CD3 antigen binding fragments and compositions comprising the same

Abstract

To provide effective bispecific antibodies for use in cancer treatment.SOLUTION: Provided is a polypeptide comprising an antigen binding fragment, wherein the antigen binding fragment comprises CDR-L, CDR-H, FR-L, and FR-H, wherein the antigen binding fragment specifically binds to cluster of differentiation 3 (CD3) T cell receptor; comprises a variable heavy chain (VH) amino acid sequence having at least 90% identity to an amino acid sequence having a specific sequence; and comprises at least 99% identity or at least 90% identity to a variable light chain (VL) amino acid sequence having a specific sequence.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on June 24, 2020, is named 32808-775_601_SL.txt and is 3,106,733 bytes in size.

[0002] Description of Cross-References This application claims the benefit of U.S. Provisional Application No. 62 / 866,746, filed Jun. 26, 2019, entitled "CD3 Antigen Binding Fragments and Compositions Comprising Same," and U.S. Provisional Application No. 63 / 041,059, filed Jun. 18, 2020, entitled "CD3 Antigen Binding Fragments and Compositions Comprising Same," each of which is hereby incorporated by reference in its entirety.

Background Art

[0003] Many approved cancer therapeutics are cytotoxic agents that kill both normal and tumor cells. The therapeutic benefit of these cytotoxic agents depends on the greater sensitivity of tumor cells compared to normal cells, thereby enabling the achievement of a clinical response using doses that do not cause unacceptable side effects. However, essentially all of these non-specific agents cause some damage, albeit not significant, to normal tissues and often limit treatment suitability.

[0004] Bispecific antibodies can provide a different approach to cytotoxic drugs by instructing 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 applications that are not possible with monospecific antibodies. In one embodiment, this approach relies on the binding of one arm of the bispecific antibody to a tumor-associated antigen or marker, while the other arm induces their cytotoxic activity by releasing effector molecules such as TNF-α, IFN-γ, interleukin 2, 4, and 10, perforin, and granzyme when binding to the CD3 molecule on T cells. Advances in antibody engineering have led to the development of several bispecific antibody formats and compositions for redirecting effector cells to tumor targets, including bispecific antibodies that function by mobilizing and activating polyclonal T cell populations at the tumor site and that function in this way without the need for costimulation or conventional MHC recognition. However, some bispecific compositions have a very short half-life, require continuous infusion for 4 to 8 weeks to maintain blood levels within the therapeutic window for a sufficient time to achieve a therapeutic effect, or have variable effects. In addition, there remains a dual problem in certain patients who experience severe side effects known as "cytokine storm" or "cytokine release syndrome" (Lee DW et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014 124(2):188-195), mediated particularly by the release of TNF-α and IFN-γ among other cytokines. Thus, there remains an unmet need in the art for the development of bispecific antibodies effective for use in cancer treatment. SUMMARY OF THE INVENTION

[0005] The present invention relates to an anti-cluster of differentiation 3 (CD3) antigen-binding fragment incorporated into a chimeric fusion protein and methods of using the same.

[0006] In one aspect, disclosed herein is a polypeptide comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a light chain complementarity determining region (CDR-L) and a heavy chain complementarity determining region (CDR-H), wherein the antigen-binding fragment a. specifically binds to the cluster of differentiation 3 (CD3) T-cell receptor, and b. comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively.

[0007] In another embodiment, a polypeptide is provided comprising an anti-CD3 antigen-binding fragment, the antigen-binding fragment comprising a light chain complementarity determining region (CDR-L) and a heavy chain complementarity determining region (CDR-H), the antigen-binding fragment a. specifically binds to CD3; b. comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10; and c. exhibits higher thermal stability as demonstrated in an in vitro assay, (i) the melting temperature (T m ), or (ii) when the anti-CD3 antigen-binding fragment is incorporated into an anti-CD3 bispecific antibody, the bispecific antibody exhibits a higher Tm compared to a control bispecific antibody, wherein the anti-CD3 bispecific antibody comprises the anti-CD3 antigen-binding fragment and a reference antigen-binding fragment that binds to an antigen other than CD3, and the control bispecific antigen-binding fragment consists of SEQ ID NO:41 and the reference antigen-binding fragment.

[0008] In some embodiments, the T of the antigen-binding fragment m T of an antigen-binding fragment consisting of the sequence of SEQ ID NO: 41 m at least 2°C higher, or at least 3°C higher, or at least 4°C higher, or at least 5°C higher.

[0009] In yet another aspect, a polypeptide comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a light chain complementarity determining region (CDR-L) and a heavy chain complementarity determining region (CDR-H), and the antigen-binding fragment: a. specifically binds to CD3; b. comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10; and c. comprises FR-H1, FR-H2, FR-H3, and FR-H4, which each exhibit at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with, or are identical to, the amino acid sequences of SEQ ID NO: 22, 23, 25, and 26, respectively, is disclosed herein. In some embodiments, the antigen-binding fragments disclosed herein are chimeric or humanized antigen-binding fragments. In other embodiments, the antigen-binding fragment is selected from the group consisting of Fv, Fab, Fab’, Fab’-SH, linear antibodies, and single-chain variable fragments (scFv).

[0010] In some embodiments, CDR-H1 and CDR-H2 each comprise the amino acid sequences of SEQ ID NO: 8 and 9, respectively. In certain embodiments, CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 1 or 2, CDR-L2 having the amino acid sequence of SEQ ID NO: 4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In another embodiment, CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 1, CDR-L2 having any one of the amino acid sequences of SEQ ID NO: 4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 or 7. In yet another embodiment, CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 2, CDR-L2 having any one of the amino acid sequences of SEQ ID NO: 4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In one embodiment, CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 1, CDR-L2 having the amino acid sequence of SEQ ID NO: 4, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In certain embodiments, CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 2, CDR-L2 having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

[0011] In certain embodiments, the antigen-binding fragment further comprises FR-L1, FR-L2, FR-L3, FR-L4, which each exhibit at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with, or are identical to, the amino acid sequences of SEQ ID NO: 12, 13, 18, and 19, respectively.

[0012] In other embodiments, the antigen-binding fragment further comprises a light-chain framework region (FR-L) and a heavy-chain framework region (FR-H), wherein the antigen-binding fragment comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 12; b. FR-L2 having the amino acid sequence of SEQ ID NO: 13; c. FR-L3 having any one of the amino acid sequences of SEQ ID NOs: 14-17; d. FR-L4 having the amino acid sequence of SEQ ID NO: 19; e. FR-H1 having the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21; f. FR-H2 having the amino acid sequence of SEQ ID NO: 23; e. FR-H3 having the amino acid sequence of SEQ ID NO: 24; and f. FR-H4 having any one of the amino acid sequences of SEQ ID NO: 26. In other embodiments, the antigen-binding fragment comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 12; b. FR-L2 having the amino acid sequence of SEQ ID NO: 13; c. FR-L3 having the amino acid sequence of SEQ ID NO: 14; d. FR-L4 having the amino acid sequence of SEQ ID NO: 19; e. FR-H1 having the amino acid sequence of SEQ ID NO: 20; f. FR-H2 having the amino acid sequence of SEQ ID NO: 23; g. FR-H3 having the amino acid sequence of SEQ ID NO: 24; and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26. In another embodiment, the antigen-binding fragment comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 12; b. FR-L2 having the amino acid sequence of SEQ ID NO: 13; c. FR-L3 having the amino acid sequence of SEQ ID NO: 15; d. FR-L4 having the amino acid sequence of SEQ ID NO: 19; e. FR-H1 having the amino acid sequence of SEQ ID NO: 21; f. FR-H2 having the amino acid sequence of SEQ ID NO: 23; g. FR-H3 having the amino acid sequence of SEQ ID NO: 24; and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26. In another embodiment, the antigen-binding fragment comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 12; b. FR-L2 having the amino acid sequence of SEQ ID NO: 13; c. FR-L3 having the amino acid sequence of SEQ ID NO: 16; d. FR-L4 having the amino acid sequence of SEQ ID NO: 19; e. FR-H1 having the amino acid sequence of SEQ ID NO: 21; f. FR-H2 having the amino acid sequence of SEQ ID NO: 23; g. FR-H3 having the amino acid sequence of SEQ ID NO: 24; and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26.In certain embodiments, the antigen-binding fragment comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO: 12; b. FR-L2 having the amino acid sequence of SEQ ID NO: 13; c. FR-L3 having the amino acid sequence of SEQ ID NO: 17; d. FR-L4 having the amino acid sequence of SEQ ID NO: 19; e. FR-H1 having the amino acid sequence of SEQ ID NO: 21; f. FR-H2 having the amino acid sequence of SEQ ID NO: 23; g. FR-H3 having the amino acid sequence of SEQ ID NO: 24; and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26.

[0013] In some embodiments, the antigen-binding fragment comprises a variable heavy chain (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or being identical to, the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 31. In certain embodiments, the antigen-binding fragment comprises a variable light chain (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or being identical to, any one of the amino acid sequences of SEQ ID NO: 27, 29, 30, 32, or 33. In other embodiments, the antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity to, or being identical to, any one of the amino acid sequences of SEQ ID NOs: 36-40.

[0014] In some embodiments, the antigen-binding fragment specifically binds to human or cynomolgus (cyno) CD3. In other embodiments, the antigen-binding fragment specifically binds to both human and cynomolgus (cyno) CD3. In certain embodiments, the antigen-binding fragment binds to a CD3 complex subunit selected from the CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta epsilon units of CD3. In other embodiments, the antigen-binding fragment binds to the CD3 epsilon fragment of CD3.

[0015] In certain embodiments, the antigen-binding fragment specifically binds to human or cynomolgus CD3 with a dissociation constant (K d ) of about 10 nM to about 400 nM as determined in an in vitro antigen-binding assay that includes the human or cynomolgus CD3 antigen. In other embodiments, the antigen-binding fragment has a dissociation constant (K d ) of less than about 10 nM, or less than about 50 nM, or less than about 100 nM, or less than about 150 nM, or less than about 200 nM, or less than about 250 nM, or less than about 300 nM, or less than about 350 nM, or less than about 400 nM as determined in an in vitro antigen-binding assay and specifically binds to human or cynomolgus CD3. In another embodiment, the antigen-binding fragment exhibits a binding affinity for CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker compared to the binding affinity of an antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 41, as determined by their respective dissociation constants (K d ) in an in vitro antigen-binding assay.

[0016] In some embodiments, the antigen-binding fragment exhibits an isoelectric point (pI) of 6.6 or less. In other embodiments, the antigen-binding fragment exhibits a pI of 6.0 to 6.6 (including the boundary values). In certain embodiments, the antigen-binding fragment exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lower than the pI of a reference antigen-binding fragment consisting of the sequence shown in SEQ ID NO: 41.

[0017] In other embodiments, the polypeptides disclosed herein further comprise a first release segment peptide (RS1), which is a substrate for cleavage by mammalian proteases. In certain embodiments, RS1 is a substrate for a protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In another embodiment, RS1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 42-660. In certain embodiments, RS1 comprises an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which is described in Table 5.

[0018] In some embodiments, the polypeptides disclosed herein further comprise a first extended recombinant polypeptide (XTEN1), which a. has at least about 36 amino acids or at least about 100 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. has at least 4 to 6 different amino acids selected from G, A, S, T, E, and P. In some embodiments, XTEN1 has at least about 36 to about 1000 amino acids or at least about 100 to 1000 amino acids. In certain embodiments, XTEN1 comprises an amino acid sequence selected from at least 3 of SEQ ID NOs: 661 - 664. In other embodiments, XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 665 - 718 and 922 - 926. In another embodiment, XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is described in Table 7.

[0019] In certain embodiments, the polypeptides disclosed herein are expressed as fusion proteins that, in the uncleaved state, have the structural arrangement of AF1-RS1-XTEN1 or XTEN1-RS1-AF1 from the N-terminus to the C-terminus, where AF1 is a first antigen-binding fragment.

[0020] In certain aspects, a polypeptide comprising RS1, RS2, AF1, AF2, XTEN1, and XTEN2, wherein: a. RS1 and RS2 are each substrates for cleavage by mammalian proteases and each 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: 42-660; b. AF1 is an antigen-binding fragment of a monoclonal antibody having binding specificity for CD3; c. AF2 is an antigen-binding fragment comprising the VL and VH of a monoclonal antibody having binding affinity for a target cell marker; d. 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 SEQ ID NOs: 665-718 and 922-926; e. XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 665-718 and 922-926; f. the polypeptide has the structural arrangement from the N-terminus to the C-terminus of: XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, or XTEN2-RS2-diabody-RS1-XTEN1, where the diabody comprises the VL and VH of AF1 and AF2; g. the polypeptide exhibits high thermal stability as determined by an increase in the melting temperature (Tm) in an in vitro assay compared to an antibody fragment consisting of the sequence shown in SEQ ID NO: 41. Polypeptides are disclosed herein.

[0021] In some embodiments, AF1 comprises heavy chain complementarity determining regions (CDR-H) CDR-H1, CDR-H2, and CDR-H3, where CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10 and exhibits high thermal stability compared to the Tm of the antigen-binding fragment consisting of the sequence shown in SEQ ID NO: 41 when determined by an increase in the melting temperature (T m )). In other embodiments, AF1 comprises light chain complementarity determining regions (CDR-L) and heavy chain complementarity determining regions (CDR-H), AF1 comprises CDR-H1, CDR-H2, and CDR-H3, where CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10 and comprises FR-H1, FR-H2, FR-H3, FR-H4, which each exhibit at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or are identical to the amino acids of SEQ ID NO: 20 or 21, 23, 24, and 26, respectively.

[0022] In certain embodiments, CDR-H1 and CDR-H2 each comprise the amino acid sequences of SEQ ID NO: 8 and SEQ ID NO: 9. In some embodiments, CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 1 or 2, CDR-L2 having the amino acid sequence of SEQ ID NO: 4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In another embodiment, CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 1, CDR-L2 having any one of the amino acid sequences of SEQ ID NO: 4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In other embodiments, CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 2, CDR-L2 having any one of the amino acid sequences of SEQ ID NO: 4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In other embodiments, CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 1, any one of the amino acid sequences of SEQ ID NO: 4 CDR-L2, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In other embodiments, CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 2, CDR-L2 having any one of the amino acid sequences of SEQ ID NO: 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6.

[0023] In some embodiments, AF1 includes a light chain framework region (FR-L) and a heavy chain framework region (FR-H), where AF1 includes a. FR-L1 having the amino acid sequence of SEQ ID NO: 12, b. FR-L2 having the amino acid sequence of SEQ ID NO: 13, c. FR-L3 having the amino acid sequence of SEQ ID NO: 14, d. FR-L4 having the amino acid sequence of SEQ ID NO: 19, e. FR-H1 having the amino acid sequence of SEQ ID NO: 20, f. FR-H2 having the amino acid sequence of SEQ ID NO: 23, g. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26. In one embodiment, AF1 includes a. FR-L1 having the amino acid sequence of SEQ ID NO: 12, b. FR-L2 having the amino acid sequence of SEQ ID NO: 13, c. FR-L3 having the amino acid sequence of SEQ ID NO: 15, d. FR-L4 having the amino acid sequence of SEQ ID NO: 19, e. FR-H1 having the amino acid sequence of SEQ ID NO: 21, f. FR-H2 having the amino acid sequence of SEQ ID NO: 23, g. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26. In another embodiment, AF1 includes a. FR-L1 having the amino acid sequence of SEQ ID NO: 12, b. FR-L2 having the amino acid sequence of SEQ ID NO: 13, c. FR-L3 having the amino acid sequence of SEQ ID NO: 16, d. FR-L4 having the amino acid sequence of SEQ ID NO: 19, e. FR-H1 having the amino acid sequence of SEQ ID NO: 21, f. FR-H2 having the amino acid sequence of SEQ ID NO: 23, g. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26. In yet another embodiment, AF1 includes a. FR-L1 having the amino acid sequence of SEQ ID NO: 12, b. FR-L2 having the amino acid sequence of SEQ ID NO: 13, c. FR-L3 having the amino acid sequence of SEQ ID NO: 17, d. FR-L4 having the amino acid sequence of SEQ ID NO: 19, e. FR-H1 having the amino acid sequence of SEQ ID NO: 21, f. FR-H2 having the amino acid sequence of SEQ ID NO: 23, g. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26.

[0024] In some embodiments, AF1 further includes FR-L1, FR-L2, FR-L3, FR-L4, and they each exhibit at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the amino acid sequences of SEQ ID NOs: 12, 13, 14-17, and 19, or are identical thereto.

[0025] In some embodiments, AF1 includes a variable heavy chain (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 31, or being identical thereto. In certain embodiments, AF1 includes a variable light chain (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 27, 29, 30, 32, or 33, or being identical thereto. In certain embodiments, AF1 includes an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 36-40, or being identical thereto.

[0026] In certain embodiments, AF1 specifically binds to human or cynomolgus (cyno) CD3. In some embodiments, AF1 specifically binds to human and cynomolgus (cyno) CD3. In some embodiments, AF1 binds to a CD3 complex subunit selected from the CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta epsilon fragments of CD3. In another embodiment, AF1 binds to CD3 epsilon. In another embodiment, when determined by an in vitro antigen binding assay, the dissociation constant (K d) binds specifically to human or cynomolgus CD3 with a constant. In certain embodiments, AF1 has a dissociation constant (K d ) that binds specifically to human or cynomolgus CD3. In other embodiments, AF1 has a binding affinity that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold lower than that of AF1 consisting of the amino acid sequence of SEQ ID NO: 41 when determined by an in vitro antigen-binding assay and each dissociation constant (K d ) binds specifically to human or cynomolgus CD3.

[0027] In some embodiments, the T m of AF1 is at least 2°C, or at least 3°C, or at least 4°C, or at least 5°C, or at least 6°C, or at least 7°C, or at least 8°C, or at least 9°C, or at least 10°C higher than the T m of the antigen-binding fragment consisting of the sequence of SEQ ID NO: 41 as determined by the increase in melting temperature in an in vitro assay.

[0028] In other embodiments, AF1 exhibits an isoelectric point (pI) of 6.6 or less. In certain embodiments, AF1 exhibits a pI of 6.0 to 6.6 (including the boundary values). In other embodiments, AF1 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 lower than the pI of the reference antigen-binding fragment consisting of the sequence shown in SEQ ID NO: 41.

[0029] In another embodiment, the polypeptide disclosed herein further comprises a second antigen-binding fragment (AF2) that specifically binds to a target cell marker other than CD3. In some embodiments, AF2 is fused to AF1 by a flexible polypeptide linker. In other embodiments, the flexible linker comprises two or three types of amino acids selected from the group consisting of glycine, serine, and proline. In certain embodiments, (1) the AF2 fragment is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, single-domain antibody, and single-chain variable fragment (scFv), or (2) AF1 and AF2 are configured as (Fab’)2 or single-chain diabody.

[0030] In some embodiments, the CDRs of AF2 are selected from the sequences of SEQ ID NOs: 719 to 918. In certain embodiments, AF2 comprises the VL and VH of a monoclonal antibody having binding affinity for a target cell marker. In other embodiments, the VL of AF2 is selected from the sequences of SEQ ID NOs: 819 to 918, and the VH is selected from the sequences of SEQ ID NOs: 719 to 818.

