day period using calipers in a vertical two-dimensional manner, and the tumor volume was measured (width). 2 The TGI% index was calculated by applying the formula ((mean tumor volume of cohort 2 vehicle controls - mean tumor volume of test substance treatment) / (mean tumor volume of cohort 2 vehicle controls) × 100). Body weight, overall appearance, and clinical findings such as seizures, tremors, lethargy, hyperresponsiveness, piloerection, labored / rapid breathing, tumor discoloration and ulceration, and death were also carefully monitored as measures of treatment-related toxicity. The tumor growth inhibition percentage (TGI%) index was calculated for each treatment group by applying the following formula: ((mean tumor volume of cohort 2 vehicle controls - mean tumor volume of test substance treatment) / mean tumor volume of cohort 2 vehicle controls) × 100. Treatment results with a TGI% of 60% or higher were considered therapeutically active.

[0270] Results: On day 33, the vehicle-treated cohort 1 mice with tumor cells measured 250 ± 113 mm. 3 They only had an average tumor burden of 238±228 mm. Cohort 2 mice treated with the vehicle in the presence of human effector cells did not exhibit inhibition of tumor progression and had an average tumor burden of 238±228 mm. 3The average tumor burden was high, indicating that human effector cells alone could not induce an antitumor effect. Treatment with 0.05 mg / kg and 0.5 mg / kg (cohorts 3 and 4, respectively) of protease-treated anti-EGFR × anti-CD3 constructs in the presence of human effector cells resulted in clear inhibition of tumor growth at 99% TGI in both treatment groups. Importantly, treatment with 0.143 mg / kg and 1.43 mg / kg (cohorts 5 and 6, respectively) of anti-EGFR × anti-CD3 XPAT in the presence of human effector cells also inhibited tumor growth in a dose-dependent manner at 70% TGI% in the 0.143 mg / kg cohort and 96% TGI% in the 1.43 mg / kg cohort. The data suggest that at doses of 0.143 mg / kg and 1.43 mg / kg, a sufficient amount of anti-EGFR × anti-CD3 construct was effectively cleaved by proteases in the in vivo tumor environment to more active non-XTENized anti-EGFR × 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, and TGI% was -20%.

[0271] Conclusion: The results suggest that the anti-EGFR × anti-CD3 bispecific construct can be effectively cleaved in vivo into an active form and is effective in inhibiting tumor progression in the EGFR BRAF mutant HT-29 tumor environment. In addition, the anti-EGFR × anti-CD3 bispecific construct was superior to the cetuximab control in terms of antitumor activity under the experimental conditions. Notably, no significant weight loss was observed in any of the test substance treatment groups, indicating that all treatments were well-tolerated.

[0272] Example 11: Cell binding evaluated by flow cytometry. The bispecific binding of the anti-EGFR × anti-CD3 bispecific antigen-binding composition is also evaluated by flow cytometry-based assays using 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 stable CHO cell lines expressing EGFR. + Cells and EGFR + Cells are incubated at 4°C for 30 minutes in a binding buffer containing HBSS with 2% BSA and 5 mM EDTA with a certain dose range of untreated anti-EGFR × anti-CD3 bispecific antigen conjugates (PJB0169, containing two XTENs and two RSs), protease-treated PJB0169, and anti-CD3 scFv and anti-EGFR scFv positive controls. After washing with binding buffer to remove unbound test material, cells are incubated at 4°C for 30 minutes with FITC conjugate anti-His tag antibody (Abcam, catalog no. ab1206). Unbound FITC conjugate antibody is removed by washing with binding buffer, cells are resuspended in binding buffer, and acquired using a FACS Calibur flow cytometer (Becton Dickerson) or equivalent instrument. All flow cytometry data are analyzed using FlowJo software (FlowJo LLC) or equivalent.

[0273] Anti-EGFR scFv is not expected to bind to Jurkat cells, while 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-conjugate anti-His tag antibody alone. Similarly, anti-EGFR scFv, protease-treated, and untreated PJB0169 are all expected to bind to EGFR-positive cells, while anti-CD3 scFv is not expected to bind to EGFR-positive cells. These data are expected to reflect the bispecific binding ability of anti-EGFR × anti-CD3 bispecific antigen-binding compositions that recognize both CD3 and EGFR antigens expressed on a panel of Jurkat cell lines and EGFR-expressing human cell lines, respectively. Furthermore, because the XTEN polymer interferes with surface binding, it is expected that the untreated anti-EGFR × anti-CD3 bispecific antigen-binding composition will bind to both the CD3 antigen and the EpCAM antigen with lower affinity than the protease-treated bispecific antigen-binding composition.