[0031] In some embodiments, the target cell marker is a tumor antigen. In some embodiments, the target cell marker is 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A kinase anchor protein 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenergic receptor β3 (ADRB3), AGS-22M6, alpha folate receptor, alpha-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoietin-binding cell surface receptor 2 (Tie 2), anthrax toxin, AOC3 (VAP-1), B cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthracis anthrax, B cell activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), brother of the regulator of imprinted sites (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCCTC-binding factor (CTCF), CCL11 (eotaxin-1), CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD171, CD179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (alpha chain of the IL-2 receptor), CD27, CD274, CD28, CD3, CD3 ε, CD30, CD300 molecule-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-related antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), claudin 18.2 (CLDN18.2), claudin 6 (CLDN6), Clostridium difficile, clamping factor A, CLCA2, colony stimulating factor 1 receptor (CSF1R), CSF2, CTLA-4, C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-X-C chemokine receptor type 4, cyclin B1, cytochrome P450 1B1 (CYP1B1), cyp-B, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, E. coli Shiga toxin type 1, E. coli Shiga toxin type 2, ecto ADP-ribosyltransferase 4 (ART4), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), EGF-like domain multiple 7 (EGFL7), elongation factor 2 variant (ELF2M), endotoxin, ephrin A2, ephrin B2, ephrin A receptor type 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithin, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane serine protease 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin IIβ-chain, fibroblast activation protein α (FAP), fibronectin extracellular domain-B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor α, folate receptor β, Fos-related antigen 1, frizzled receptor, fucosyl GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5 member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), glypican-3 (GPC3), GMCSF receptor α-chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, heat shock protein 70-2 variant (mut hsp70-2), hemagglutinin, hepatitis A virus cell receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, hexasaccharide protein of globoH glycosphingolipid (GloboH), HGF, HHGFR, high molecular weight melanoma-associated antigen (HMW-MAA), histone complex, HIV-1, HLA-DR, HNGF, Hsp90, HST-2 (FGF6), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), human TNF, ICAM-1 (CD54), iCE, IFN-α, IFN-β, IFN-γ, IgE, IgEFc region, IGF-1, IGF-1 receptor, IGHE, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-1β, IL-20, IL-22, IL-23, IL-31, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin α4β7, integrin β2, integrin α2, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor, interferon γ-inducible protein, interleukin 11 receptor α (IL-11Rα), interleukin 13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, kinase domain region (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), lymphocyte antigen 6 (Ly-6), Lewis Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex locus K9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-α (LT-α) or tumor necrosis factor-β (TNF-β), lysosome-associated membrane protein 1 (LAMP1), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, mammary differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma apoptosis inhibitory factor melanoma (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, cell surface-bound mucin 1 (MUC1), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, mucin CanAg, myelin-associated glycoprotein, myostatin, N-acetylglucosaminyl-transferase V (NA17), NCA-90 (granulocyte antigen), nectin-4, nerve growth factor (NGF), nerve apoptosis regulatory protease 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitor (e.g., NOGO-A, NOGO-B, NOGO-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), tumor fetal antigen (h5T4), a cancer gene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia virus cancer gene homolog 1 (Abl) (bcr-abl), anasakis, OX-40, oxLDL, p53 variant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), P-cadherin, sodium phosphate cotransporter, phosphatidylserine placenta-specific 1 (PLAC1), platelet-derived growth factor receptor α (PDGF-Rα), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), T cell recognition melanoma antigen 1 (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate cancer cells, prostain, protease serine 21 (testisin or PRSS21), proteasome (prososome, macropain) subunit beta type 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), receptor activator of nuclear factor kappa-B ligand (RANKL), receptor for advanced glycation end products (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), respiratory syncytial virus, Rh blood group D antigen, Rh factor, sarcoma translocation breakpoint, sclerostin (SOST), selectin P, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), sphingosine-1-phosphate, T cell recognition squamous cell carcinoma antigen 1, 2, and 3 (SART1, SART2, and SART3), stage-specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1, syndecan 1 (SDC1)+A314, SOX10, survivin, survivin-2B, synovial sarcoma, X breakpoint 2 (SSX2), T cell receptor, TCR gamma alternative reading frame protein (TARP), telomerase, TEM1, tenascin C, TGF-β (e.g., TGF-β1, TGF-β2, TGF-β3),Thyroid-stimulating hormone receptor (TSHR), Tissue factor pathway inhibitor (TFPI), Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), TNF-α, TRAIL-R1, TRAIL-R2, TRG, Transglutaminase 5 (TGS5), Tumor antigen CTAA16.88, Tumor endothelial marker 1 (TEM1 / CD248), Tumor endothelial marker 7-related (TEM7R), Tumor protein p53 (p53), Tumor-specific glycosylation of MUC1, Tumor-associated calcium signal transducer 2 (TROP-2), Tumor-associated glycoprotein 72 (TAG72), Tumor-associated glycoprotein 72 (TAG-72)+A327, TWEAK receptor, Tyrosinase, Tyrosinase-related protein 1 (TYRP1 or glycoprotein 75), Tyrosinase-related protein 2 (TYRP2), Uroplakin 2 (UPK2), Vascular endothelial growth factor (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF), Vascular endothelial growth factor receptor 1 (VEGFR1), Vascular endothelial growth factor receptor 2 (VEGFR2), Vimentin, v-myc myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), von Willebrand factor (VWF), Wilms tumor protein (WT1), X antigen family member 1A (XAGE1), β-Amyloid, κ-Light chain, Fibroblast growth factor receptor 2 (FGFR2), LIV-1 protein estrogen-regulated (LIV1, also known as SLC39A6), Neurotrophic tyrosine kinase receptor type 1 (NTRK1, also known as TRK), Ret proto-oncogene (RET), B cell maturation antigen (BCMA, also known as TNFRSF17), Transferrin receptor (TFRC, also known as CD71), Activated leukocyte cell adhesion molecule (ALCAM, also known as CD166), Somatostatin receptor 2 (SSTR2), KIT proto-oncogene receptor tyrosine kinase (cKIT), V-Set immunoregulatory receptor (VSIR, also known as VISTA), Glycoprotein Nmb (GPNMB), Delta-like canonical Notch ligand 3 (DLL3), Interleukin-3 receptor subunit alpha (IL3RA, also known as CD123), Lysosome-associated membrane protein 1 (LAMP1), Cadherin 3, type 1, P-cadherin (CDH3), Ephrin A4 (EFNA4)Selected from Protein Tyrosine Kinase 7 (PTK7), Solute Carrier Family 34 Member 2 (SLC34A2, also known as NaPi-2b), GCC, PLAUR Domain-Containing 3 (LYPD3, also known as LY6 or C4.4a), Cell Surface-Bound Mucin 17 (MUC17), Fms-Related Receptor Tyrosine Kinase 3 (FLT3), NKG2D Ligand (e.g., ULBP1, ULBP2, ULBP3, H60, Rae-1α, Rae-1β, Rae-1δ, Rae-1γ, MICA, MICB, hHLA-A), SLAM Family Member 7 (SLAMF7), Interleukin 13 Receptor Subunit Alpha 2 (IL13RA2), C-Type Lectin Domain Family 12 Member A (CLEC12A, also known as CLL-1), CEA Cell Adhesion Molecule 5 (CEACAM, also known as CD66e), Interleukin 3 Receptor Subunit Alpha (IL3RA), CD5 Molecule (CD5), UL16 Binding Protein 1 (ILBP1), V-Set Domain-Containing T Cell Activation Inhibitor 1 (VTCN1, also known as B7-H4), Chondroitin Sulfate Proteoglycan 4 (CSPG4), Syndecan 1 (SDC1, also known as CD138), Interleukin 1 Receptor Accessory Protein (IL1RAP), Baculovirus IAP Repeat-Containing 5 (BIRC5, also known as Survivin), CD74 Molecule (CD74), Hepatitis A Virus Cellular Receptor 1 (HAVCR1, also known as TIM1), SLIT and NTRK-Like Family Member 6 (SILTRK6), CD37 Molecule (CD37), Coagulation Factor III, Tissue Factor (CD142, also known as F3), AXL Receptor Tyrosine Kinase (AXL), Endothelin Receptor Type B (EDNRB, also known as ETBR), Cadherin 6 (CDH6), Fibroblast Growth Factor Receptor 3 (FGFR3), Carbonic Anhydrase 6 (CA6), CanAg Glycoform of MUC1, Integrin Subunit Alpha V (ITGAV), Teratocarcinoma-Derived Growth Factor 1 (TDGF1, also known as Crypto 1), SLAM Family Member 6 (SLAMF6, also known as CD352), and Notch Receptor 3 (NOTCH3).

[0032] In some embodiments, the CDRs of AF2 are selected from the CDR sequences of the sequences of SEQ ID NOs: 719-918. In certain embodiments, AF2 comprises the VL and VH of a monoclonal antibody having binding affinity for a target cell marker. In certain embodiments, the VL sequence is selected from the sequences of SEQ ID NOs: 719-818, and the VH sequence is selected from the sequences of SEQ ID NOs: 819-918.

[0033] In some embodiments, AF2 specifically binds to a target cell marker with a K of about 0.1 nM to about 100 nM as determined in an in vitro antigen-binding assay that includes the target cell marker. d In certain embodiments, the binding affinity of AF2 for the target cell marker is at least 10-fold higher, or at least 100-fold higher, or at least 1000-fold higher than the binding affinity of AF1 for CD3 as measured in an in vitro antigen-binding assay. In certain embodiments, AF2 comprises the CDRs of a monoclonal antibody having binding affinity for a target cell marker.

[0034] In certain embodiments, the polypeptides disclosed herein further comprise a second release segment (RS2), which is a substrate for cleavage by mammalian proteases. In some embodiments, RS2 is a substrate for a protease selected from legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other embodiments, RS2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a sequence selected from SEQ ID NOs: 42-660. In another embodiment, the sequence of RS1 and the sequence of RS2 are identical. In yet another embodiment, the sequence of RS1 and the sequence of RS2 are not identical. In some embodiments, RS1 and RS2 are each substrates for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence.

[0035] In some embodiments, the polypeptides disclosed herein further comprise a second extended recombinant polypeptide (XTEN2), wherein the XTEN2 has a. at least about 36 amino acids or at least about 100 amino acids, b. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), and c. it has at least 4-6 different amino acids selected from G, A, S, T, E, and P. In other embodiments, XTEN2 comprises an amino acid sequence, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence thereof comprises a non-overlapping sequence selected from at least 3 of SEQ ID NOs: 661-664. In another embodiment, XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 665-718 and 922-926. In certain embodiments, XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is listed in Table 7.

[0036] In other embodiments, the polypeptide has, from the N-terminus to the C-terminus, the following structural arrangements: 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, or XTEN1-RS1-diabody-RS2-XTEN2, where the diabody comprises the VL and VH of AF1 and AF2, AF1 specifically binds to CD3, AF2 specifically binds to a target cell marker, and XTEN1 and XTEN2 are of different amino acid lengths or sequences.

[0037] In some other embodiments, AF1 is fused to AF2 by a flexible peptide linker, where a. AF2 specifically binds to a second reference antigen other than CD3, such that the polypeptide becomes a bispecific antigen-binding fragment capable of binding to both CD3 and the second reference antigen, and b. the bispecific antigen-binding fragment exhibits high thermal stability as determined by an increase in the melting temperature (T m ) in an in vitro assay, compared to a control bispecific antigen-binding fragment comprising SEQ ID NO: 41 and AF2.

[0038] In certain embodiments, the second reference antigen is 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A kinase anchor protein 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenergic receptor β3 (ADRB3), AGS-22M6, α-folate receptor, α-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoietin-binding cell surface receptor 2 (Tie 2), anthrax toxin, AOC3 (VAP-1), B cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthracis anthrax, B cell activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), brother of the imprinting site regulator (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCCTC-binding factor (CTCF), CCL11 (eotaxin-1), CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD171, CD179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (α-chain of the IL-2 receptor), CD27, CD274, CD28, CD3, CD3 ε, CD30, CD300 molecule-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-related antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), claudin 18.2 (CLDN18.2), claudin 6 (CLDN6), Clostridium difficile, clamping factor A, CLCA2, colony stimulating factor 1 receptor (CSF1R), CSF2, CTLA-4, C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-X-C chemokine receptor type 4, cyclin B1, cytochrome P450 1B1 (CYP1B1), cyp-B, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, E. coli Shiga toxin type 1, E. coli Shiga toxin type 2, ecto ADP-ribosyltransferase 4 (ART4), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), EGF-like domain multiple 7 (EGFL7), elongation factor 2 variant (ELF2M), endotoxin, ephrin A2, ephrin B2, ephrin A receptor type 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithin, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane serine protease 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin IIβ-chain, fibroblast activation protein α (FAP), fibronectin extracellular domain-B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor α, folate receptor β, Fos-related antigen 1, frizzled receptor, fucosyl GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5 member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), glypican-3 (GPC3), GMCSF receptor α chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, heat shock protein 70-2 variant (mut hsp70-2), hemagglutinin, hepatitis A virus cell receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, hexasaccharide protein of globoH glycosphingolipid (GloboH), HGF, HHGFR, high molecular weight melanoma-associated antigen (HMW-MAA), histone complex, HIV-1, HLA-DR, HNGF, Hsp90, HST-2 (FGF6), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), human TNF, ICAM-1 (CD54), iCE, IFN-α, IFN-β, IFN-γ, IgE, IgEFc region, IGF-1, IGF-1 receptor, IGHE, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-1β, IL-20, IL-22, IL-23, IL-31, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin α4β7, integrin β2, integrin α2, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor, interferon γ-induced protein, interleukin 11 receptor α (IL-11Rα), interleukin 13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, kinase domain region (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), lymphocyte antigen 6 (Ly-6), Lewis Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex locus K9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-α (LT-α) or tumor necrosis factor-β (TNF-β), lysosome-associated membrane protein 1 (LAMP1), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, mammary differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma apoptosis inhibitory factor melanoma (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, cell surface-bound mucin 1 (MUC1), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, mucin CanAg, myelin-associated glycoprotein, myostatin, N-acetylglucosaminyl-transferase V (NA17), NCA-90 (granulocyte antigen), nectin-4, nerve growth factor (NGF), nerve apoptosis regulatory protease 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitor (e.g., NOGO-A, NOGO-B, NOGO-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), tumor fetal antigen (h5T4), a cancer gene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia virus cancer gene homolog 1 (Abl) (bcr-abl), anasarca, OX-40, oxLDL, p53 variant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), P-cadherin, sodium phosphate cotransporter, phosphatidylserine placenta-specific 1 (PLAC1), platelet-derived growth factor receptor α (PDGF-Rα), Platelet-derived growth factor receptor beta (PDGFR-β), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostein, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), T cell recognition melanoma antigen 1 (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate cancer cells, prostain, protease serine 21 (testisin or PRSS21), proteasome (prososome, macropain) subunit beta type 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), receptor activator of nuclear factor kappa-B ligand (RANKL), receptor for advanced glycation end products (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), respiratory syncytial virus, Rh blood group D antigen, Rh factor, sarcoma translocation breakpoint, sclerostin (SOST), selectin P, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), sphingosine-1-phosphate, T cell recognition squamous cell carcinoma antigen 1, 2, and 3 (SART1, SART2, and SART3), stage-specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1, syndecan 1 (SDC1)+A314, SOX10, survivin, survivin-2B, synovial sarcoma, X breakpoint 2 (SSX2), T cell receptor, TCR gamma alternative reading frame protein (TARP), telomerase, TEM1, tenascin C, TGF-β (e.g., TGF-β1, TGF-β2, TGF-β3), thyroid-stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), TNF-α, TRAIL-R1, TRAIL-R2, TRG, transglutaminase 5 (TGS5),Tumor antigen CTAA16.88, Tumor endothelial marker 1 (TEM1 / CD248), Tumor endothelial marker 7 related (TEM7R), Tumor protein p53 (p53), Tumor-specific glycosylation of MUC1, Tumor-associated calcium signal transducer 2 (TROP-2), Tumor-associated glycoprotein 72 (TAG72), Tumor-associated glycoprotein 72 (TAG-72)+A327, TWEAK receptor, Tyrosinase, Tyrosinase-related protein 1 (TYRP1 or glycoprotein 75), Tyrosinase-related protein 2 (TYRP2), Uroplakin 2 (UPK2), Vascular endothelial growth factor (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF), Vascular endothelial growth factor receptor 1 (VEGFR1), Vascular endothelial growth factor receptor 2 (VEGFR2), Vimentin, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), von Willebrand factor (VWF), Wilms tumor protein (WT1), X antigen family member 1A (XAGE1), β-amyloid, κ-light chain, Fibroblast growth factor receptor 2 (FGFR2), LIV-1 protein estrogen-regulated (LIV1, also known as SLC39A6), Neurotrophic tyrosine kinase receptor type 1 (NTRK1, also known as TRK), Ret proto-oncogene (RET), B-cell maturation antigen (BCMA, also known as TNFRSF17), Transferrin receptor (TFRC, also known as CD71), Activated leukocyte cell adhesion molecule (ALCAM, also known as CD166), Somatostatin receptor 2 (SSTR2), KIT proto-oncogene receptor tyrosine kinase (cKIT), V-Set immunoregulatory receptor (VSIR, also known as VISTA), Glycoprotein Nmb (GPNMB), Delta-like canonical Notch ligand 3 (DLL3), Interleukin-3 receptor subunit alpha (IL3RA, also known as CD123), Lysosome-associated membrane protein 1 (LAMP1), Cadherin 3, type 1, P-cadherin (CDH3), Ephrin A4 (EFNA4), Protein tyrosine kinase 7 (PTK7), Solute carrier family 34 member 2 (SLC34A2, also known as NaPi-2b), GCC, PLAUR domain-containing 3 (LYPD3, also known as LY6 or C4.4a), Cell surface-bound mucin 17 (MUC17), Fms-related receptor tyrosine kinase 3 (FLT3), NKG2D ligand (e.g.,Target cell markers selected from ULBP1, ULBP2, ULBP3, H60, Rae-1α, Rae-1β, Rae-1δ, Rae-1γ, MICA, MICB, hHLA-A), SLAM family member 7 (SLAMF7), interleukin 13 receptor subunit alpha 2 (IL13RA2), C-type lectin domain family 12 member A (CLEC12A, also known as CLL-1), CEA cell adhesion molecule 5 (CEACAM, also known as CD66e), interleukin 3 receptor subunit alpha (IL3RA), CD5 molecule (CD5), UL16 binding protein 1 (ILBP1), V-Set domain-containing T cell activation inhibitor 1 (VTCN1, also known as B7-H4), chondroitin sulfate proteoglycan 4 (CSPG4), syndecan 1 (SDC1, also known as CD138), interleukin 1 receptor accessory protein (IL1RAP), baculovirus IAP repeat-containing 5 (BIRC5, also known as survivin), CD74 molecule (CD74), hepatitis A virus cellular receptor 1 (HAVCR1, also known as TIM1), SLIT and NTRK-like family member 6 (SILTRK6), CD37 molecule (CD37), coagulation factor III, tissue factor (CD142, also known as F3), AXL receptor tyrosine kinase (AXL), endothelin receptor type B (EDNRB, also known as ETBR), cadherin 6 (CDH6), fibroblast growth factor receptor 3 (FGFR3), carbonic anhydrase 6 (CA6), the CanAg glycoform of MUC1, integrin subunit alpha V (ITGAV), teratocarcinoma-derived growth factor 1 (TDGF1, also known as Crypto 1), SLAM family member 6 (SLAMF6, also known as CD352), and Notch receptor 3 (NOTCH3).

[0039] In some embodiments, (1) the AF2 fragment disclosed herein is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibodies, single-domain antibodies, and single-chain variable fragments (scFv), or (2) the AF1 and AF2 disclosed herein are configured as (Fab’)2 or single-chain diabodies.

[0040] In certain embodiments, the binding affinity of AF2 for the target cell marker is at least 10-fold higher, or at least 100-fold higher, or at least 1000-fold higher than the binding affinity of AF1 for CD3 as measured in an in vitro antigen-binding assay.

[0041] In some embodiments, AF1 and AF2 each exhibit an isoelectric point (pI) of 6.6 or less. In another embodiment, AF1 and AF2 each exhibit a pI in the range of 5.5 to 6.6 (including the boundary values). In certain embodiments, the pI of AF1 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 AF2.

[0042] In yet another aspect, a polypeptide comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a light-chain complementarity-determining region (CDR-L) and a heavy-chain complementarity-determining region (CDR-H), and the antigen-binding fragment specifically binds to the epsilon subunit of CD3 and comprises a VH amino acid sequence comprising SEQ ID NO: 920, is disclosed herein. In some embodiments, the antigen-binding fragment comprises a VL amino acid sequence comprising SEQ ID NO: 919. In certain embodiments, the antigen-binding fragment consists of SEQ ID NO: 921.

[0043] In one aspect, a pharmaceutical composition comprising a polypeptide disclosed herein and one or more pharmaceutically acceptable excipients is disclosed herein. In some embodiments, the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intracavitary, intrathecal, or intramuscular administration. In another embodiment, the pharmaceutical composition is in liquid form. In another embodiment, the pharmaceutical composition is in a prefilled syringe for single injection. In yet another embodiment, the pharmaceutical composition is formulated as a lyophilized powder that is reconstituted prior to administration.

[0044] In another aspect, the use of the polypeptides disclosed herein in the preparation of a medicament for treating a disease in a subject is disclosed herein. In some embodiments, the disease is selected from the group consisting of carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, diffuse large 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, vaginal cancer, vulvar cancer, Ewing sarcoma, peritoneal carcinomatosis, uterine serous cancer, parathyroid cancer, endometrial cancer, cervical cancer, colorectal cancer, epithelial intraperitoneal malignancy with malignant ascites, uterine cancer, peritoneal mesothelioma, kidney cancer, lung cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular carcinoma, retinoblastoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, testicular cancer, cholangiocarcinoma, bone cancer, salivary gland cancer, thyroid cancer, craniopharyngioma, carcinoid tumor, epithelial cancer, masculinizing tumor, adenocarcinoma, sarcoma of any origin, primary hematological malignancies (including acute or chronic lymphocytic leukemia, acute or chronic myelogenous leukemia, B-cell derived chronic lymphocytic leukemia, hairy cell leukemia, myeloproliferative neoplasm disorders, or myelodysplastic disorders), myasthenia gravis, Graves' disease, Kaposi sarcoma, neuroblastoma, Hashimoto's thyroiditis, Wilms tumor, or Goodpasture syndrome.

[0045] In yet another aspect, a method of treating a disease in a subject, the method comprising administering to a subject in need thereof one or more therapeutically effective doses of the pharmaceutical composition disclosed herein, is disclosed herein. In certain embodiments, the subject is selected from the group consisting of mice, rats, monkeys, and humans. In some embodiments, the disease is selected from the group consisting of carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, urogenital cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous cancer, endometrial cancer, cervical cancer, colorectal cancer, epithelial intraperitoneal malignancy with malignant ascites, uterine cancer, peritoneal mesothelioma, kidney cancer, lung cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular cancer, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, salivary gland cancer, thyroid cancer, epithelial cancer, adenocarcinoma, sarcoma of any origin, primary hematological malignancies (including acute or chronic lymphocytic leukemia, acute or chronic myelogenous leukemia, myeloproliferative neoplasm disorders, or myelodysplastic disorders), myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpasture's syndrome.

[0046] In other embodiments, the pharmaceutical composition is administered to the subject as one or more therapeutically effective doses administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, or once a month. 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, intraarterially, intraperitoneally, intra-abdominally, intrathecally, or intramuscularly.

[0047] In one aspect, an isolated nucleic acid is disclosed herein, which comprises (a) a polynucleotide encoding a polypeptide disclosed herein, or (b) a complement of the polynucleotide of (a).

[0048] In related aspects, an expression vector is disclosed herein that comprises a polynucleotide sequence disclosed herein and a recombinant regulatory sequence operably linked to the polynucleotide sequence.

[0049] In another aspect, an isolated host cell is disclosed herein that comprises an expression vector disclosed herein. In some embodiments, the host cell is a prokaryote. In one embodiment, the host cell is E. coli. Incorporation by reference

[0050] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0051] The various features of the present disclosure are described in detail in the appended claims. A further understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description of the invention and the accompanying drawings, which illustrate exemplary embodiments in which the principles of the invention are utilized.

Brief Description of the Drawings

[0052]

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[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 by way of example only. Without departing from the present invention, those skilled in the art will envision 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 be covered thereby.

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

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

[0056] As used throughout this specification and the claims, the terms "a," "an," and "the" are used in the sense of meaning "at least one," "at least a first," "one or more," or "a plurality" of the referenced element or step, unless the upper limit is specifically recited thereafter. Thus, "emission segment," as used herein, means "at least a first emission segment," but includes a plurality of emission segments. As with the amount of any single agent, the operable limits and parameters of the combination will be known to those skilled in the art in light of the present disclosure.

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

[0058] The term "monomer" as applied to a polypeptide refers to the state of a polypeptide that is a single continuous amino acid sequence 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 non-natural or synthetic amino acid, including but not limited to both D and L optical isomers, as well as amino acid analogs and peptidomimetics. Amino acids can be designated using the standard one-letter or three-letter codes.

[0060] The term "natural L-amino acid" or "L-amino acid" means the L-enantiomeric 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, so long as they exhibit the desired antigen-binding or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein. Full-length antibodies can be, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies. Antibodies represent a large family of molecules that include several types of molecules such as IgD, IgG, IgA, IgM, and IgE. The term "immunoglobulin molecule" includes, for example, hybrid antibodies, or modified antibodies, and fragments thereof. It has been shown that the antigen-binding function of an antibody can be carried out by fragments of a native or monoclonal antibody.

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

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

[0064] As used herein, the term "antigen-binding fragment" refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that specifically binds to (and "immunoreacts" 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 camelid antibodies, single-chain fragment variable (scFv) antibody molecules, and multispecific antibodies formed from antibody fragments that retain the ability to specifically bind to an antigen. The term "antigen-binding fragment" also encompasses any polypeptide chain-containing molecular structure having a specific shape that fits, recognizes, and binds to an epitope, in which one or more non-covalent interaction(s) stabilize the complex between the molecular structure and the epitope. An antigen-binding fragment "specifically binds to" an antigen or is "immunoreactive" with an antigen if it binds with greater affinity or binding activity than it binds to other reference antigens, including polypeptides or other substances.

[0065] "scFv" or "single-chain fragment variable" are used interchangeably herein and refer to an antibody fragment format that contains the variable regions of the heavy (VH) and light (VL) chains of an antibody, or two copies of the VH or VL chain, linked together by a short flexible polypeptide linker, which enables the scFv to form a structure desirable for antigen binding. An 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 (about 5-10 residues) between the VH and VL domains, thereby achieving inter-chain pairing rather than intra-chain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. A bispecific diabody is a heterodimer of two "crossover" scFv fragments where the VH and VL domains of two antibodies are present on different polypeptide chains. Diabodies are more fully described, for example, in US7635475.

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

[0068] The terms "antigen", "target antigen", and "immunogen" are used interchangeably herein and refer to a structure or binding determinant to which an antibody, antibody fragment, or antibody fragment-based molecule binds or has specificity. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound, or a portion thereof. An antigen is also a ligand for an antibody or antibody fragment having binding affinity for the antigen. Non-limiting exemplary antigens include CD3, HER2, EGFR, and EpCAM (and portions thereof) from human, non-human primate, mouse, and other homologs thereof.

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

[0070] The "target cell marker" refers to a molecule expressed by target cells that may function as a ligand for an antibody and includes, but is not limited to, cell surface receptors, antigens, glycoproteins, oligonucleotides, enzyme substrates, antigenic determinants, or binding sites that may be present on or within the target tissue or cell.

[0071] The "target tissue" or "target cell" refers to a tissue or cell that is the cause of, or a part of, a disease state, such as cancer or an inflammatory condition, but is not limited to these. The diseased target tissue or cell source includes body organs, tumors, cancerous cells or cancerous cell populations, or cells that form a matrix or are found in association with cancerous cell populations, bone, skin, and cells that produce cytokines or factors contributing to the disease state.

[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 total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., between an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K d ). As used herein, "greater binding affinity" means a lower K d value. For example, 1 × 10 -9 M has a higher binding affinity than 1 × 10 -8 M. An antibody that binds to a desired antigen, such as a tumor-associated target cell antigen, 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 " are used interchangeably and refer to the affinity between a ligand "L" and a protein "P", i.e., how strongly the ligand binds to a particular protein. This can be calculated using the equation K d = [L][P] / [LP], where [P], [L], and [LP] represent the molar concentrations of the protein, ligand, and complex, respectively.

[0075] The terms "hypervariable region", "HVR", or "CDR", as used herein, refer interchangeably to regions of the antibody variable domain that are hypervariable in sequence and / or form structurally defined loops and / or are involved in antigen recognition. Generally, an antibody contains six hypervariable regions, three (H1, H2, H3) in VH and three (L1, L2, L3) in VL. Several CDR descriptions are used and included herein, e.g., 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, and so on. The 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] "Framework" or "FR" residues are variable domain residues in the antigen-binding fragment other than the hypervariable region residues as defined herein, and generally lie between or adjacent to the CDRs. Several FR descriptions are used and included herein, e.g., FR-L1 refers to the first FR region of the light chain, and FR-H2 refers to the second FR region of the heavy chain, and so on.

[0077] "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 the statistical mean. The official name for representing the isoelectric point is pH, and thus the unit is in pH units. For example, an antigen-binding fragment having a pI of 6.3 will have a neutral charge in a solution at pH 6.3. The isoelectric point can be determined mathematically and includes several algorithms for estimating the isoelectric points of peptides and proteins, such as the Henderson-Hasselbalch equation with different pK values. The isoelectric point can also be determined experimentally by in vitro assays such as capillary isoelectric focusing electrophoresis.

[0078] The term "release segment" or "RS" refers to one or more sites within a sequence that can be recognized and cleaved by one or more proteases, and to peptides in a composition that affect the release of antigen-binding fragments and XTEN from the subject composition. As used herein, "mammalian protease" means a protease that is normally present in body fluids, cells, and tissues and can be found at higher levels in certain target tissues or cells of a mammal, such as diseased tissue (e.g., tumor). The RS sequence can be engineered to be cleaved by various mammalian proteases or multiple mammalian proteases that are present in or near the target tissue in a subject or introduced in an in vitro assay. Other equivalent proteases (endogenous or exogenous) that can recognize defined cleavage sites can be utilized. It is particularly contemplated that the RS sequence can be tailored and individualized to the protease utilized and can incorporate linker amino acids to bind to adjacent polypeptides.

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

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

[0081] As used herein, "active" as applied to the forms of the compositions provided herein refers to an action or effect that includes, but is not limited to, antigen binding, antagonist activity, agonist activity, cell or physiological response, cytolysis, cell death, or an effect generally known in the art for effector components of the composition, whether measured by in vitro assays, ex vivo assays, or in vivo assays, or by clinical effects.

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

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

[0084] As used herein, "CD3" or "cluster of differentiation 3" means the T cell surface antigen CD3 complex, including all known CD3 subunits, e.g., CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta, in individual forms or in independently combined forms. The extracellular domains of CD3 epsilon, gamma, and delta contain immunoglobulin-like domains and are thus considered part of the immunoglobulin superfamily. CD3 includes, for example, the 207 amino acid-long human CD3 epsilon protein (NCBI reference sequence number NP_000724) and the 182 amino acid-long human CD3 gamma protein (NCBI reference sequence number NP_000064).

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

[0086] "Host cell" includes any individual cell or cell culture that can serve as or is a recipient of a target vector into which an exogenous nucleic acid, such as those described herein, has been introduced. Host cells include the progeny of a single host cell. The progeny need not be identical to the original parent cell (either in terms of the form of the total DNA complement or in terms of the genome) due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo using the vectors of the present invention.

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

[0088] "Isolated nucleic acid" refers to a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the natural source of the nucleic acid encoding the polypeptide. For example, a nucleic acid molecule encoding an isolated polypeptide is other than in the form or context in which it is found in nature. Thus, a nucleic acid molecule encoding an isolated polypeptide is distinguished from a nucleic acid molecule encoding a particular polypeptide because it is present in a natural cell. However, a nucleic acid molecule encoding an isolated polypeptide includes, for example, a nucleic acid molecule encoding a polypeptide that is included in a cell that normally expresses the polypeptide and is at a chromosomal location or extrachromosomal location different from the location in the natural cell.

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

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

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

[0092] "Operably linked" means that the DNA sequences being linked are in the reading phase or in frame. "In-frame fusion" refers to joining two or more open reading frames (ORFs) in a manner that maintains the reading frame of the original ORF to form a continuous longer ORF. For example, a promoter or enhancer is operably 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 that contains two or more segments corresponding to the polypeptides encoded by the original ORFs (these segments are not normally joined in that way in nature).

[0093] In the context of a polypeptide, "linear sequence" or "sequence" refers to the order of amino acids in a polypeptide in the direction from the amino terminus to the carboxyl terminus (N-terminus to C-terminus), and the residues adjacent to each other within that sequence are continuous in the primary structure of the polypeptide. A "subsequence" is a linear sequence of a part of a polypeptide that is known to contain additional residues in one or both directions.

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

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

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

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

[0098] The terms "gene" and "gene fragment" are used interchangeably herein. These refer to polynucleotides that contain at least one open reading frame capable of encoding a specific protein after transcription and translation. A gene or gene fragment can be genomic or cDNA as long as the polynucleotide contains at least one open reading frame that can encompass the entire coding region or a segment thereof. A "fusion gene" is a gene composed of at least two heterologous polynucleotides linked together.

[0099] As used herein, a "coding region" or "coding sequence" is a portion of a polynucleotide consisting of codons that can be translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, but it can be considered part of the coding region. However, any adjacent sequences such as, for example, a promoter, ribosome binding site, transcription terminator, intron, etc. are not part of the coding region. The boundaries of the coding region are typically determined by the start codon at the 5' end that encodes the amino terminus of the resulting polypeptide and the translation stop codon at the 3' end that encodes the carboxyl terminus of the resulting polypeptide. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. A single vector can then contain only a single coding region or can contain two or more coding regions. For example, a single vector can encode binding domain-A and binding domain-B separately as described below. In addition, a vector, polynucleotide, or nucleic acid of the present invention can encode a heterologous coding region, either fused or not fused to a nucleic acid encoding a binding domain of the present invention. Heterologous coding regions include, but are not limited to, special elements or motifs such as a secretion signal peptide or a heterologous functional domain.

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

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

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

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

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

[0105] The terms "percent identity", "sequence identity percentage", and "identity %" as applied to polynucleotide sequences refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such algorithms insert gaps in the sequences being compared in a standardized, reproducible way in order to optimize the alignment between the two sequences, and thus can achieve a more meaningful comparison of the two sequences. The percent identity can be measured over the length of the defined polynucleotide sequence as a whole, or over a shorter length, for example, over the length of a fragment taken from a larger defined polynucleotide sequence, such as a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210, or at least 450 contiguous residues. Such lengths are merely exemplary, and it is understood that any fragment length supported by the sequences shown herein in a table, figure, or sequence listing can be used to describe the length over which the percent identity can be measured. The sequence identity percentage is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which identical residues occur in both polypeptide sequences, dividing the number of positions at which there is identity by the total number of positions in 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 the polypeptide sequences specified in this specification, the terms "percent identity", "sequence identity percentage", and "identity %" refer to the percentage of amino acid residues in a query sequence that are identical to the amino acid residues in a second reference polypeptide sequence of equal length or a portion thereof, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage, and without considering any conservative substitutions as part of the sequence identity. The alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve an optimal alignment over the entire length of the sequences being compared. The percent identity can be measured over the length of the entire defined polypeptide sequence or over a shorter length, for example, a fragment taken from a larger defined polypeptide sequence, such as a fragment of at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. Such lengths are merely illustrative, and it is understood that any fragment length supported by the sequences shown in this specification in a table, figure, or sequence listing can be used to describe the length over which the percent identity can be measured.

[0107] "Repeatability", as used in connection with a polynucleotide sequence, refers to the degree of internal homology in a sequence, such as, for example, the frequency of the same nucleotide sequence of a given length. Repeatability can be measured, for example, by analyzing the frequency of the same sequence.

[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, transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or other RNA products, and translation of the mRNA into polypeptide. An "expression" results in the production of a "gene product". As used herein, a gene product can be either a nucleic acid, such as messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products 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] The terms "vector" or "expression vector" are used interchangeably and preferably refer to a nucleic acid molecule that can self-replicate in a suitable host and transfer an inserted nucleic acid molecule into and / or between host cells. This term includes vectors that function primarily for insertion of DNA or RNA into cells, replication of vectors that function primarily for replication of DNA or RNA, and expression vectors that function for 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 can be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" generally implies a suitable host cell containing an expression vector that can function to produce a desired expression product.

[0110] "Serum stability" as applied to a polypeptide typically refers to the ability of the polypeptide to resist degradation in blood or its components, involving proteases in serum or plasma. Serum stability can typically be measured by combining the protein with human (or mouse, rat, dog, monkey if necessary) serum or plasma at about 37°C for a typical range of days (e.g., 0.25, 0.5, 1, 2, 4, 8, 16 days). Samples at these time points can be electrophoresed in a Western blot assay, and the protein is detected using an antibody. The antibody can be against a tag in the protein. If the protein shows a single band on the Western (where the size of the protein is the same as the size of the injected protein), no degradation has occurred. In this exemplary method, the time point at which 50% of the protein, as determined by Western blot or equivalent techniques, is degraded is the serum half-life or "serum stability" of the protein.

[0111] "t 1 / 2 ", "half-life", "terminal half-life", "elimination half-life", and "circulating half-life" are used interchangeably herein and, when used herein, mean the terminal half-life calculated as ln(2) / K el where K el is the terminal phase elimination rate constant calculated by linear regression of the log concentration on the time curve for the terminal linear portion. Half-life typically refers to the time required for half of the amount of an administered substance deposited in a living organism to be metabolized or removed by normal biological processes. 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 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. The molecular weight can be theoretically determined by summing the atomic masses of the constituent atoms in the molecule. When applied in the context of polypeptides, the molecular weight is calculated by adding the molecular weights of each type of amino acid in the composition based on the amino acid composition, or by estimation from comparison with molecular weight standards in an SDS electrophoresis gel. The calculated molecular weight of a molecule may differ from the "apparent molecular weight" of the molecule, which generally refers to the molecular weight of the molecule determined by one or more analytical techniques. "Apparent molecular weight coefficient" and "apparent molecular weight" are related terms, and when used in the context of polypeptides, these terms refer to a measure of the relative increase or decrease in the apparent molecular weight presented by a particular amino acid or polypeptide sequence. The apparent molecular weight can be determined, for example, by comparison with globular protein standards measured in "apparent kD" units using size exclusion chromatography (SEC) or similar methods. The apparent molecular weight coefficient is the ratio between the apparent molecular weight and the "molecular weight", the latter being calculated by addition based on the amino acid composition as described above, or by estimation from comparison with molecular weight standards in an SDS electrophoresis gel. The determination of the apparent molecular weight and the apparent molecular weight coefficient is described in U.S. Patent No. 8,673,860.

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

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

[0115] As used herein, "treatment" or "treating", or "alleviating", or "ameliorating" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including, but not limited to, therapeutic and / or prophylactic benefits. Therapeutic benefit means eradication or amelioration of the underlying disease being treated. Also, therapeutic benefit can be achieved by eradication or amelioration of one or more of the physiological symptoms or improvement of one or more clinical parameters associated with the underlying disease, even though the subject may still be susceptible to the underlying disease and improvement is observed in the subject. In the case of prophylactic benefit, the composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the 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, reduction, amelioration, or prevention of a disease, or improvement of one or more clinical parameters associated with the underlying disease in a subject, or alternatively enhancement of the physical or mental health of the subject resulting from administration of the polypeptide of the invention other than the ability to induce production of an antibody against an antigenic epitope of a biologically active protein. In the case of prophylactic benefit, the composition can be administered to a subject at risk of developing a particular disease, a recurrence of a previous disease, or a condition or symptom of that disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease has not been diagnosed.

[0117] As used herein, the terms "therapeutically effective amount" and "therapeutically effective dosage" refer to an amount of a drug or biologically active protein, either alone or as part of a composition, that when administered to a subject, either singly or repeatedly, can produce some detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of a medical condition or state. Such an effect need not be absolute in order to be beneficial. Determination of a therapeutically effective amount is well within the ability of one of ordinary skill in the art, particularly in light of the detailed disclosure provided herein.

[0118] As used herein, the term "therapeutically effective non-toxic dosage" refers to a tolerable dosage of a composition as defined herein that is high enough to cause depletion of tumors or cancer cells, elimination of tumors, shrinkage of tumors, or stabilization of a disease in a subject without causing or essentially without causing a major toxic effect. Such a therapeutically effective non-toxic dosage may be determined by dose escalation studies as described in the art and should be below the dosage 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 becomes toxic to the concentration at which the drug is effective. One exemplary ratio for the therapeutic index is LD 50 :ED 50 where LD 50 is the dosage that results in a 50% mortality rate in a population of subjects and ED 50 is the dosage that results in effectiveness in a population of subjects.

[0120] As used herein, the term "dosage regimen" refers to a schedule of multiple (i.e., at least two or more) consecutive dosages of a composition, where the dosages are administered at a therapeutically effective amount and produce a sustained beneficial effect on any symptom, aspect, measured parameter, endpoint, or characteristic of a medical condition or state in a subject.

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

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

[0123] The terms "cancer" and "cancerous" typically refer to or describe a physiological state in a mammal characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, tumors, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, urogenital cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous cancer, endometrial cancer, cervical cancer, colorectal cancer, epithelial intraperitoneal malignancy with malignant ascites, uterine cancer, peritoneal mesothelioma, kidney cancer, lung cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular cancer, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, salivary gland cancer, thyroid cancer, epithelial cancer, adenocarcinoma, sarcoma of any origin, primary hematological malignancies (including acute or chronic lymphocytic leukemia, acute or chronic myelogenous leukemia, myeloproliferative neoplasm disorders, or myelodysplastic disorders), myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpasture's syndrome.

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

[0125] As used herein, the term "tumor-specific marker" refers to an antigen present on or in cancer cells that can be found more often on or in cancer cells as compared to normal cells or normal tissues, but not necessarily so.

[0126] I). General Techniques The practice of the present invention employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art, unless otherwise indicated. Sambrook J et al., “Molecular Cloning: A Laboratory Manual,” 3 rd edition, Cold Spring Harbor Laboratory Press, 2001, “Current protocols in molecular biology”, F.M. Ausubel, et al. eds., 1987, the series “Methods in Enzymology,” Academic Press, San Diego, CA., “PCR 2: a practical approach”, M.J. MacPherson, B.D. Hames and G.R. Taylor 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,” 11 thEdition, McGraw-Hill, 2005, and Freshney, R.I., “Culture of Animal Cells: A Manual of Basic Technique,” 4 th edition, John Wiley & Sons, Somerset, NJ, 2000 are hereby incorporated by reference in their entirety for all purposes, and these contents are hereby incorporated by reference in their entirety into this specification.

[0127] Host cells can be cultured in various media. Commercially available media such as Ham's F10 (Sigma), Minimal 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 grow in defined media that lack serum but are supplemented with hormones, growth factors, or any other factors necessary for the survival and / or growth of a particular cell type. Defined media that support cell survival maintain viability, morphology, metabolic capacity, and potentially the differentiation capacity of the cells, while defined media that promote cell growth provide all the chemicals necessary for cell proliferation or multiplication. 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 are, for example, pH, pO2, temperature, and osmolarity. The nutritional requirements of the cells are usually provided in standard media formulations developed to provide an optimal environment. Nutrients can be divided into several categories: amino acids and their derivatives, carbohydrates, sugars, fatty acids, complex lipids, nucleic acid derivatives, and vitamins. Apart from nutrients for maintaining cell metabolism, most cells may require one or more hormones derived from at least one of the following groups: steroids, prostaglandins, growth factors, pituitary hormones, and peptide hormones for growth in serum-free media (Sato, G.H., et al. in “Growth of Cells in Hormonally Defined Media”, Cold Spring Harbor Press, N.Y., 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 survival and growth in vitro. The optimal set of hormones or transport proteins varies for each cell type. Most of these hormones or transport proteins are added exogenously or, in rare cases, mutant cell lines that do not require specific factors have been found.One of ordinary skill in the art will know other factors necessary to maintain cell culture without undue experimentation.