[0274] Example 12: Cell lysis evaluated by flow cytometry. Cell lysis by an anti-EGFR × anti-CD3 bispecific antigen-binding composition is evaluated 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 no. 88-0870-16) according to the manufacturer's instructions. Alternatively, PKH26 (Sigma, catalog no. MINI26 and PKH26GL) can be used. Briefly, HCT-116 cells are washed twice with PBS and then 2 × 10⁶ cells are lysed in 0.1 mL of dilution C provided in the CellVue Maroon labeling kit. 6Resuspend the cells. In a separate tube, mix 2 μL of CellVue Maroon dye with 0.5 mL of diluent C, then add 0.1 mL to the HCT-116 cell suspension. Mix t...

Claims

1. A polypeptide comprising (i) a first antigen-binding fragment (AF1), (ii) a second antigen-binding fragment (AF2), (iii) a first extended recombinant polypeptide (XTEN1), (iv) a first release segment (RS1), (v) a second release segment (RS2), and (vi) a second extended recombinant polypeptide (XTEN2), (i) The AF1 specifically binds to the epidermal growth factor receptor (EGFR), The AF1 comprises CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. The AF1 comprises CDR-L1, CDR-L2, and CDR-L3, each containing the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. The AF1 comprises a variable heavy chain (VH) amino acid sequence having the amino acid sequence of SEQ ID NO: 36, and a variable light chain (VL) amino acid sequence having the amino acid sequence of SEQ ID NO: 35; (ii) The AF2 specifically binds to cluster of difference 3 (CD3), and the AF2 comprises a variable heavy chain (VH) amino acid sequence having the amino acid sequence of SEQ ID NO: 773, and a variable light chain (VL) amino acid sequence having the amino acid sequence of SEQ ID NO: 772; (iii) The above XTEN1 is a. It has at least 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 is characterized by having at least 4 to 6 different amino acids selected from G, A, S, T, E, and P, and The aforementioned XTEN1 contains the amino acid sequence of SEQ ID NO: 732; (iv) RS1 is a substrate for cleavage by mammalian proteases; (v) RS2 is a substrate for cleavage by mammalian proteases; (vi) The XTEN2 is a. It has at least 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 XTEN2 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), and c. It is characterized by having at least 4 to 6 different amino acids selected from G, A, S, T, E, and P, and The aforementioned XTEN2 contains the amino acid sequence of SEQ ID NO: 729; and Here, the polypeptide has the structural configuration XTEN1-RS1-AF1-AF2-RS2-XTEN2 from the N-terminus to the C-terminus. Polypeptide.

2. The polypeptide according to claim 1, wherein the difference between the isoelectric point (pI) of AF2 and the pI of AF1, when determined by an in vitro assay, is in the range of 0 to 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.

3. The polypeptide according to claim 1 or 2, wherein AF1 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with, or being identical to, the amino acid sequence of Sequence ID No.

52.

4. The polypeptide according to any one of claims 1 to 3, wherein the AF1 specifically binds to human or cynomolgus monkey (cyno) EGFR.

5. The polypeptide according to any one of claims 1 to 3, wherein AF1 specifically binds to human and cynomolgus monkey (cyno) EGFR.

6. The polypeptide according to any one of claims 1 to 5, wherein RS1 is a substrate for a protease selected from the group consisting of regmine, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase.

7. The polypeptide according to any one of claims 1 to 6, wherein AF1 is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, linear antibody, and single-chain variable fragment (scFv).

8. The polypeptide according to claim 1, wherein the CD3 comprises a CD3 composite subunit.

9. The polypeptide according to claim 8, wherein AF2 specifically binds to human or cynomolgus monkey (cyno) CD3.

10. The polypeptide according to claim 8, wherein AF2 specifically binds to human and cynomolgus monkey (cyno) CD3.

11. The aforementioned AF2 is fused to the aforementioned AF1 by a mobile peptide linker, The polypeptide according to any one of claims 1 to 10, wherein the mobile linker comprises two or three types of amino acids selected from the group consisting of glycine, serine, and proline.

12. (1) The polypeptide according to any one of claims 1 to 11, wherein AF2 is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, linear antibodies, and single-stranded variable fragments (scFv), or (2) AF1 and AF2 are configured as (Fab')2 or single-stranded diabodies.

13. The polypeptide according to any one of claims 1 to 12, wherein RS2 is a substrate for a protease selected from regmine, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase.

14. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 13 and one or more pharmaceutically suitable excipients.

15. The use of a polypeptide according to any one of claims 1 to 13 in the preparation of a drug for the treatment of a disease in a subject, The aforementioned diseases include anaplastic and medullary thyroid carcinoma, appendiceal cancer, allenoblastoma, biliary tract cancer, bladder cancer, breast cancer, bile duct cancer, carcinoid tumors, cervical cancer, bile duct cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignant tumors with malignant ascites, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, follicular cancer, gallbladder cancer, and gastric cancer. Cancer, gastrointestinal stromal tumor (GIST), GE-associated 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 cancer, 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, stomach cancer Use is selected from the group of cancers consisting of cancer, testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial carcinoma, uterine cancer, serous uterine carcinoma, vaginal cancer, vulvar cancer, and Wilms' tumor.

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