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

[0129] II). CD3 Antigen-Binding Fragment Composition In a first aspect, the present disclosure provides a polypeptide comprising an antigen-binding fragment (AF1) having specific binding affinity for an effector cell antigen expressed on the surface of an effector cell selected from plasmablasts, 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. In one embodiment, the antigen-binding fragment has binding affinity for an effector cell antigen expressed on the surface of a T cell. In another embodiment, the present disclosure provides a polypeptide comprising an antigen-binding fragment having binding affinity for CD3. In another embodiment, the antigen-binding fragment has binding affinity for members of the CD3 complex, including all known CD3 subunits of the CD3 complex, such as CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta, in individual forms or in a combined form independently.

[0130] An antigen-binding fragment that binds to the CD3 antigen is particularly useful for pairing with a second antigen-binding fragment (AF2) that has binding affinity for a target cell marker or an antigen of an affected cell or tissue in a composition format that results in cell death of the affected cell or tissue. Binding specificity can be determined by complementarity-determining regions, or CDRs such as light-chain CDRs or heavy-chain CDRs. In many cases, binding specificity is determined by both light-chain CDRs and heavy-chain CDRs. A given combination of heavy-chain CDRs and light-chain CDRs provides a given binding pocket that confers higher affinity and / or specificity for CD3 as compared to other reference antigens.

[0131] The antigen-binding fragments contemplated by the present disclosure can be derived from naturally occurring antibodies or fragments thereof, non-naturally occurring antibodies or fragments thereof, humanized antibodies or fragments thereof, synthetic antibodies or fragments thereof, hybrid antibodies or fragments thereof, or engineered antibodies or fragments thereof. Methods for generating antibodies against a given target marker are well known in the art. For example, monoclonal antibodies may be produced using the hybridoma method described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (U.S. Patent No. 4,816,567). The structures of antibodies and fragments thereof, the variable regions of the heavy and light chains of antibodies (VH and VL), single-chain variable regions (scFv), complementarity-determining regions (CDR), and domain antibodies (dAb) are well understood. Methods for generating polypeptides having a desired antigen-binding fragment of a target cell marker are known in the art.

[0132] Certain CD3-binding antigen-binding fragments of the present disclosure are specifically modified to enhance their stability in the polypeptide embodiments described herein as compared to CD3 antibodies and antigen-binding fragments known in the art. Protein aggregation of monoclonal antibodies and other antibodies remains a significant problem in their developability and is still a major area of focus in antibody production. Antibody aggregation is induced by partial unfolding of its domains and can lead to nucleation and aggregate growth following monomer-monomer association. The tendency of antibodies and antibody-based proteins to aggregate can be affected by external experimental conditions, but they strongly depend on the intrinsic antibody properties determined by their sequences and structures. It is well known that proteins are slightly stable in their folded state, but in many cases, most proteins are inherently prone to aggregation in their unfolded or partially unfolded states, and there is a poorly understood possible isomer in which the resulting aggregates are extremely stable and long-lived. It has also been shown that a decrease in the tendency to aggregate is accompanied by an increase in the expression titer, indicating that a decrease in protein aggregation is beneficial throughout the development process and may lead to a more efficient path to clinical trials. In the case of therapeutic proteins, aggregates are a significant risk factor for adverse immune reactions in patients and can occur via various mechanisms. By controlling aggregation, the stability, manufacturability, attrition rate, safety, formulation, titer, immunogenicity, and solubility of the protein can be improved. Intrinsic properties of proteins such as size, hydrophobicity, electrostatics, and charge distribution play important roles in the solubility of proteins. It has been shown that when the solubility of a therapeutic protein is low due to surface hydrophobicity, formulation development becomes more difficult and can lead to poor in vivo distribution, undesirable pharmacokinetic behavior, and immunogenicity in vivo. Reducing the overall surface hydrophobicity of a candidate monoclonal antibody can also provide benefits and cost savings related to purification and dosing regimens. Individual amino acids can be identified as contributing to the aggregation ability of an antibody by structural analysis and can be located in both the CDRs and the framework regions.In particular, residues can be predicted to have a high risk of causing hydrophobic problems for a given antibody. In one embodiment, the present disclosure provides an AF1 that has the ability to specifically bind to CD3, where the AF1 has at least one amino acid substitution of a hydrophobic amino acid within the framework region, for a parent antibody or antibody fragment, and the hydrophobic amino acid is selected from isoleucine, leucine, or methionine. In another embodiment, the CD3 AF1 has at least two amino acid substitutions of hydrophobic amino acids within one or more framework regions, and the hydrophobic amino acids are selected from isoleucine, leucine, or methionine.

[0133] In relation to the antigen-binding fragment, the isoelectric point (pI) is the pH at which the antibody fragment has no net charge. When the pH is below the pI of the antibody fragment, it has a net positive charge. The greater the positive charge, the more likely it is to correlate with increased blood clearance and tissue retention, and generally has a shorter half-life. When the pH is above the pI of the antibody fragment, it has a negative charge. Negative charges generally result in decreased tissue uptake and a longer half-life. It is possible to manipulate this charge on the framework residues via mutations. These considerations provided information for the design of the sequences of the antigen-binding fragments of the embodiments described herein, where individual amino acid substitutions were made relative to the parental antibody 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 charges of specific amino acids in the protein sequence. Estimates of charge, called acid dissociation constants or pKa values, are used to calculate the pI. The pI can be determined in vitro by methods such as capillary isoelectric focusing (see Datta-Mannan, A., et al. The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies. mAbs 7:1084 (2015), Li, B., et al. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge. mAbs 6, 1255-1264 (2014)) or other methods known in the art

[0134] In some aspects of any 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), and AF1 specifically binds to a cluster of differentiation 3 (CD3) T cell receptor, which may include (a) all known CD3 subunits, such as CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta, in individual forms or in combination independently. In one embodiment, the antigen-binding fragment according to any of the target composition embodiments described herein is a chimeric or humanized antigen-binding fragment. In another embodiment, the antigen-binding fragment according to any of the target composition embodiments described herein is selected from the group consisting of Fv, Fab, Fab’, Fab’-SH, linear antibody, and single-chain variable fragment (scFv). The antigen-binding fragment having CDR-H and CDR-L may be constructed in the (CDR-H)-(CDR-L) or (CDR-L)-(CDR-H) orientation from the N-terminus to the C-terminus.

[0135] In one embodiment, the present disclosure provides a polypeptide comprising an AF1 comprising CDR-L and CDR-H, wherein AF1 specifically binds to a cluster of differentiation 3 (CD3) T cell receptor and (b) comprises a CDR-H3 having the amino acid sequence of SEQ ID NO: 10. In some embodiments of the present disclosure, AF1 comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO: 8, 9, and 10, respectively. In another embodiment, a polypeptide according to any of the target composition embodiments described herein can comprise AF1, wherein AF1 comprises CDR-L and CDR-H, and AF1 (a) specifically binds to CD3, (b) comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10, and (c) comprises heavy chain framework regions (FR-H) FR-H1, FR-H2, FR-H3, FR-H4, each of 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 SEQ ID NOs: 22, 23, 25, and 26, respectively. In another embodiment, the present disclosure provides a polypeptide comprising AF1, wherein AF1 comprises CDR-L and CDR-H, AF1 (a) specifically binds to CD3, (b) comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10, and (c) comprises heavy chain framework regions (FR-H) FR-H1, FR-H2, FR-H3, FR-H4, each of 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 SEQ ID NOs: 22, 23, 25, and 26, respectively, and further comprises light chain framework regions (FR-L) FR-L1, FR-L2, FR-L3, FR-L4, each of 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 SEQ ID NOs: 12, 13, 18, and 19, respectively.

[0136] In another embodiment, the polypeptide of the subject composition embodiments described herein comprises AF1, which comprises CDR-L and CDR-H, and AF1 (a) specifically binds to CD3, (b) comprises CDR-H1, CDR-H2, and CDR-H3, and CDR-H1, CDR-H2, and CDR-H3 each comprise the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively. In another embodiment as described above, the polypeptide comprising AF1 further comprises (a) CDR-L1 having the amino acid sequence of SEQ ID NO: 1 or 2, (b) CDR-L2 having the amino acid sequence of SEQ ID NO: 4 or 5, and (c) CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In yet another embodiment, the polypeptide described in any of the subject composition embodiments described herein can comprise AF1 comprising CDR-L and CDR-H, and AF1 (a) specifically binds to CD3, (b) comprises CDR-H1, CDR-H2, and CDR-H3, CDR-H1, CDR-H2, and CDR-H3 each comprise the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively, (c) CDR-L1 having the amino acid sequence of SEQ ID NO: 1, (d) CDR-L2 having the amino acid sequence of any one of SEQ ID NOs: 4 or 5, (e) further comprises CDR-L3 having the amino acid sequence of SEQ ID NO: 6 or 7. In yet another embodiment, the present disclosure provides a polypeptide comprising AF1 comprising CDR-L and CDR-H, wherein AF1 (a) specifically binds to CD3, (b) comprises CDR-H1, CDR-H2, and CDR-H3, CDR-H1, CDR-H2, and CDR-H3 each comprise the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively, (c) CDR-L1 having the amino acid sequence of SEQ ID NO: 2, (d) CDR-L2 having the amino acid sequence of any one of SEQ ID NOs: 4 or 5, (e) further comprises CDR-L3 having the amino acid sequence of SEQ ID NO: 6.In another embodiment, the polypeptide described in any of the subject composition embodiments described herein can include AF1, which includes CDR-L and CDR-H, and AF1: (a) specifically binds to CD3; (b) includes CDR-H1, CDR-H2, and CDR-H3, where CDR-H1, CDR-H2, and CDR-H3 each include the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively; (c) includes CDR-L1 having the amino acid sequence of SEQ ID NO: 1; (d) further includes CDR-L2 having any one of the amino acid sequences of SEQ ID NO: 4; and (e) includes CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In another embodiment, the disclosure provides a polypeptide including AF1, which includes CDR-L and CDR-H, where AF1: (a) specifically binds to CD3; (b) includes CDR-H1, CDR-H2, and CDR-H3;... CDR-H1, CDR-H2, and CDR-H3 each contain the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively, and further include (c) CDR-L1 having the amino acid sequence of SEQ ID NO: 2, (d) CDR-L2 having any one of the amino acid sequences of SEQ ID NO: 5, and (e) CDR-L3 having the amino acid sequence of SEQ ID NO: 6, to provide a polypeptide. In the foregoing embodiments of this paragraph, AF1 can further include a light chain framework region (FR-L) and a heavy chain framework region (FR-H) that link the respective CDR regions.In some cases of the foregoing embodiments of this paragraph, AF1 further includes FR-L1 that exhibits 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: 12 or is identical thereto, FR-L2 that exhibits 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: 13 or is identical thereto, FR-L3 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 14 to 17 or is identical thereto, FR-L4 that exhibits 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: 19 or is identical thereto, FR-H1 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 20 and 21 or is identical thereto, FR-H2 that exhibits 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: 23 or is identical thereto, FR-H3 that exhibits 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: 24 or is identical thereto, and FR-H4 that exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with any one of the amino acid sequences of SEQ ID NO: 26 or is identical thereto.In other examples of the foregoing embodiments of this paragraph, AF1 further includes FR-L1 that exhibits 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: 12 or is identical thereto, FR-L2 that exhibits 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: 13 or is identical thereto, FR-L3 that exhibits 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: 14 or is identical thereto, FR-L4 that exhibits 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: 19 or is identical thereto, FR-H1 that exhibits 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: 20 or is identical thereto, FR-H2 that exhibits 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: 23 or is identical thereto, FR-H3 that exhibits 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: 24 or is identical thereto, and FR-H4 that exhibits 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: 26 or is identical thereto.In other examples of the foregoing embodiments of this paragraph, AF1 further includes an FR-L1 that exhibits 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: 12 or is identical thereto, an FR-L2 that exhibits 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: 13 or is identical thereto, an FR-L3 that exhibits 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: 15 or is identical thereto, an FR-L4 that exhibits 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: 19 or is identical thereto, an FR-H1 that exhibits 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: 21 or is identical thereto, an FR-H2 that exhibits 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: 23 or is identical thereto, an FR-H3 that exhibits 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: 24 or is identical thereto, and an FR-H4 that exhibits 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: 26 or is identical thereto.In other examples of the foregoing embodiments of this paragraph, AF1 comprises an FR-L1 that exhibits 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: 12 or is identical thereto, an FR-L2 that exhibits 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: 13 or is identical thereto, an FR-L3 that exhibits 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: 16 or is identical thereto, an FR-L4 that exhibits 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: 19 or is identical thereto, an FR-H1 that exhibits 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: 21 or is identical thereto, an FR-H2 that exhibits 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: 23 or is identical thereto, an FR-H3 that exhibits 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: 24 or is identical thereto, and an FR-H4 that exhibits 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: 26 or is identical thereto.In still other examples of the foregoing embodiments of this paragraph, the polypeptide comprising AF1 exhibits 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: 12 or is identical thereto, FR-L1; 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: 13 or is identical thereto, FR-L2; 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: 17 or is identical thereto, FR-L3; 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: 19 or is identical thereto, FR-L4; 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: 21 or is identical thereto, FR-H1; 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: 23 or is identical thereto, FR-H2; 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: 24 or is identical thereto, FR-H3; 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: 26 or is identical thereto, FR-H4 and comprises.

[0137] In some aspects according to any of the embodiments disclosed herein, the target polypeptide can include AF1 that binds to CD3, and AF1 includes a VL region and a VH region that confer the ability to specifically bind to CD3. AF1 can be configured in a VL-VH or VH-VL orientation and is fused by a linker peptide.

[0138] In one example, the present disclosure provides a polypeptide comprising an AF1 comprising a VH amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or identical to the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 31. In another example, the present disclosure provides a polypeptide comprising an AF1 comprising a VL amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or identical to any one of the amino acid sequences of SEQ ID NO: 27, 29, 30, 32, or 33. In another example, the polypeptide according to any of the subject composition embodiments described herein comprises an AF1 that binds to CD3, the AF1 confers the ability to specifically bind to CD3, and comprises a VL region and a VH region, each of which has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or is identical to the amino acid sequences of SEQ ID NO: 27 and SEQ ID NO: 28, respectively. In another example, the present disclosure provides a polypeptide comprising an AF1 that binds to CD3, the AF1 confers the ability to specifically bind to CD3, and comprises a VL region and a VH region, each of which has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or is identical to the amino acid sequences of SEQ ID NO: 29 and SEQ ID NO: 28, respectively. In another example, the present disclosure provides a polypeptide comprising an AF1 that binds to CD3, the AF1 confers the ability to specifically bind to CD3, and comprises a VL region and a VH region, each of which has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with or is identical to the amino acid sequences of SEQ ID NO: 30 and SEQ ID NO: 31, respectively.In yet another example, a polypeptide described in any of the subject composition embodiments described herein includes an AF1 that binds CD3, where the AF1 confers the ability to specifically bind CD3 and includes a VL region and a VH region, each of which has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequences of SEQ ID NO: 32 and 31, respectively, or is identical thereto. In other examples, the present disclosure provides a polypeptide that includes an AF1 that binds CD3, where the AF1 confers the ability to specifically bind CD3 and includes a VL region and a VH region, each of which has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the amino acid sequences of SEQ ID NO: 33 and 31, respectively, or is identical thereto.

[0139] In some aspects of any of the embodiments disclosed herein, the subject polypeptide includes an AF1 that binds CD3, where the AF1 is configured as a scFv having the ability to specifically bind CD3. In one embodiment, the AF1 includes an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity to or being identical to any one of the amino acid sequences of SEQ ID NOs: 36 - 40.

[0140] In some examples, the CD3 AF1 of the polypeptide embodiments described herein specifically binds human or cynomolgus (cyno) CD3. In other examples, the CD3 AF1 of the polypeptide embodiments described herein specifically binds human and cynomolgus (cyno) CD3. In one embodiment, the CD3 AF1 of the polypeptide embodiments described herein binds to a CD3 complex subunit selected from CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta epsilon units of CD3. In one embodiment, the AF1 of the polypeptide embodiments described herein binds to a CD3 epsilon fragment of CD3.

[0141] In another aspect, the present disclosure provides a polypeptide comprising an AF1 that binds to a CD3 protein complex and has enhanced stability compared to a CD3-binding antibody or AF1 known in the art. Additionally, certain CD3 AF1s of the present disclosure are designed to confer high stability to chimeric bispecific antigen-binding compositions in 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, certain CD3 AF1s of the present disclosure are designed to have higher thermal stability compared to certain CD3-binding antibodies and antigen-binding fragments known in the art. As a result, the CD3 AF1s utilized as components of chimeric bispecific antigen-binding fragment compositions in which they are incorporated exhibit favorable pharmaceutical properties, including high thermal stability and low tendency to aggregate, resulting in improved expression and recovery during manufacture and storage, and promoting a long serum half-life. Biophysical properties such as thermal stability are often limited by antibody variable domains, and their inherent properties vary widely. 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 determined by measuring the "melting temperature" (T m ), defined as the temperature at which half of the molecules are denatured. The melting temperature of each heterodimer indicates its thermal stability. The T mIn vitro assays for determining this 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).

[0142] 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 show that various constructs of the present disclosure are more resistant to thermal denaturation than the antigen - binding fragment consisting of the sequence shown in SEQ ID NO: 41 or a control bispecific antibody, where the control bispecific antigen - binding fragment comprises a reference antigen - binding fragment that binds to an antigen other than CD3 and SEQ ID NO: 41. In one embodiment, the polypeptide of the embodiments described herein comprises anti - CD3 AF1, AF1 comprises CDR - L and CDR - H, AF1 specifically binds to CD3, comprises CDR - H1, CDR - H2, and CDR - H3, where CDR - H3 comprises the amino acid sequence of SEQ ID NO: 10 and exhibits higher thermal stability as demonstrated by in vitro assays, (i) the polypeptide has a higher Tm compared to the melting temperature (T m ) of the antigen - binding fragment consisting of the sequence shown in SEQ ID NO: 41, or (ii) when the anti - CD3 AF1 is incorporated into an anti - CD3 bispecific antibody, the bispecific antibody has a higher T mexhibits, wherein the anti-CD3 bispecific antibody comprises the anti-CD3 antigen fragment and a reference antigen-binding fragment that binds to an antigen other than CD3, and the control bispecific antigen-binding fragment consists of SEQ ID NO: 41 and the reference antigen-binding fragment. For example, in some situations, the control bispecific antibody is identical to the target polypeptide except that AF1 is replaced with the antigen-binding fragment of SEQ ID NO: 41. The reference antigen-binding fragment of the embodiments is intended to include antigen-binding fragments that bind to any of the target cell markers described herein, including but not limited to EGFR, HER2, EpCAM, and CD19 among other disclosed target cell markers. In one embodiment, the present disclosure provides a polypeptide comprising an anti-CD3 AF1, wherein the T m of the antigen-binding fragment consisting of the sequence of SEQ ID NO: 41 m is at least 2 °C higher, or at least 3 °C higher, or at least 4 °C higher, or at least 5 °C higher, or at least 6 °C higher, or at least 7 °C higher, or at least 8 °C higher, or at least 9 °C higher, or at least 10 °C higher. In another embodiment, the present disclosure provides a polypeptide comprising an anti-CD3 AF1, wherein the T m of the antigen-binding fragment consisting of the sequence of SEQ ID NO: 41 m is at least 2 - 10 °C higher, or at least 3 - 9 °C higher, or at least 4 - 8 °C higher, or at least 5 - 7 °C higher. In yet another embodiment, a bispecific antigen-binding polypeptide comprising an anti-CD3 AF1, wherein AF1 comprises CDR-L and CDR-H, AF1 specifically binds to CD3, comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10, comprises a second antigen-binding fragment that binds to an antigen other than CD3, and exhibits higher thermal stability as demonstrated in an in vitro assay, and the bispecific antigen-binding polypeptide has a higher melting temperature (T m ) compared to a control bispecific antibody control comprising the sequence shown in SEQ ID NO: 41 and a reference antigen-binding fragment that binds to an antigen other than CD3.

[0143] In related aspects, the present disclosure provides various polypeptides comprising AF1 that binds to CD3, which are incorporated into chimeric bispecific antigen-binding fragment compositions designed to have an isoelectric point (pI) that confers enhanced stability to the compositions of the present disclosure compared to corresponding compositions comprising CD3-binding antibodies or antigen-binding fragments known in the art. In one embodiment, the polypeptide embodiments described herein can comprise an antigen-binding fragment that binds to CD3, and AF1 exhibits a pI of 5.8 to 6.6 (including the boundary values). In other embodiments, the present disclosure provides a polypeptide comprising AF1 that binds to CD3, wherein AF1 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units lower than the pI of a reference antigen-binding fragment consisting of the sequence shown in SEQ ID NO: 41. In another embodiment, any of the polypeptide embodiments of the subject composition embodiments described herein can comprise AF1 that binds to CD3 fused to a second antigen-binding fragment that binds to an antigen other than CD3, and the CD3 AF1 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units of the pI of the antigen-binding fragment that does not bind to CD3. In another embodiment, the present disclosure provides a polypeptide comprising AF1 that binds to CD3 fused to a second antigen-binding fragment that binds to an antigen other than CD3, wherein the CD3 AF1 exhibits a pI within at least about 0.1 to about 1.5, or at least about 0.3 to about 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 to about 0.9 pH units of the pI of the second antigen-binding fragment, as demonstrated by calculation (see Examples) or in vitro. In one embodiment, the second antigen-binding fragment is selected from the group consisting of, but not limited to, EpCAM, EGFR, HER2, CD19, or any of the target cell marker embodiments disclosed herein including those in Table 8, and has specific binding affinity for a non-CD3 antigen.By designing the pI of these two antigen-binding fragments to be within such a range, the resulting fused antigen-binding fragment confers high stability to the chimeric bispecific antigen-binding fragment composition into which they are incorporated, improving the expression and enhancing the recovery of the fusion protein in a soluble, non-aggregated form, increasing the shelf life of the formulated chimeric bispecific polypeptide composition, and specifically intended to result in enhanced stability when the composition is administered to a subject.

[0144] In some aspects of any of the embodiments disclosed herein, the subject polypeptide has a dissociation constant (K d ) of about ~ about 10 nM to about 400 nM, or about 50 nM to about 350 nM, or about 100 nM to 300 nM when determined in an in vitro antigen-binding assay that includes the human or cynomolgus CD3 antigen. In another embodiment, the polypeptide of any of the subject composition embodiments described herein has a dissociation constant (K d ) 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 when determined in an in vitro antigen-binding assay and may include AF1 that specifically binds to human or cynomolgus CD3. For clarity, an antigen-binding fragment having a K d of 400 nM binds its ligand less strongly than one having a K d of 10 nM.

[0145] In another embodiment, the present disclosure provides a polypeptide comprising AF1 that exhibits a binding affinity for CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker compared to the binding affinity of an antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 41 when determined by their respective dissociation constants (K d ) in an in vitro antigen-binding assay. In another embodiment, the present disclosure provides a polypeptide comprising AF1 that exhibits a binding affinity for CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker compared to the binding affinity of an antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 41 when determined by their respective dissociation constants (K dWhen determined by , the binding affinity for CD3 is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold weaker compared to the binding affinity of a second antigen-binding fragment incorporated into a polypeptide that specifically binds to an antigen other than CD3. Provided is a polypeptide comprising AF1 that exhibits such a binding affinity for CD3. In the foregoing embodiments, the antigen other than CD3 is selected from, but not limited to, HER2, EGFR, EpCAM, or CD19, or any of the target cell marker embodiments disclosed herein including, but not limited to, those in Table 8. The binding affinity of the subject composition for the target ligand can be assayed using a binding assay or a competitive binding assay, such as a Biacore assay using a chip-bound receptor or binding protein, or an ELISA assay as described in U.S. Patent No. 5,534,617, an assay described in the examples herein, a radioligand assay, or other assays known in the art. The binding affinity constant can then be determined using standard methods such as Scatchard analysis as described by van Zoelen, et al., Trends Pharmacol Sciences (1998) 19(12):487, or other methods known in the art. The same methodology will be used to generate bispecific antigen-binding fragment constructs having antigen-binding fragments for CD3 and target cell markers described herein in any combination or orientation (i.e., AF1-AF2 or AF2-AF1 in an N-terminal to C-terminal orientation).

Table 1

Table 2

Table 3

Table 4-1

Table 4-2

[0146] III). Release segment In another aspect, the present disclosure relates to a release segment (RS) peptide suitable for inclusion into the subject compositions described herein that is a substrate for one or more mammalian proteases associated with or produced by diseased tissue or cells found near or in diseased tissue. Such proteases include, but are not limited to, classes of proteases such as metalloproteases, cysteine proteases, aspartic proteases, and serine proteases. The RS is useful, inter alia, for imparting a prodrug form to a subject composition that can be activated, in particular, by cleavage of the RS by a mammalian protease. As described herein, the RS is incorporated into a subject composition embodiment described herein, and the incorporated antigen-binding fragment is linked to XTEN (the structure of which is more fully described below), such that upon cleavage of the RS by the action of one or more proteases of which the RS is a substrate, the antigen-binding fragment and XTEN are released from the composition, and the antigen-binding fragment, no longer shielded by XTEN, regains the full ability to bind to their respective ligands. In a specific feature, the RS is found in close association with or co-localizes with diseased tissue or cells such as, but not limited to, tumors, cancer cells, and inflamed tissue, or functions as a substrate for a protease associated with the diseased tissue or cells, and upon cleavage of the RS, an antigen-binding fragment that is otherwise shielded by XTEN of the subject composition (and thus has a lower binding affinity for its respective ligand) is released from the composition and regains the full ability to bind to target and / or effector cell ligands. In another embodiment, the RS of the subject 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 subject compositions described herein, the RS, which is a substrate for two or three classes of proteases, is designed using a sequence that can be cleaved at different positions of the RS sequence by different proteases, and a representative example is shown in FIG. 6.Thus, RS, which is a substrate for two, three, or more classes of proteases, has two, three, or multiple distinct cleavage sites in the RS sequence, yet cleavage by a single protease results in the release of antigen-binding fragments and XTEN from the composition containing RS.

[0147] In one embodiment, the present disclosure provides an activatable polypeptide comprising one or more release segments, wherein the release segment is a substrate for cleavage by one or more mammalian proteases. In another embodiment, the present 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 legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other cases, a polypeptide according to any of the target composition embodiments described herein comprises a first release segment (RS1) sequence, wherein RS1 is meprin, neprilysin (CD10), PSMA, BMP-1, A disintegrin and metalloprotease (ADAM), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM with thrombospondin motif (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloprotease-1 (MMP-1), matrix metalloprotease-2 (MMP-2, gelatinase A), matrix metalloprotease-3 (MMP-3, stromelysin 1), matrix metalloprotease-7 (MMP-7, matrilysin 1), matrix metalloprotease-8 (MMP-8, collagenase 2), matrix metalloprotease-9 (MMP-9, gelatinase B), matrix metalloprotease-10 (MMP-10, stromelysin 2), matrix metalloprotease-11 (MMP-11, stromelysin 3), matrix metalloprotease-12 (MMP-12, macrophage elastase), matrix metalloprotease-13 (MMP-13, collagenase 3), matrix metalloprotease-14 (MMP-14, MT1-MMP), matrix metalloprotease-15 (MMP-15, MT2-MMP), matrix metalloprotease-19 (MMP-19), matrix metalloprotease-23 (MMP-23, CA-MMP),It is a substrate for cleavage by one or more mammalian proteases selected from the group consisting of matrix metalloprotease-24 (MMP-24, MT5-MMP), matrix metalloprotease-26 (MMP-26, matrilysin 2), matrix metalloprotease-27 (MMP-27, CMMP), legumain, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X, cathepsin D, cathepsin E, secretase, urokinase (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, prostate-specific antigen (PSA, KLK3), human neutrophil elastase (HNE), elastase, tryptase, type II transmembrane serine protease (TTSP), DESC1, hepsin (HPN), matriptase, matriptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast activation protein (FAP), kallikrein-related peptidase (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14.,

[0148] In another embodiment, the present 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 to a sequence selected from SEQ ID NOs: 42-660. In another embodiment, RS1 comprises an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which is described in Table 5. As described in more detail below in the description of the construction 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.

[0149] In other embodiments, the present disclosure provides a polypeptide comprising a first release segment (RS1) sequence and a second release segment (RS2) for incorporation into the subject polypeptide compositions described herein, wherein RS1 and RS2 are identical. In another embodiment, the present disclosure provides a polypeptide comprising a first release segment (RS1) sequence and a second release segment (RS2) for incorporation into the subject polypeptide compositions, wherein RS1 and RS2 are different. In some instances of the foregoing embodiments, RS1 and RS2 are each substrates for cleavage by mammalian proteases selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In another embodiment, the present disclosure provides a polypeptide comprising RS1 and RS2 sequences for incorporation into the subject polypeptide compositions described herein, wherein RS1 and RS2 are each substrates for cleavage by one or more mammalian proteases, and wherein RS1 and RS2 each comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 42-660. In another embodiment, RS1 and RS2 each comprise an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which is described in Table 5. As described in more detail in the paragraphs below relating to the construction and properties of the subject polypeptide compositions, the release segments are fused between the antigen-binding fragment and the XTEN polypeptide such that upon cleavage of each release segment, the adjacent XTEN is released from the composition.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

[0150] In another aspect, the release segment (either RS1 and / or RS2) for incorporation into a polypeptide as described in any of the subject composition embodiments described herein can be designed to be selectively sensitive such that they have different cleavage rates and different cleavage efficiencies for the various proteases that they are substrates for. Since a given protease can be found at different concentrations in diseased tissues including, but not limited to, tumors, blood cancers, or inflamed tissues or sites of inflammation as compared to healthy tissue or circulating blood, the present disclosure provides for RSs that ensure that when the released antigen-binding fragment is near the target cell or tissue and its co-localized protease, the polypeptide is preferentially converted from the prodrug form to the active form (i.e., by separation and release of the antigen-binding fragment and XTEN from the polypeptide after cleavage of the release segment) such that the ability of the released antigen-binding fragment to bind to the ligand in the diseased tissue is higher as compared to the prodrug form remaining in the circulating blood. By such selective design, the therapeutic index of the resulting composition is improved and side effects can be reduced as compared to conventional therapeutics that do not incorporate such site-specific activation.

[0151] As used herein, cleavage efficiency is defined as the log2 value of the ratio of the percentage of a test substrate containing a cleaved release segment when subjected to a protease enzyme in a biochemical assay (more particularly detailed in the Examples) in which the reaction is carried out, to the percentage of the cleaved control substrate AC1611, where the initial substrate concentration is 6 μM, the reaction is stopped by the addition of EDTA after incubation at 37 °C for 2 hours, and the amount of digestion products 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

[0152] Therefore, a cleavage efficiency of -1 means that the amount of cleavage of the test substrate was 50% compared to the amount of cleavage of the control substrate, and a cleavage efficiency of +1 means that the amount of cleavage of the test substrate was 200% compared to the amount of cleavage of the control substrate. A higher cleavage rate by the test protease compared to the control results in a higher cleavage efficiency, and a lower cleavage rate by the test protease compared to the control results in a lower cleavage efficiency. As detailed in the examples, when tested in an in vitro biochemical assay for the cleavage rate by individual proteases, the control RS sequence AC1611 (RSR-1517) having the amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO: 42) was established as having appropriate baseline cleavage efficiencies by the proteases legumain, MMP-2, MMP-7, MMP-9, MMP-14, uPA, and matriptase. By selective substitution of amino acids at individual positions in the RS peptide, a library of RSs was generated and evaluated against a panel of seven proteases (more fully detailed in the examples) to obtain a profile that was used to establish guidelines for appropriate amino acid substitutions to achieve an RS with the desired cleavage efficiency. When generating an RS with the desired cleavage efficiency, substitutions using the hydrophilic amino acids A, E, G, P, S, and T are preferred, but other L-amino acids can be substituted at a given position to adjust the cleavage efficiency as long as the release segment retains at least some sensitivity to cleavage by the protease.

[0153] IV). XTEN polypeptide In another aspect, the present disclosure relates to a polypeptide comprising at least a first extended recombinant polypeptide (XTEN) incorporated into the subject composition embodiments described herein, thereby increasing the mass and size of the construct and also serving to significantly reduce the ability of the antigen-binding fragment to bind to the ligand when the molecule is in an intact state where it has not been cleaved, as described more fully below. In some embodiments, the present disclosure provides a polypeptide comprising a single XTEN fused to the terminus of an RS located between the antigen-binding fragment and the XTEN. In other embodiments, the present disclosure provides a polypeptide 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.

[0154] Without being bound by theory, the incorporation of XTEN imparts certain properties to the polypeptide composition: 1) when the polypeptide composition is in its intact prodrug form, the property of providing an XTEN that shields the antigen-binding fragment and reduces their binding affinity to target cell markers and effector cell antigens; ii) the property of providing an XTEN to the polypeptide composition that provides an enhanced half-life when administered to a subject; iii) the property of contributing to the solubility and stability of the intact composition, thereby enhancing the pharmaceutical properties of the subject composition; and iv) the property of reducing extravasation in normal tissues and organs in the polypeptide composition, but permitting some degree of extravasation in diseased tissues (e.g., tumors) with larger pore sizes in the vasculature, and yet being released from the tissue by the action of certain mammalian proteases, whereby the antigen-binding fragment of the composition can readily penetrate diseased tissues, e.g., tumors, and bind to target cell markers on effector cells and tumor cells and permit them to be ligated together. To meet these needs, the present disclosure provides a composition comprising one or more XTENs, wherein the XTENs provide a composition that results in an increased mass and hydrodynamic radius. The XTEN polypeptide of embodiments not only provides an increased mass and hydrodynamic radius to the composition, but its unstructured nature of mobility can also provide a shielding effect on the antigen-binding fragment of the composition, thereby reducing binding to antigens in normal tissues or the vasculature of normal tissues that do not express or express at reduced levels the target cell marker and / or effector cell antigen, providing certain advantages in the design of the subject composition. In addition, the incorporation of XTEN into the subject composition can enhance the solubility and proper folding of single-chain antibody binding fragments during expression and recovery.

[0155] XTEN is a polypeptide having a substantially non-repetitive sequence that does not occur naturally and has or does not have a secondary or tertiary structure to some extent under physiological conditions, as well as one or more additional properties described in the following paragraphs. In some embodiments, the present disclosure provides a polypeptide comprising one or more XTENs having at least about 36, 72, 96, 100, 144, 200, 288, 292, 293, 300, 576, 584, 800, 864, 867, 868, 900, or at least about 1000 or more amino acids. In one embodiment, the present disclosure provides a polypeptide comprising XTEN1, wherein XTEN1 has at least about 36 or 100 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 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 present disclosure provides a polypeptide comprising XTEN1 having at least about 36 to about 1000, at least about 100 to 1000, or at least 100 to about 900, or at least about 144 to about 868, or at least about 288 to 868 amino acid residues. In other cases, the present disclosure provides a polypeptide comprising XTEN1 having at least about 36 to about 1000, at least about 100 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 cases, the present disclosure provides a polypeptide comprising XTEN1, wherein XTEN1 has at least about 36 to about 1000 amino acid residues or at least about 100 to about 1000 amino acid residues, and 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).

[0156] In another embodiment, the present disclosure provides a polypeptide as described in any of the embodiments described herein that includes XTEN1, wherein XTEN1 has at least about 36 to about 1000, at least about 100 to about 1000, or at least about 100 to about 900, or at least 144 to about 868 amino acid residues, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from at least three of the sequences of SEQ ID NOs: 661-664. In some cases, the XTEN1 sequence can be constructed by any combination of 12-amino acid units of SEQ ID NOs: 661-664 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, a polypeptide as described in any of the subject composition embodiments described herein can include XTEN1, wherein XTEN1 has at least about 36 to about 1000, at least about 100 to about 1000, or at least about 100 to about 900, or at least 144 to about 868 amino acid residues, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from the sequences of SEQ ID NOs: 665-718 and 922-926. In another embodiment, XTEN as described in any of the subject composition embodiments described herein can have an affinity tag of HHHHHH (SEQ ID NO: 1150), HHHHHHHH (SEQ ID NO: 1151), or sequence EPEA (SEQ ID NO: 1149) added to the N-terminus or C-terminus of the XTEN of the composition to facilitate purification of the composition to a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% by chromatography methods known in the art, such as IMAC chromatography or C-tagXL chromatography, or by the methods described in the following examples.

[0157] In another embodiment, the present disclosure provides a polypeptide comprising XTEN1. 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 AE36 (comprising a sequence selected from any three of the sequences of SEQ ID NOs: 661-664), or a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is described in Table 7.

[0158] In some aspects described in any of the embodiments disclosed herein, the target polypeptide includes XTEN1 and XTEN2. Among other components, the composition of the polypeptide including XTEN1 and XTEN2 in particular is described in the following specification. In one embodiment, the present disclosure provides a polypeptide including XTEN1 and XTEN2, wherein XTEN1 and XTEN2 each have at least about 36 to about 1000 amino acid residues or at least about 100 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 XTEN1 and XTEN2 are selected from at least three of the sequences of SEQ ID NOs: 661 to 664. In another embodiment, the present disclosure provides a polypeptide including XTEN1 and XTEN2, wherein XTEN1 and XTEN2 each have at least about 36 to about 1000 amino acid residues or at least about 100 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 XTEN1 and XTEN2 are selected from the sequences of SEQ ID NOs: 665 to 718 and 922 to 926. In another embodiment, the polypeptide described in any of the target composition embodiments described herein can include XTEN1 and XTEN2, and XTEN1 and XTEN2 each include at least about 90% of the amino acid sequence and have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an amino acid sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, and these are each described in Table 7.In some cases of the foregoing embodiments of this paragraph, XTEN1 and XTEN2 are the same. In other cases of the foregoing embodiments of this paragraph, XTEN1 and XTEN2 of the foregoing embodiments of this paragraph have different amino acid sequences. In some cases, XTEN1 described in any of the polypeptide composition embodiments having two XTENs is fused to the C-terminus of the polypeptide and is selected from the group consisting of AE293, AE300, AE584, and AE868. In other cases, XTEN2 described in any of the polypeptide composition embodiments having two XTENs is fused to the N-terminus of the polypeptide and is selected from the group consisting of AE144_7A, AE292, AE576, and AE864. In other cases, XTEN1 described in any of the polypeptide composition embodiments having two XTENs is fused to the C-terminus of the polypeptide and is selected from the group consisting of AE293, AE300, AE584, and AE868, 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

Table 7-12

[0159] The present disclosure contemplates a composition according to any of the embodiments described herein, including an XTEN of intermediate length relative to those of Table 7, and an XTEN of a length longer than those of Table 7, for example, where a 12-amino acid motif of Table 6 is added to the N-terminus or C-terminus of an XTEN of Table 7.

[0160] In another embodiment, the present disclosure contemplates a polypeptide composition according to any of the embodiments described herein, including XTEN1 and XTEN2, which can further include His tags of HHHHHH (SEQ ID NO: 1150) or HHHHHHHH (SEQ ID NO: 1151) at the N-terminus and / or sequence EPEA (SEQ ID NO: 1149) at the C-terminus of the polypeptide composition, so as to promote the generation of a composition to a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% by chromatography methods known in the art, including, but not limited to, IMAC chromatography, C-tagXL affinity matrix, and other such methods (including, but not limited to, those described in the following embodiments).

[0161] Additional examples of XTEN arrays that can be used in accordance with the present disclosure are described in U.S. Patent Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1, or International Patent Publication Nos. WO2010 / 091122A1, WO2010 / 144502A2, WO2010144508A1, WO2011 / 028228A1, WO2011 / 028229A1, WO2011 / 028344A2, WO2014 / 011819A2, or WO2015 / 023891.

[0162] V). Target cell marker antigen-binding fragment In another aspect, the present disclosure relates to antigen-binding fragments having specific binding affinity for target cell markers other than CD3 that can be incorporated into any of the subject composition embodiments described herein. A bispecific composition resulting from linking a first antigen-binding fragment (AF1) having binding affinity for CD3 to a second antigen-binding fragment (AF2) having binding affinity for a second non-CD3 antigen using a short flexible peptide linker is bispecific such that each antigen-binding fragment has specific binding affinity for its respective ligand. In such a composition, it will be understood that an antigen-binding fragment directed against a target cell marker of an affected tissue is used in combination with a second antigen-binding fragment directed towards an effector cell marker in order to bring effector cells into close proximity to the cells of the affected tissue and effect cell lysis of the cells of the affected tissue. Further, AF1 and AF2 can be incorporated into a specifically designed polypeptide containing a cleavable release segment and XTEN, such that when in the vicinity of an affected tissue having a protease capable of cleaving the release segment at one or more positions of the release segment sequence, prodrug properties can be imparted to the composition that is activated by the release of the fused AF1 and AF2 upon cleavage of the release segment.

[0163] In one embodiment, the polypeptide described in any of the subject composition embodiments described herein may include AF2 having specific binding affinity for a target cell marker expressed on the cell surface, within the cytoplasmic membrane, or within a target cell associated with cancer, autoimmune diseases, inflammatory diseases, and other conditions where local activation of the polypeptide is desired. In one embodiment, the antigen for which AF2 has specific binding affinity is 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A kinase anchor protein 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenergic receptor β3 (ADRB3), AGS-22M6, α-folate receptor, α-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoietin-binding cell surface receptor 2 (Tie 2), anthrax toxin, AOC3 (VAP-1), B cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthracis anthrax, B cell-activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), brother of the regulator of imprinted sites (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCCTC-binding factor (CTCF), CCL11 (eotaxin-1), CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD171, CD179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (α-chain of the IL-2 receptor), CD27, CD274, CD28, CD3, CD3 ε, CD30, CD300 molecule-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44 v6,CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-related antigen, CFD, ch4D5, Chromosome X open reading frame 61 (CXORF61), Claudin 18.2 (CLDN18.2), Claudin 6 (CLDN6), Clostridium difficile, Clamping factor A, CLCA2, Colony stimulating factor 1 receptor (CSF1R), CSF2, CTLA-4, C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-X-C chemokine receptor type 4, Cyclin B1, Cytochrome P450 1B1 (CYP1B1), cyp-B, Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran, DLL4, DPP4, DR5, E. coli Shiga toxin type 1, E. coli Shiga toxin type 2, Ecto ADP-ribosyltransferase 4 (ART4), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), EGF-like domain multiple 7 (EGFL7), Elongation factor 2 variant (ELF2M), Endotoxin, Ephrin A2, Ephrin B2, Ephrin A receptor 2, Epidermal growth factor receptor (EGFR), Epidermal growth factor receptor variant III (EGFRvIII), Epicyanin, Epithelial cell adhesion molecule (EpCAM), Epithelial glycoprotein 2 (EGP-2), Epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (Transmembrane serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), Respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), Fetal acetylcholine receptor, Fibrin II β chain, Fibroblast activation protein α (FAP), Fibronectin extracellular domain-B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), Folic acid binding protein (FBP), Folic acid hydrolase, Folic acid receptor 1, Folic acid receptor α, Folic acid receptor β, Fos-related antigen 1, Frizzled receptor, Fucosyl GM1, G250, G protein-coupled receptor 20 (GPR20),G-protein coupled receptor class C group 5 member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), glypican-3 (GPC3), GMCSF receptor alpha chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, heat shock protein 70-2 variant (mut hsp70-2), hemagglutinin, hepatitis A virus cell receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, globoH glycosphingolipid (GloboH) hexasaccharide protein, HGF, HHGFR, high molecular weight melanoma associated antigen (HMW-MAA), histone complex, HIV-1, HLA-DR, HNGF, Hsp90, HST-2 (FGF6), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), human TNF, ICAM-1 (CD54), iCE, IFN-alpha, IFN-beta, IFN-gamma, IgE, IgE Fc region, IGF-1, IGF-1 receptor, IGHE, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-1beta, IL-20, IL-22, IL-23, IL-31, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin alpha4beta7, integrin beta2, integrin alpha2, integrin alpha4, integrin alpha5beta1, integrin alpha7beta7, integrin alphaIIbbeta3, integrin alphavbeta3, interferon alpha / beta receptor, interferon gamma-induced protein, interleukin 11 receptor alpha (IL-11Ralpha), interleukin 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, kinase domain region (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), lymphocyte antigen 6 (Ly-6)Lewis Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex locus K 9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-α (LT-α) or tumor necrosis factor-β (TNF-β), lysosome-associated membrane protein 1 (LAMP1), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, mammary differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma apoptosis inhibitory factor melanoma (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, cell surface-bound mucin 1 (MUC1), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, mucin CanAg, myelin-associated glycoprotein, myostatin, N-acetylglucosaminyl-transferase V (NA17), NCA-90 (granulocyte antigen), nectin-4, nerve growth factor (NGF), nerve apoptosis regulatory protease 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitor (e.g., NOGO-A, NOGO-B, NOGO-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), tumor fetal antigen (h5T4), cancer gene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia virus cancer gene homolog 1 (Abl) (bcr-abl), anasarca, OX-40, oxLDL, p53 variant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), P-cadherin, sodium phosphate cotransporter, phosphatidylserine placenta-specific 1 (PLAC1), platelet-derived growth factor receptor α (PDGF-Rα), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1)Proprotein convertase subtilisin / kexin type 9 (PCSK9), prostase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), T cell recognition melanoma antigen 1 (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate cancer cells, prostatin, protease serine 21 (testisin or PRSS21), proteasome (prososome, macropain) subunit beta type 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), receptor activator of nuclear factor kappa-B ligand (RANKL), receptor for advanced glycation end products (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), respiratory syncytial virus, Rh blood group D antigen, Rh factor, sarcoma translocation breakpoint, sclerostin (SOST), selectin P, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), sphingosine-1-phosphate, T cell recognition squamous cell carcinoma antigen 1, 2, and 3 (SART1, SART2, and SART3), stage-specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1, syndecan 1 (SDC1)+A314, SOX10, survivin, survivin-2B, synovial sarcoma, X breakpoint 2 (SSX2), T cell receptor, TCR gamma alternative reading frame protein (TARP), telomerase, TEM1, tenascin C, TGF-beta (e.g., TGF-beta1, TGF-beta2, TGF-beta3), thyroid-stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)), TNF receptor family member B cell maturation antigen (BCMA), TNF-α, TRAIL-R1, TRAIL-R2, TRG, transglutaminase 5 (TGS5), tumor antigen CTAA16.88, Tumor Endothelial Marker 1 (TEM1 / CD248), Tumor Endothelial Marker 7-Related (TEM7R), Tumor Protein p53 (p53), Tumor-Specific Glycosylation of MUC1, Tumor-Associated Calcium Signal Transducer 2 (TROP-2), Tumor-Associated Glycoprotein 72 (TAG72), Tumor-Associated Glycoprotein 72 (TAG-72)+A327, TWEAK Receptor, Tyrosinase, Tyrosinase-Related Protein 1 (TYRP1 or Glycoprotein 75), Tyrosinase-Related Protein 2 (TYRP2), Uroplakin 2 (UPK2), Vascular Endothelial Growth Factor (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF), Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), Vimentin, v-myc Avian Myelocytomatosis Virus Oncogene Neuroblastoma-Derived Homolog (MYCN), von Willebrand Factor (VWF), Wilms Tumor Protein (WT1), X Antigen Family Member 1A (XAGE1), β-Amyloid, κ-Light Chain, Fibroblast Growth Factor Receptor 2 (FGFR2), LIV-1 Protein Estrogen-Regulated (LIV1, also known as SLC39A6), Neurotrophic Receptor Tyrosine Kinase 1 (NTRK1, also known as TRK), Ret Proto-Oncogene (RET), B-Cell Maturation Antigen (BCMA, also known as TNFRSF17), Transferrin Receptor (TFRC, also known as CD71), Activated Leukocyte Cell Adhesion Molecule (ALCAM, also known as CD166), Somatostatin Receptor 2 (SSTR2), KIT Proto-Oncogene Receptor Tyrosine Kinase (cKIT), V-Set Immunoregulatory Receptor (VSIR, also known as VISTA), Glycoprotein Nmb (GPNMB), Delta-Like Canonical Notch Ligand 3 (DLL3), Interleukin 3 Receptor Subunit Alpha (IL3RA, also known as CD123), Lysosome-Associated Membrane Protein 1 (LAMP1), Cadherin 3, Type 1, P-Cadherin (CDH3), Ephrin A4 (EFNA4), Protein Tyrosine Kinase 7 (PTK7), Solute Carrier Family 34 Member 2 (SLC34A2, also known as NaPi-2b), GCC, PLAUR Domain-Containing 3 (LYPD3, also known as LY6 or C4.4a), selected from antigens including, but not limited to, cell surface-binding mucin 17 (MUC17), Fms-related receptor tyrosine kinase 3 (FLT3), NKG2D ligands (e.g., ULBP1, ULBP2, ULBP3, H60, Rae-1α, Rae-1β, Rae-1δ, Rae-1γ, MICA, MICB, hHLA-A), SLAM family member 7 (SLAMF7), interleukin 13 receptor subunit alpha 2 (IL13RA2), C-type lectin domain family 12 member A (CLEC12A, also known as CLL-1), CEA cell adhesion molecule 5 (CEACAM, also known as CD66e), interleukin 3 receptor subunit alpha (IL3RA), CD5 molecule (CD5), UL16-binding protein 1 (ILBP1), V-Set domain-containing T cell activation inhibitor 1 (VTCN1, also known as B7-H4), chondroitin sulfate proteoglycan 4 (CSPG4), syndecan 1 (SDC1, also known as CD138), interleukin 1 receptor accessory protein (IL1RAP), baculovirus IAP repeat-containing 5 (BIRC5, also known as survivin), CD74 molecule (CD74), hepatitis A virus cellular receptor 1 (HAVCR1, also known as TIM1), SLIT and NTRK-like family member 6 (SILTRK6), CD37 molecule (CD37), coagulation factor III, tissue factor (CD142, also known as F3), AXL receptor tyrosine kinase (AXL), endothelin receptor type B (EDNRB, also known as ETBR), cadherin 6 (CDH6), fibroblast growth factor receptor 3 (FGFR3), carbonic anhydrase 6 (CA6), the CanAg glycoform of MUC1, integrin subunit alpha V (ITGAV), teratocarcinoma-derived growth factor 1 (TDGF1, also known as Crypto 1), SLAM family member 6 (SLAMF6, also known as CD352), and Notch receptor 3 (NOTCH3).

[0164] Therapeutic monoclonal antibodies that can be obtained for incorporation into any of the polypeptide embodiments of the subject compositions described herein are known in the art. Such therapeutic antibodies include rituximab, IDEC / Genentech / Roche (see, e.g., U.S. Patent No. 5,736,137), a chimeric anti-CD20 antibody used in the treatment of many lymphomas, leukemias, and some autoimmune disorders, ofatumumab, an anti-CD20 antibody approved for use in chronic lymphocytic leukemia and being developed for follicular non-Hodgkin lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing-remitting multiple sclerosis, lucatumumab (HCD122), an anti-CD40 antibody for non-Hodgkin lymphoma or Hodgkin lymphoma (see, e.g., U.S. Patent No. 6,899,879), AME-133, an antibody that binds to cells expressing CD20 for treating non-Hodgkin lymphoma, belzutuzumab (hA20), an antibody that binds to cells expressing CD20 for treating immune thrombocytopenic purpura, HumaLYM developed for the treatment of low-grade B-cell lymphoma, and ocrelizumab, an anti-CD20 monoclonal antibody for the treatment of rheumatoid arthritis (see, e.g., U.S. Patent Application No. 2009 / 0155257), trastuzumab (see, e.g., U.S. Patent No. 5,677,171), a humanized anti-HER2 / neu antibody approved for the treatment of breast cancer, pertuzumab, an anti-HER2 dimerization inhibitor antibody developed for the treatment of prostate cancer, breast cancer, and ovarian cancer (see, e.g., U.S. Patent No. 4,753,894), cetuximab, an anti-EGFR antibody used in the treatment of KRAS wild-type metastatic colorectal cancer and head and neck cancer expressing epidermal growth factor receptor (EGFR) (see, e.g., U.S. Patent No. 4,943,533, PCT WO96 / 40210), panitumumab, a fully human monoclonal antibody specific for epidermal growth factor receptor (also known as EGF receptor, EGFR, ErbB-1, and HER1, currently sold for the treatment of metastatic colorectal cancer (U.S. Patent No. 6,235,See U.S. Patent No. 7,247,301), cetuximab, a fully human IgG1 monoclonal antibody directed against the epidermal growth factor receptor (EGFR) for the treatment of head and neck squamous cell carcinoma (e.g., see U.S. Patent No. 7,247,301), nimotuzumab, a chimeric antibody against EGFR developed for the treatment of head and neck squamous cell carcinoma, nasopharyngeal carcinoma, and glioma (e.g., see U.S. Patent No. 5,891,996, U.S. Patent No. 6,506,883), matuzumab, a humanized monoclonal antibody directed against the epidermal growth factor receptor (EGFR) developed for the treatment of colorectal cancer, lung cancer, esophageal cancer, and gastric cancer (e.g., see U.S. Patent Application No. 2009 / 0175858A1), cetuximab, a chimeric (mouse / human) monoclonal antibody directed against the epidermal growth factor receptor (EGFR) used in the treatment of metastatic colorectal cancer, metastatic non-small cell lung cancer, and head and neck cancer (e.g., see U.S. Patent No. 6,217,See No. 866), alemtuzumab, a humanized monoclonal antibody against CD52 marketed for the treatment of chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL), and T-cell lymphoma, ibritumomab tiuxetan, an anti-CD20 monoclonal antibody developed for the treatment of several forms of B-cell non-Hodgkin lymphoma, gemtuzumab ozogamicin, an anti-CD33 (p67 protein) antibody conjugated to the cytotoxic chelating agent tiuxetan linked to a radioisotope used in the treatment of acute myeloid leukemia, ABX-CBL, an anti-CD147 antibody, ABX-IL8, an anti-IL8 antibody, ABX-MA1, an anti-MUC18 antibody, pemtumomab (R1549, 90Y-muHMFG1), anti-MUC1 under development, Therex (R1550), an anti-MUC1 antibody, AngioMab (AS1405) developed by Antisoma, HuBC-1 developed by Antisoma, Thioplatin (AS1407) developed by Antisoma, ANTEGREN (natalizumab), an anti-alpha-4-beta-1 (VLA4) and alpha-4-beta-7 antibody, VLA-1 mAb, an anti-VLA-1 integrin antibody, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody, CAT-152, an anti-TGF-β2 antibody, J695, an anti-IL-12 antibody, CAT-192, an anti-TGFβ1 antibody developed, CAT-213, an anti-eotaxin 1 antibody developed, LYMPHOSTAT-B, an anti-Blys antibody, TRAIL-R1mAb, an anti-TRAIL-R1 antibody, Herceptin, an anti-HER receptor family antibody, anti-tissue factor (ATF), an anti-tissue factor antibody, Xolair (omalizumab), an anti-IgE antibody, MLN-02 antibody (formerly LDP-02), HuMax CD4 (registered trademark), an anti-CD4 antibody, tocilizumab, and an anti-IL6R antibody, HuMax-IL15, an anti-IL15 antibody, HuMax-inflammation, HuMax-cancer, an anti-heparanase I antibody, HuMax-lymphoma,, HuMax-TAC, IDEC-131, anti-CD40, IDEC-151 (Crenoliximab), anti-CD4 antibody, IDEC-114, anti-CD80 antibody, IDEC-152, anti-CD23, anti-KDR antibody, DC101, anti-flk-1 antibody, anti-VE cadherin antibody developed by Imclone, CEA-CIDE (Rabetuzumab), anti-carcinoembryonic antigen (CEA) antibody developed by Immunomedics, Yervoy (Ipilimumab), anti-CTLA4 antibody used in the treatment of melanoma, Lumphocide (registered trademark) (Epratuzumab), anti-CD22 antibody, AFP-Cide developed by Immunomedics, MyelomaCide developed by Immunomedics, LkoCide developed by Immunomedics, ProstaCide developed by Immunomedics, MDX-010, anti-CTLA4 antibody, MDX-060, anti-CD30 antibody, MDX-070, MDX-018 developed by Medarex, OSIDEM (IDM-1), anti-HER2 antibody, HuMax (registered trademark)-CD4, anti-CD4 antibody, HuMax-IL15, anti-IL15 antibody, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibody, MOR201, Tremelimumab, anti-CTLA-4 antibody, anti-α5β1 integrin developed by Protein Design Labs, anti-IL-12 developed by Protein Design Labs, ING-1, anti-Ep-CAM antibody developed by Xoma, and MLN01, anti-Beta2 integrin antibody may be included, but are not limited to, and all of the above antibody references in this paragraph are expressly incorporated herein by reference. The sequences of the above antibodies can be obtained from publicly available databases, patents, or literature references. In addition, non-limiting examples of monoclonal antibodies and VH and VL sequences (and, in some cases, CDR sequences indicated to be incorporable into AF2) against cancers, tumors, or target cell markers suitable for incorporation into the compositions of the present disclosure are presented in Table 8.,

[0165] According to the above antigen-binding fragment embodiments, it can be advantageous when having a high binding affinity to capture target cells in which the binding site that recognizes the target cell marker antigen is efficiently destroyed. In a preferred embodiment, when determined by an in vitro binding assay, the AF2 target cell antigen-binding fragment has a K -7 in the range of 10 -10 to 10 d M. Therefore, the subject polypeptides according to any of the embodiments of the present disclosure have the advantage that they can be used multiple times to kill tumor cells. If the affinity of the bispecific antigen-binding fragment for binding to the target cell marker is too high, the composition binds to the expressed target cells and remains on their surface, preventing it from being released and binding to other cells. In one embodiment, the polypeptides according to any of the subject composition embodiments described herein comprise AF2, and AF2 has a K of about 0.1 nM to about 100 nM, or about 0.5 to about 50 nM, or about 1.0 to about 10 nM when determined by an in vitro antigen-binding assay including the target cell marker. d Specifically binds to the target cell marker. In another embodiment, AF2 specifically binds to the target cell marker with 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 (when determined by K d in an in vitro binding assay). In another embodiment, the present disclosure provides a polypeptide comprising AF2, wherein the binding affinity of AF2 for the target cell marker is at least 10 times, or at least 100 times, or at least 1000 times higher than the binding affinity of AF1 for CD3 when measured by an in vitro antigen-binding assay. In another embodiment, the AF1 antigen-binding fragment according to any of the subject embodiments of the present disclosure has a lower binding affinity for CD3 that is at least one order of magnitude, at least two orders of magnitude, or at least three orders of magnitude lower compared to the greater binding affinity of AF2 for the target cell marker antigen when determined as the K d constant in an in vitro assay. A greater binding affinity corresponds to a lower K dis understood to mean a value, for example, 1×10 -9 M is 1×10 -8 has a binding affinity greater than M.

[0166] In another embodiment, the present disclosure provides a polypeptide comprising AF2, wherein AF2 comprises a CDR of a monoclonal antibody having a binding affinity for a target cell marker antigen. In another embodiment, a polypeptide according to any of the target composition embodiments described herein comprises AF2, wherein AF2 comprises a CDR derived from a monoclonal antibody having a binding affinity for a target cell marker antigen, and the CDR of AF2 is selected from the CDRs within the VL and VH sequences of SEQ ID NOs: 719-918.

[0167] In some aspects according to any of the embodiments disclosed herein, the subject polypeptide comprises AF2, wherein AF2 comprises the VL and VH of a monoclonal antibody having a binding affinity for a target cell marker antigen. In some cases, a polypeptide according to any of the target composition embodiments described herein can comprise AF2, wherein AF2 comprises the VL and VH of a monoclonal antibody having a binding affinity for a target cell marker antigen, and the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or being identical to, the amino acid sequence of SEQ ID NOs: 719-918, and the VH comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to, or being identical to, the amino acid sequence of SEQ ID NOs: 719-818.

[0168] It will be understood that the use of the term "antigen-binding fragment" with respect to the disclosed composition embodiments is intended to include portions or fragments of an antibody that retain the ability to bind to an antigen that is a ligand of the corresponding intact antibody. In such embodiments, antigen-binding fragments can include, but are not limited to, 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 (CDR), domain antibodies (dAb), BHH- or BNAR-type single-domain heavy-chain immunoglobulins, single-domain light-chain immunoglobulins, or other polypeptides known in the art that can bind to an antigen. The VL and VH of two antigen-binding fragments can also be configured in a single-chain diabody construct, i.e., the VL and VH of AF1 and AF2 are configured with a linker of appropriate length to allow for diabody placement.

[0169] In certain embodiments, the VL and VH of the antigen-binding fragment are fused by a relatively long linker consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids that, when linked together, have mobility characteristics. In one embodiment, the VL and VH as described in any of the scFv embodiments described herein are linked by a linker of hydrophilic amino acids selected from the sequences GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1142), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 1143), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 1144), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 1145). In other cases, the AF1 and AF2 of the subject composition are linked together by a short linker of hydrophilic amino acids having 3, 4, 5, 6, or 7 amino acids. In one embodiment, the short linker sequence is selected from the group consisting of the sequences SGGGGS (SEQ ID NO: 1146), GGGGS (SEQ ID NO: 1147), GGSGGS (SEQ ID NO: 1148), GGS, or GSP. In another embodiment, the present disclosure provides a composition comprising a single-chain diabody that, upon folding, has the first domain (VL or VH) paired with the last domain (VH or VL) to form one scFv, and these two domains paired in the middle to form the other scFv, where the first and second domains and the third and last domains are fused together by one of the short linkers described above, and the second and third variable domains are fused by one of the long linkers described above. The selection of the short and long linkers can prevent mispairing of adjacent variable domains, thereby facilitating the formation of a single-chain diabody configuration comprising the VL and VH of the first and second antigen-binding fragments.

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 8-13

Table 8-14

Table 8-15

Table 8-16

Table 8-17

Table 8-18

Table 8-19

Table 8-20

Table 8-21

Table 8-22

Table 8-23

Table 8-24

[0170] VI). Bispecific Antigen-Binding Compositions - Composition and Functional Characteristics In another aspect, the present disclosure relates to novel chimeric bispecific antigen-binding compositions that bind to an antigen or epitope of the CD3 protein complex of effector cells (e.g., T cells) and a second target cell marker associated with diseased cells or tissues. Thus, they can be referred to as T cell engagers. As described in more detail below, the bispecific antigen-binding compositions that confer advantages over bispecific T cell engagers and related compounds known in the art are composed in an activatable prodrug form. The various compositions of the present disclosure have enhanced stability during their manufacture and purification, enhanced stability when administered to a subject and increased half-life in the circulating blood, the ability to be activated at the intended site of therapy but not in normal healthy tissues, and when activated by proteolytic cleavage of the release segment and release of the fused AF1 and AF2, exhibit binding affinities for target cells and effector cells that are at least comparable to those of the corresponding conventional bispecific IgG antibodies. When binding effector cells and target cells by AF1 and AF2, an immunological synapse is formed that results in activation of the effector cells and promotes subsequent destruction of the target cells by apoptosis or cytolysis.

[0171] The various bispecific antigen-binding compositions of the present disclosure described herein are specifically designed to be in prodrug form in that the XTEN component shields the antigen-binding fragment and reduces its ability to bind to its ligand until it is released from the composition by proteolytic cleavage of any of the protease cleavage sites located within the release segment. Proteases known to be associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine proteases, aspartic proteases, and metalloproteases, including the specific proteases described herein. This prodrug property of the bispecific antigen-binding composition improves the specificity of the composition for the diseased tissue or cells as compared to bispecific T cell engager therapeutics that are not in prodrug form. In contrast, by specifically activating the bispecific antigen-binding composition in the microenvironment of target cells or diseased tissues where target cell markers and proteases capable of cleaving the release segment are highly expressed, the XTEN of the bispecific antigen-binding fragment and its construct are released upon cleavage of the release segment, and the fused antigen-binding fragment crosslinks cytotoxic effector cells with cells expressing the target cell marker in a highly specific manner, thereby enabling the cytotoxic performance of T cells to be directed towards the target cells. After protease cleavage, the antigen-binding fragment is no longer shielded and effectively regains its full ability to bind to target cells having the target cell marker and effector cells such as cytotoxic T cells by binding to the CD3 antigen, thereby forming part of the T cell receptor complex and triggering T cell activation that mediates the subsequent lysis of target cells expressing a specific target cell marker. Thus, the bispecific antigen-binding compositions of the present disclosure are contemplated to exhibit potent, specific, and efficient target cell killing. In such cases, the cells are selectively removed, thereby reducing the potential for toxic side effects.

[0172] In one aspect, the present disclosure provides an activatable bispecific antigen-binding fragment composition comprising two antigen-binding fragments, a first antigen-binding fragment that targets effector cells and a second antigen-binding fragment that targets a cell marker associated with diseased tissue or cells, each having specific binding affinity for their respective ligands. The design of the subject composition having the first and second antigen-binding fragments (AF1 and AF2, respectively) is based on at least three properties: 1) the composition has a bispecific antigen-binding fragment that has the ability to bind to effector cells and target cells and link them together, resulting in the formation of an immunological synapse; 2) the composition has an XTEN that i) shields both of these antigen-binding fragments, reducing the ability of the composition to bind to target cell ligands and effector cell ligands when the composition is in the intact prodrug form, ii) provides an enhanced half-life when administered to a subject, iii) reduces extravasation of the intact composition from the bloodstream in normal tissues and organs compared to diseased tissue (e.g., a tumor), and iv) confers an increased safety profile compared to conventional bispecific cytotoxic antibody therapeutics, and 3) the RS is activated when cleaved by one or more mammalian proteases near the diseased tissue, thereby releasing the fused bispecific antigen-binding fragments, whereby they regain their full binding affinity for the target ligands. The design of the subject composition utilizes the properties of the XTEN and release segment (RS) components and, as demonstrated by the results of the following exemplary examples, by their positioning relative to the bispecific antigen-binding fragments, the aforementioned properties are achieved.

[0173] In one embodiment, the polypeptide according to any of the bispecific antigen-binding fragment composition embodiments described herein having two antigen-binding fragments (AF1 and AF2), a single RS, and a single XTEN, in an uncleaved state, from the N-terminus to the C-terminus, may have a structural arrangement of AF2-AF1-RS1-XTEN1, AF1-AF2-RS1-XTEN1, XTEN1-RS1-AF2-AF1, XTEN1-RS1-AF1-AF2, or diabody-RS1-XTEN1, or XTEN1-RS1-diabody, where the diabody comprises the VL and VH of AF1 and AF2.

[0174] In another aspect, the present disclosure provides bispecific antigen-binding compositions having two antigen-binding fragments (AF1 and AF2), two RSs, and two XTENs. The design of these compositions was informed by considering that the addition of a second XTEN further reduces the binding affinity of the intact composition for each ligand of the AF1 and AF2 antibody fragments, further reducing unintended binding of the composition to healthy tissue or cells of a subject when administered to the subject, 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 results in a surprising decrease in the binding affinity of the intact polypeptide for each ligand of the AF1 and AF2 antibody fragments compared to a composition having a single RS and XTEN when assayed in vitro, and as described in the examples below, also results in a decrease in toxicity in an animal model of the disease when administered at a therapeutically effective dose. In embodiments of the subject composition having two antigen-binding fragments, two RSs, and two XTENs, the composition may have a structural arrangement of 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 (where the diabody comprises the VL and VH of AF1 and AF2), or XTEN1-RS1-diabody-RS2-XTEN2 (where the diabody comprises the VL and VH of AF1 and AF2) from the N-terminus to the C-terminus in an uncleaved state.

[0175] When the RS of the bispecific antigen-binding composition is cleaved by mammalian proteases in the environment of target cells and converted from the prodrug form to the active form or the apoprotein form, upon cleavage and release of the bispecific antigen-binding fragment and XTEN from the composition, the fused AF1 and AF2 bind to effector cells (e.g., T cells having CD3) and diseased cells (e.g., tumor or cancer cells) having a target cell marker antigen that can bind to AF2 and link them together, at which time the effector cells are activated, which is a feature of various designed compositions. In one embodiment, the RS of the bispecific antigen-binding composition is cleaved, the antigen-binding fragment is released, and the subsequent simultaneous binding to effector cells and target cells can result in activation of at least 3-fold, or 10-fold, or 30-fold, or 100-fold, or 300-fold, or 1000-fold of the effector cells, and this activation is evaluated by cytokine, production of cytolytic proteins, or lysis of target cells, as evaluated in an in vitro cell-based assay. In another embodiment, the simultaneous binding of the released fused AF1 and AF2 to T cells having the CD3 antigen and diseased cells having a target cell marker antigen forms an immunological synapse, and this binding results in the release of effector molecules derived from T cells that can lyse the diseased 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, ELISA, radiometric 51Cr release assays, fluorometric europium release assays, CalceinAM release assays, photometric MTT assays, XTT assays, WST-1 assays, alamar blue assays, radiometric 3H-Thd incorporation assays, clonogenic assays for measuring cell proliferation activity, fluorometric Rhodamine123 assays for measuring mitochondrial membrane potential, apoptosis assays monitored by FACS-based phosphatidylserine exposure, ELISA-based TUNEL assay, caspase activity assays, and cell morphology assays, or other assays known in the art for assays of cytokines, cytolytic proteins, or cell lysis, or the methods described in the following examples.

[0176] Without being bound by a particular theory, it is believed that by using the above-described bispecific antigen-binding composition format, upon cleavage of RS, the released fusion AF1 and AF2 can kill target cells by mobilization of cytotoxic effector cells without the need for pre-stimulation and / or co-stimulation. Furthermore, the independence from pre-stimulation and / or co-stimulation of effector cells can substantially contribute to the very high cytotoxicity mediated by the released fusion AF1 and AF2 antigen-binding fragments. In some embodiments, the released AF1 and AF2 in which AF1 remains fused to AF2 by a linker peptide are designed with binding specificities such that they can bind to a target cell marker pre-selected by AF2 having binding specificity for a target cell marker associated with a cytotoxic effector cell (e.g., a T cell, NK cell, cytokine-induced killer cell (CIK cell), tumor cell, cancer cell, or cell associated with diseased tissue), bringing them close together and linking them, thereby resulting in an immunological synapse of released cytokines and effector molecules against the target tumor or cancer cells as well as selective, directed, and local effects, and as a result, the tumor or cancer cells are damaged or destroyed, providing a therapeutic benefit to the subject. The released AF1 that binds to an effector cell antigen can modulate one or more functions of the effector cell, bringing about or contributing to a cytolytic effect on the target cells, such as tumor cells, to which AF2 binds. The effector cell antigen can be expressed by an effector cell or other cells. In one embodiment, the effector cell antigen is expressed on the cell surface of the effector cell. Non-limiting examples of effector cell antigens are CD3, CD4, CD8, CD16, CD25, CD38, CD45RO, CD56, CD57, CD69, CD95, CD107, and CD154. Thus, one of ordinary skill in the art will understand that the composition of the subject composition is intended to selectively or disproportionately deliver the active form of the composition to the target tumor tissue or cancer cells as compared to healthy tissue or healthy cells in the subject to which the composition is administered, thereby providing a therapeutic benefit.As is apparent from the foregoing, the present disclosure provides a large polypeptide family in a configuration designed to provide desired properties.

[0177] The design of a target bispecific antigen-binding composition in which the masking effect is conferred by XTEN of a circulating composition intact and at the same time a decrease in the possibility of binding to effector cells and target tissues occurs, so that the overall side effect and safety profile (e.g., therapeutic index) is improved compared to a bispecific antigen-binding composition not linked to a masking moiety such as XTEN, is to result in a decrease in the production of Th1 T cell-related cytokines or other inflammation-promoting mediators during systemic exposure when administered to a subject. As important components of cellular immunity, the production of IL-2, TNF-alpha, and IFN-gamma is a prominent feature of the Th1 response, especially in T cells stimulated by anti-CD3 (Yoon, S.H. 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), and (Romagnani S. T-cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol. 2000. 85(1):9-18). Il-4, IL-6, and IL-10 are also inflammation-promoting cytokines important for 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 (registered trademark)) antibody composition blinatumomab as a potential therapy. Int. Immunol. (2015) 27(1):31-37).In one embodiment, the intact bispecific antigen-binding composition of the embodiments described herein, when the intact polypeptide contacts effector cells and target cells in an in vitro cell-based cytokine stimulation assay, under equivalent conditions, e.g., in an in vitro cell-based cytokine stimulation assay performed at equivalent molar concentrations, exhibits at least a 3-fold, or at least a 4-fold, or at least a 5-fold, or at least a 6-fold, or at least a 7-fold, or at least an 8-fold, or at least a 9-fold, or at least a 10-fold, or at least a 20-fold, or at least a 30-fold, or at least a 50-fold, or at least a 100-fold, or at least a 1000-fold reduction in the production of Th1 and / or pro-inflammatory cytokines compared to the corresponding released AF1 and AF2 (which remain fused together after release by RS proteolysis) of the corresponding protease-treated composition that stimulates Th1 and / or cytokine levels. Non-limiting examples of Th1 and / or pro-inflammatory cytokines are IL-2, IL-4, IL-6, IL-10, TNF-alpha, and IFN-gamma. In the aforementioned one embodiment, the production of Th1 cytokines is assayed in an in vitro assay comprising effector cells such as PBMC or CD3+ T cells and target cells having a target cell marker antigen disclosed herein. In another embodiment, the cytokines can be evaluated from blood, body fluids, or tissue samples taken from a subject to whom the polypeptide composition has been administered. In the aforementioned embodiments, the subject can be a mouse, a rat, a monkey, and a human. However, as an advantage of the subject bispecific antigen-binding composition of the embodiments described herein, it has been found that the cytolytic properties of the composition do not require pre-stimulation by cytokines, and the formation of an immunological synapse of effector cells bound to target cells by antigen-binding fragments is sufficient to affect cytolysis or apoptosis in target cells.Nevertheless, the production of pro-inflammatory cytokines is a useful marker for assessing the efficacy or effectiveness of a subject polypeptide composition, whether by in vitro assay or by monitoring the treatment of a subject having an affected tissue (e.g., a tumor, etc.) after administration of the subject bispecific antigen-binding composition.

[0178] In the context of the use of bispecific antigen-binding compositions in a subject, for the purposes of the present disclosure, the subject bispecific antigen-binding compositions are designed to take advantage of the difference in pore size of the vasculature in tumors or inflamed tissues compared to healthy vasculature by the addition of XTEN such that extravasation of the intact bispecific antigen-binding compositions in normal tissues is reduced, but in the leaky environment of tumor vasculature or other inflamed portions of the diseased tissue, the intact construct can leak proteases in the diseased tissue environment and thereby be activated to release antigen-binding fragments to effector and target cells (see, for example, FIG. 5). In the case of the RS of the bispecific antigen-binding composition, this design takes advantage of the situation where the RS sequence, which is sensitive to one or more proteases produced by the diseased tissue, such as a tumor, can be cleaved by the proteases when the bispecific antigen-binding composition is in the vicinity of the tumor (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 appreciated, the reduction in the molecular weight and hydrodynamic radius of the composition allows the released fusion AF1 and AF2 to move more freely in solution, move through smaller pore spaces in tissues and tumors, leak more easily from the larger pores of tumor vasculature, penetrate more easily into tumors, and confers the property of increasing the ability to bind to and link effector and tumor cells together. Such properties can be measured by different assays. Thus, one of ordinary skill in the art will understand that in the context of treating a subject with the subject composition, the bispecific antigen-binding composition exists in prodrug form and is converted to a more active form when it enters a particular cellular environment by the action of proteases co-localized with the diseased tissue or cells.Upon release from the composition by the action of proteases in the target tissue, AF1 having binding specificity for an effector cell antigen and the fused AF2 having binding specificity for a target cell marker antigen of the diseased cell regain the ability to bind to effector cells and target cells and link them together, forming an immunological synapse. Formation of the immunological synapse activates the effector cells, activating various signaling pathways to activate new gene transcription and, by exocytosis, release the effector molecule contents of the vesicles. 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 "effector molecules") such as perforin and granzyme that kill the target. It is particularly contemplated that when the released bispecific antigen-binding fragment of the bispecific antigen-binding composition binds to effector cells and target tumor cells and links them together simultaneously, at a very low effector:target (E:T) ratio, the tumor cells are acted upon by the effector molecules released into the intercellular immunological synapse by the effector cells, resulting in tumor cell damage, perforin-mediated lysis, granzyme B-induced cell death, and / or apoptosis. Thus, in another aspect, when an activatable bispecific antigen-binding fragment composition is administered to a subject having a disease such as a tumor, the prodrug form remains in the circulatory system in normal tissue, but the prodrug form of the construct is activated by proteases co-localized with the tumor and the released antigen-binding fragment binds to effector cells (e.g., T cells) and tumor cells expressing the target cell marker targeted by AF2 of the composition and links them together, leaking into the more permeable vascular structures of the tumor, at which time the effector cells are activated, resulting in lysis of the tumor cells.In other words, in some cases, due to the more permeable vasculature in the tumor tissue, the bispecific antigen-binding polypeptide can leak into the tissue, where tumor-associated proteases act on the release segment (RS), cleave it, release the binding moiety, and then can bind to effector cells and tumor-associated cells and link them together. In the case of normal tissue, extravasation can be blocked by a more tight vasculature barrier, or if the bispecific antigen-binding polypeptide leaks to some extent, the bispecific antigen-binding polypeptide may mainly remain in the "pro" form because there may be insufficient proteases in healthy tissue to release the binding moiety with a net effect that an immunological synapse is not formed. In some cases, the released fusion AF1 and AF2 in the target tumor bound to both tumor cells and effector cells exhibit an increase in the ability to activate effector cells by 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 compared to the corresponding intact bispecific antigen-binding composition that has not been cleaved. In other cases, the released linked AF1 and AF2 in the target tumor bound to both tumor cells and effector cells exhibit an increase in the ability to lyse tumor cells by 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 compared to the corresponding intact bispecific antigen-binding composition that has not been cleaved in the tumor. In the foregoing embodiments, effector cell activation and / or cytotoxicity can be assayed by conventional methods known in the art such as measurement of the number of activated effector cells, assay of cytokines, measurement of tumor size, or histopathology. In the foregoing embodiments, the subject can be a mouse, a rat, a dog, a monkey, and a human.Specifically, when the target composition is administered to a subject having a disease with a target cell marker to which AF2 can bind, the bispecific antigen-binding composition is designed to exhibit enhanced therapeutic indices and a 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 and steric hindrance in the prodrug form. In particular, it is contemplated that the bispecific AF1 and AF2 can be released near or within a target tissue (e.g., a tumor) that produces a protease for which RS is a substrate (achieved by inclusion of the cleavage sequence into RS).

[0179] VII). Methods and Uses of Bispecific Antigen-Binding Compositions In another aspect, the present disclosure provides an activatable bispecific antigen-binding composition, and a pharmaceutical composition comprising a bispecific antigen-binding composition that is particularly useful in the prevention, treatment, and / or amelioration of certain diseases, such as, but not limited to, medical conditions such as cancer, tumors, or inflammatory diseases. For use in the treatment of diseases, the bispecific antigen-binding compositions of the invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to the medical practitioner.

[0180] In particular, several therapeutic strategies involving the modulation of T cell responses by targeting TcR signaling, using the VL and VH portions of anti-human CD3 monoclonal antibodies that are clinically widely used in immunosuppressive regimens, have been used to design polypeptide compositions for use in methods of treating subjects having a cancerous disease. The CD3-specific monoclonal OKT3 was the first such monoclonal to be approved for use in humans (Sgro, Toxicology 105(1995), 23-29) and is clinically widely used as an immunosuppressant at the time of 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 recognition structure of T cells (TCR) that is essential for signaling. 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 bound by theory, it is thought that cytotoxic T cells require direct cell-to-cell contact to effect target cell lysis, and the TCR on cytotoxic T cells must recognize and associate with an appropriate antigen on the target cell. This creates an immunological synapse, which then initiates a signaling cascade within the cytotoxic T cell, leading to T cell activation and the production of various cytotoxic cytokines and effector molecules.Perforin and granzyme are highly toxic molecules stored in pre-formed 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.

[0181] The subject bispecific antigen-binding composition described herein, in which AF1 having specific binding affinity for CD3 of T cells is closely fused to AF2 having specific binding affinity for a target cell marker, is a T cell engager capable of, upon release from its intact prodrug form by cleavage of a release segment, fully regaining its ability to bind to T cells and target cells, promoting activation of the T cells, and forming an immunological synapse that promotes subsequent destruction of the tumor cells by apoptosis or cytolysis.

[0182] The present disclosure contemplates a method of using a bispecific antigen-binding composition engineered to target a wide range of malignant cells, such as tumors, in addition to effector cells, in order to bring about beneficial therapeutic results in that the bispecific antigen-binding composition is designed to initiate target cell lysis and, while AF1 binds and associates with CD3 to activate cytotoxic T cells, AF2 targets various different target cell markers characteristic of a particular malignancy and brings them together to form an immunological synapse. A particular advantage of this design is that the physical binding of cytotoxic effector cells to cells having target cell markers eliminates the need for antigen processing, MHCI / β2-microglobulin, and costimulatory molecules. Examples of important target cell markers include, but are not limited to, the markers disclosed herein. Due to the range of such target cell markers (described more broadly above) that can be engineered into various embodiments of the subject bispecific antigen-binding composition, it will be understood that the resulting composition will be useful for a variety of diseases, including blood cancers and solid tumors. In one embodiment, the present disclosure provides a method of treating a subject having a tumor. The tumors to be treated can include tumor cells resulting from cells selected from the group consisting of stromal cells, fibroblasts, myofibroblasts, glial cells, epithelial cells, adipocytes, lymphocyte cells, vascular cells, smooth muscle cells, mesenchymal cells, breast tissue cells, prostate cells, kidney cells, brain cells, colon cells, ovarian cells, uterine cells, bladder cells, skin cells, stomach cells, urogenital tract cells, cervical cells, uterine cells, small intestine cells, liver cells, pancreatic cells, gallbladder cells, bile duct cells, esophageal cells, salivary gland cells, lung cells, and thyroid cells. A further advantage of the composition is that the cytotoxic effector cells are not consumed during the damage / destruction of the cross-linked target cancer cells, so that after causing the lysis of one target cell, the activated effector cells are released and can migrate through the local tissue towards other target cancer cells, re-bind to AF1-AF2 and the target antigen, and initiate additional cell lysis.In addition, in a local environment such as a solid tumor, the release of effector cell molecules such as perforin and granzyme results in damage to tumor cells adjacent to a given molecule of the bispecific binding domain but not bound thereby, which is intended to result in stasis of tumor growth or regression.

[0183] Accordingly, 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 a target cell marker, 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 linking of the target cells and effector cells, and as a result, effector molecules derived from effector cells capable of lysing the target cells are released into the synapse, resulting in apoptosis, cytolysis, or death of the target cancer or tumor cells. The utility of the present disclosure will be understood. Furthermore, one of ordinary skill in the art will understand that the use of the bispecific antigen-binding composition can result in a more beneficial therapeutic effect systemically and over a longer period than a "single hit kill" when an immunological synapse is formed by the binding of the released binding domain to effector cells and target cancer cells.

[0184] In one aspect, the present disclosure relates to a method of treating a disease in a subject such as cancer or an inflammatory disorder. In some embodiments, the present disclosure provides a method of treating a disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a bispecific antigen-binding composition according to any of the embodiments described herein. 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 such that the therapeutically beneficial effects outweigh 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 of time necessary to achieve the desired prophylactic result.

[0185] The therapeutically effective dosage of the bispecific antigen-binding compositions described herein generally provides a therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the bispecific antigen-binding compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal experiments are used to determine the LD 50 (lethal dose for 50% of the population) and ED 50 (therapeutically effective dose in 50% of the population). The dosage ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the ratio of LD 50 / ED 50 . Bispecific antigen-binding compositions with a high therapeutic index are preferred. In one aspect, the bispecific antigen-binding molecules according to the present disclosure exhibit a high therapeutic index. Using the data obtained from cell culture assays and animal experiments, a dosage range suitable for use in humans can be formulated. The dosage is preferably within the range of blood concentrations that include little or no toxicity and the ED 50 . The dosage can vary within this range depending on various factors such as the dosage form used, the route of administration utilized, the condition of the subject, etc. The exact formulation, route of administration, and dosage can be selected by the individual physician considering the condition of the patient (see, for example, Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Ch. 1, p. 1). One of ordinary skill in the art will readily recognize that in many cases, the bispecific antigen-binding compositions may only provide a partial benefit rather than a cure. In some aspects, any physiological change that has some benefit is also considered therapeutically beneficial. Thus, in some aspects, the amount of the bispecific antigen-binding composition that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount". The subject, patient, or individual in need of treatment is typically a mouse, rat, dog, monkey, or human.

[0186] The bispecific antigen-binding composition of the present invention can be administered in combination with one or more other agents during treatment. For example, the bispecific antigen-binding molecule according to any of the embodiments described herein can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents can include any active ingredient suitable for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulatory agent, an immuno-oncology antibody, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, a cell apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis-inducing substances. In certain embodiments, the additional therapeutic agent is an anti-cancer agent, such as a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, hormone therapy, a kinase inhibitor, a receptor antagonist, a tumor cell apoptosis activator, or an anti-angiogenic agent.

[0187] In one embodiment of a method for treating a disease in a subject, the disease to be treated is a cancer tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, genitourinary cancer, ovarian cancer, ovarian cancer with malignant ascites, vaginal cancer, vulvar cancer, Ewing's sarcoma, peritoneal carcinomatosis, uterine serous cancer, parathyroid cancer, endometrial cancer, cervical cancer, colorectal cancer, epithelial intraperitoneal malignancy with malignant ascites, uterine cancer, peritoneal mesothelioma, kidney cancer, lung cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular cancer, retinoblastoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, testicular cancer, bile duct cancer, bone cancer, salivary gland cancer, thyroid cancer, craniopharyngioma, carcinoid tumor, epithelial cancer, masculinizing tumor, adenocarcinoma, sarcoma of any origin, primary hematological malignancies (including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, B-cell-derived chronic lymphocytic leukemia, hairy cell leukemia, myeloproliferative neoplasm disorders, or myelodysplastic disorders), myasthenia gravis, Graves' disease, Kaposi's sarcoma, neuroblastoma, Hashimoto's thyroiditis, Wilms' tumor, or Goodpasture syndrome. A therapeutically effective amount can produce a beneficial effect in assisting in the treatment (e.g., cure or reduction in severity) or prevention (e.g., reduction in the likelihood of recurrence) of cancer or a tumor. In another embodiment of a method for treating a disease in a subject, the pharmaceutical composition is administered to the subject as two or more therapeutically effective doses administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, or once a month. In another embodiment of the method, the pharmaceutical composition is administered to the subject as two or more therapeutically effective doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months.In another embodiment of the method, a first low priming dose is administered to a subject, followed by one or more higher maintenance doses over a dosing schedule of at least 2 weeks, or at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months. The initial priming dose administered is selected from the group consisting of at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, at least about 0.1 mg / kg, and one or more subsequent maintenance doses administered are selected from the group consisting of at least about 0.02 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.22 mg / kg, 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 the method, the pharmaceutical composition is administered to the subject intradermally, subcutaneously, intravenously, intraarterially, intraperitoneally, intracavitary, intrathecally, or intramuscularly. In another embodiment of the method, the pharmaceutical composition is administered to the subject as one or more therapeutically effective bolus doses or by infusion for 5 minutes to 96 hours, as long as it shows tolerance to maximum safety and efficacy.In another embodiment of the 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 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 0.22 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, selected from the group consisting of. In another embodiment of the 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 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 or more of the intact, un-cleaved bispecific antigen-binding composition that is maintained in the subject 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 amount 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 0.22 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. 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, at least about 0.1 mg / kg, and subsequent doses are selected from the group consisting of at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.14 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.22 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.In the foregoing embodiments, administration to a subject results in a plasma concentration of the polypeptide of at least about 0.1 ng / mL to at least about 2 ng / mL or more in the subject over at least about 3 days, at least about 7 days, at least about 10 days, at least about 14 days, or at least about 21 days. In the foregoing embodiments of the method, the subject can be a mouse, rat, dog, monkey, or human.

[0188] VIII). Nucleic Acid Sequence In another aspect, the invention relates to an isolated polynucleotide sequence encoding a polypeptide or bispecific antigen-binding composition according to any of the embodiments described herein, and a sequence complementary to the polynucleotide molecule encoding the polypeptide composition embodiment.

[0189] In some embodiments, the invention provides an isolated polynucleotide sequence encoding an AF1 sequence, or an AF2 sequence, or a release segment sequence (RS1 and RS2), or an XTEN sequence according to any of the embodiments described herein, or a complement of the polynucleotide sequence thereof. In one embodiment, the invention provides an isolated polynucleotide sequence encoding a polypeptide or bispecific antigen-binding composition according to any of the embodiments described herein, or a complement of the polynucleotide sequence thereof. In one embodiment, the invention provides an isolated polynucleotide sequence encoding a polypeptide or bispecific antigen-binding composition, the polynucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the polynucleotide sequence set forth in Table 9.

[0190] In another aspect, the present disclosure relates to a polynucleotide sequence encoding a polypeptide or bispecific antigen-binding composition described in any of the embodiments described herein, or a method of generating a sequence complementary to the polynucleotide sequence (including homologous variants thereof), and a method of expressing a protein expressed by the polynucleotide sequence. Generally, the method includes generating a polynucleotide sequence encoding a proteinaceous polypeptide or bispecific antigen-binding composition described in any of the embodiments described herein, and incorporating the coding gene into an appropriate expression vector in a host cell. For the production of the encoded polypeptide or bispecific antigen-binding composition described in any of the embodiments described herein, the method includes transforming an appropriate host cell with the expression vector and culturing the host cell under conditions that cause or permit the expressed polypeptide or bispecific antigen-binding composition described in any of the embodiments described herein to be expressed in the transformed host cell, thereby producing the polypeptide or bispecific antigen-binding composition, which is recovered by the methods described herein or by standard protein purification methods known in the art. Standard recombinant techniques in molecular biology are used to generate the polynucleotides and expression vectors of the present disclosure.

[0191] According to the present disclosure, a nucleic acid sequence (or its complement) encoding a polypeptide or bispecific antigen-binding composition described in any of the embodiments described herein is used to generate a recombinant DNA molecule directed to expression in a suitable host cell. Several cloning strategies are suitable for the practice of the present disclosure, and many of them are used to generate a construct or its complement that contains a gene encoding a composition of the present disclosure. In one embodiment, a cloning strategy is used to create a gene encoding a construct that contains nucleotides encoding a polypeptide or bispecific antigen-binding composition used to transform a host cell to express the composition. In the foregoing embodiments described above in this paragraph, the gene may contain nucleotides encoding an antigen-binding fragment, a release segment, and XTEN in the configuration disclosed herein.

[0192] In one approach, a construct containing a DNA sequence encoding a polypeptide or bispecific antigen-binding composition construct is first prepared. Exemplary methods for preparing such constructs are described in the Examples. This construct is then used to create an expression vector suitable for transforming a host cell, such as a prokaryotic or eukaryotic host cell, to express and recover the polypeptide construct. If desired, the host cell is E. coli. In another embodiment, the host cell is selected from BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, NIH3T3 cells, COS, HeLa, CHO, or yeast cells. Exemplary methods for creating the expression vector, transforming the host cell, and expressing and recovering XTEN are described in the Examples.

[0193] Genes encoding polypeptides or bispecific antigen-binding composition constructs can be made in one or more steps, either completely synthetically or synthetically in combination with enzymatic processes such as restriction enzyme-mediated cloning, PCR, and overlap extension, including the methods fully described by the examples. The methods disclosed herein can be used, for example, to ligate the sequences of polynucleotides encoding various components (e.g., binding domains, linkers, release segments, and XTEN) of the desired length and sequence of the polypeptide composition. 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 the E. coli or mammalian host cells utilized for the production of the polypeptide or bispecific antigen-binding composition. In one method of the present disclosure, as described above, a library of polynucleotides encoding the components of the construct is made and then constructed. The resulting gene is then constructed as described herein, the resulting gene is used to transform host cells, and the polypeptide composition is produced and recovered to evaluate its properties.

[0194] Next, the polynucleotide resulting from encoding the polypeptide or bispecific antigen-binding composition sequence can be individually cloned into an expression vector. The nucleic acid sequence is inserted into the vector 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 a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components uses standard ligation techniques known to those skilled in the art. Such techniques are well known in the art and are fully described in the scientific and patent literature. Various vectors are publicly available. The vector can be in the form of, for example, a plasmid, cosmid, viral particle, or phage that is conveniently used in recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is introduced. Thus, the vector can be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication does not depend on chromosomal replication, such as a plasmid. Alternatively, the vector can be a vector that, when introduced into a host cell, integrates into the host cell genome and is replicated along with the chromosome into which it is integrated. When introduced into a suitable host cell, 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 the transcribed mRNA of the light chain CDR or heavy chain CDR, the antigen-binding fragment, or the bispecific antigen-binding composition can be detected and / or quantified by conventional hybridization assays (e.g., Northern blot analysis), amplification procedures (e.g., RT-PCR), SAGE (U.S. Patent No. 5,695,937), and array-based technologies (see, e.g., U.S. Patents Nos. 5,405,783, 5,412,087, and 5,445,934) using a probe complementary to any region of the antigen-binding unit polynucleotide.

[0195] The present disclosure provides for the use of a plasmid expression vector that is compatible with, recognized by, and operably linked to a gene encoding a polypeptide for the controlled expression of the polypeptide, comprising a replication sequence and a control sequence. The vector typically carries a replication site and a sequence encoding a protein that can provide phenotypic selection within the transformed cell. Such vector sequences are well known in various bacteria, yeasts, and viruses. Useful expression vectors that can be used include, for example, chromosomal segments, extrachromosomal segments, and synthetic DNA sequences. An "expression vector" refers to a DNA construct comprising a DNA sequence operably linked to suitable control sequences capable of effecting the expression of DNA encoding a polypeptide in a suitable host. It is a requirement that the vector be replicable and viable within the selected host cell. Vectors with low or high copy numbers may be used as desired.

[0196] Suitable vectors include SV40 as well as derivatives of pcDNA and known bacterial plasmids, such as col E1, pCR1, pBR322, pMal-C2, pET, pGEX (described in Smith, et al., Gene 57:31-40 (1988)), pMB9, and their derivatives, plasmids such as RP4, phage DNA such as numerous derivatives of phage λ such as NM989, and other phage DNA such as M13 and filamentous single-stranded phage DNA; yeast plasmids such as the 2 micron plasmid or derivatives of the 2 micron plasmid, and centromere and integrating yeast shuttle vectors; vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA such as phage DNA or plasmids modified to use expression control sequences, etc., but are not limited thereto. Similarly, yeast expression systems that can be used in the present disclosure include, but are not limited to, non-fusion pYES2 vectors (Invitrogen), fusion pYESHisA, B, C (Invitrogen), pRS vectors, etc. The control sequences of the vector include a promoter that causes transcription, any operator sequence that controls such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control the termination of transcription and translation. The promoter can be any DNA sequence that exhibits transcriptional activity in the selected host cell and can be derived from a gene encoding a protein that is either homologous or heterologous to the host cell.Promoters suitable for use in expression vectors with prokaryotic hosts include, for example, the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978), Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, the tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980), European Patent No. 36,776], 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 linked to DNA encoding the XTEN polypeptide. Promoters for use in bacterial systems may also include a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the polypeptide polypeptide.

[0197] Expression of the vector can also be determined by examining the antigen-binding fragment or the components of the expressed bispecific antigen-binding composition. A variety of techniques for protein analysis are available in the art. These include, but are not limited to, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoradiometric assay, in situ immunoassay (e.g., using colloidal gold, enzyme, or radioisotope labeling), Western blot analysis, immunoprecipitation assay, immunofluorescence assay, and SDS-PAGE.

[0198] IX). Methods for Producing Polypeptides and Bispecific Antigen-Binding Compositions In another aspect, the present disclosure provides a method of manufacturing a target composition. In one embodiment, the method comprises culturing a host cell comprising a nucleic acid construct encoding a polypeptide or bispecific antigen-binding composition according to 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 standard purification methods (e.g., column chromatography, HPLC, etc.) by 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 correctly folded. In another embodiment of the method of making, the expressed polypeptide or bispecific antigen-binding composition is recovered, wherein at least or at least 90%, or at least 95%, or at least 97%, or at least 99% of the polypeptide or bispecific antigen-binding composition is recovered in a monomeric soluble form.

[0199] In another aspect, the present disclosure relates to methods for producing polypeptides and bispecific antigen-binding compositions at high fermentative expression levels of functional proteins using E. coli or mammalian host cells, as well as methods for providing expression vectors encoding constructs useful for generating cytotoxic polypeptide construct compositions at high expression levels. In one embodiment, the method comprises: 1) preparing a polynucleotide encoding a polypeptide according to any of the embodiments disclosed herein; 2) cloning the polynucleotide into an expression vector, wherein the expression vector can be a plasmid or other vector under the control of transcriptional and translational sequences suitable for high-level protein expression in a biological system; 3) transforming a suitable host cell with the expression vector; and 4) culturing the host cell in a conventional nutrient medium under conditions suitable for the expression of the polypeptide composition. Optionally, the host cell is E. coli. By this method, the expression of the polypeptide results in a fermentation titer of at least 0.05 g / L, or at least 0.1 g / L, or at least 0.2 g / L, or at least 0.3 g / L, or at least 0.5 g / L, or at least 0.6 g / L, or at least 0.7 g / L, or at least 0.8 g / L, or at least 0.9 g / L, or at least 1 g / L of 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 correctly folded. As used herein, the term "correctly 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 present disclosure provides a method for producing a polypeptide or a bispecific antigen-binding composition, comprising culturing, under a fermentation reaction, a host cell comprising a vector encoding a polypeptide, wherein the polypeptide or the polypeptide comprising the bispecific antigen-binding composition is present at a concentration of greater than about 10 milligrams per gram (mg / g), or at least about 250 mg / g, or about 300 mg / g, or about 350 mg / g, or about 400 mg / g, or about 450 mg / g, or about 500 mg / g of dry weight host cells when the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, under conditions effective to express the polypeptide product, and wherein the antigen-binding fragment of the expressed protein is correctly folded. In another embodiment, the present disclosure provides a method for producing a polypeptide or a bispecific antigen-binding composition, comprising culturing, under a fermentation reaction, a host cell comprising a vector encoding a composition, wherein the polypeptide or the polypeptide comprising the bispecific antigen-binding composition is present at a concentration of greater than about 10 milligrams per gram (mg / g), or at least about 250 mg / g, or about 300 mg / g, or about 350 mg / g, or about 400 mg / g, or about 450 mg / g, or about 500 mg / g of dry weight host cells when the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, under conditions effective to express the polypeptide product, and wherein the expressed polypeptide product is soluble.

[0200] The following are examples of the compositions of the present disclosure and the evaluation of the compositions. It is understood that various other embodiments can be implemented in view of the general description provided above.

Examples

[0201] Example 1: Construction of a bispecific antigen-binding polypeptide having two release segments. To generate a plasmid from which individual scFvs can be removed 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: 1150), 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 with silent point mutations 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 the In-Fusion kit (New England Biolabs) to construct pJB0035. Subsequently, 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 annealing 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: 1150).

[0202] To generate plasmids with various CD3 binding domain variants, pCW1880 was digested with Bsu36I and NheI to remove the UCHT1 anti-CD3 scFv. DNA fragments encoding the designed CD3 variants were synthesized. Each gene fragment contained 30 nucleotides with restriction sites at the 5’ and 3’ ends to function as DNA overlaps for Gibson DNA constructs. The synthetic DNA fragments were cloned into the digested backbones using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to construct pJB0205, pJB0206, pJB0207, and pJB0208.

[0203] 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’ homology region to the N-terminal backbone DNA and the uncleavable release segment at the C-terminus (RSR3058, amino acid sequence TTGEAGEAAGATSAGATGP (SEQ ID NO: 100)). A second PCR product encoding the light chain of the anti-EpCAM antibody 4D5MOCB and a portion of its heavy chain was amplified using primers containing a 16-21 bp 5’ homology region to the N-terminal RSR3058 and the heavy chain of the C-terminal 4D5MOCB. These PCR fragments were cloned into a backbone vector digested with BsiWI-SacII encoding the remaining portion of the 4D5MOCB heavy chain / anti-CD3 tandem scFv, a second copy of the uncleavable release segment RSR3058, and AE837 XTEN having a 6xHIS (SEQ ID NO: 1150) affinity tag 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 the STII secretion leader, and its components are (from N-terminus to C-terminus) AE292 XTEN, the uncleavable release segment RSR3058, the anti-EpCAM-anti-CD3 bispecific tandem scFv, with RSR3058 fused to AE867 XTEN having a 6xHIS (SEQ ID NO: 1150) affinity tag. The resulting construct is pJB0084 (Table 9).

[0204] Using pJB0084 as a template, a bispecific antigen-binding polypeptide construct encoding AE292 XTEN, a cleavable release segment RSR2295, and an anti-EpCAM-anti-CD3 bispecific tandem scFv was prepared with RSR2295 fused to AE868 XTEN. The plasmid utilized two PCR products using pJB0084 as a template. The first encoded AE292 XTEN with a 6xHIS (SEQ ID NO: 1150) affinity tag, a 5' homologous region to the vector backbone, and a 3' homologous region encoding the first RSR2295. The second encoded an anti-EpCAM-anti-CD3 bispecific tandem scFv with 5' and 3' homologous regions encoding the release segments RSR2295 of the tandem scFv. The third fragment encoded AE868 XTEN with a C-tag affinity tag (amino acid sequence EPEA (SEQ ID NO: 1149)) having a 5' homologous region encoding the second RSR2295 and a 3' homologous region to the backbone vector. These three PCR fragments were cloned into pJB0084 digested with BsiWI-NotI using the In-Fusion Plasmid Assembly Kit. The final vector, pJB0169, encodes a bispecific antigen-binding polypeptide molecule under the control of the PhoA promoter and the STII secretion leader, and its components are (from N-terminus to C-terminus) a 6xHIS affinity tag (SEQ ID NO: 1150), AE292 XTEN, release segment RSR2295, an anti-EGFR-anti-CD3 bispecific tandem scFv, RSR2295, and AE868 XTEN with a C-tag affinity tag, along with its DNA sequence.

[0205] To construct pJB0163 and pJB0179, pJB0169 was digested with DraIII and BtsI to remove 5’RSR2295, the anti-EGFR-anti-CD3 bispecific tandem scFv, RSR2295, and the first 72 amino acids of AE868XTEN. For pJB0163, DNA fragments encoding RSR3058, the anti-CD3 light chain, the anti-EGFR light and heavy chains, the anti-CD3 heavy chain, RSR3058, and the first 72 amino acids of AE868 XTEN were synthesized. For pJB0179, DNA fragments encoding RSR2295, the anti-CD3 light chain, the anti-EGFR light and heavy chains, the anti-CD3 heavy chain, RSR2295, and the first 72 amino acids of AE868 XTEN were synthesized. Each of these gene fragments also contained 30 nucleotides with restriction sites at the 5’ and 3’ ends to function as DNA overlaps for Gibson DNA Assembly. The synthetic DNA fragments were cloned into the digested pJB0169 backbone using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to construct pJB0163 and pJB0179.

[0206] pJB0179 was digested with BsaI and BbvCI to remove the sequences encoding the anti-CD3 and anti-EGFR binding domains. PCR products encoding the anti-HER2 light and heavy chains were amplified with primers containing an 18bp 5’ homologous region to the backbone DNA at the N-terminus and a 21bp 3’ homologous region to the second PCR product. A second PCR product encoding the anti-CD3 scFv sequence variant (CD3.23) was amplified using pJB0205 as a template with primers containing an 18bp 5’ homologous region to the first PCR product at the N-terminus and a 23bp 3’ homologous region to the vector backbone. These two PCR products were cloned into the digested backbone using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to construct pAH0011.

[0207] pJB0163 was digested with BsaI and BstEII to remove the sequences encoding the anti-CD3 and anti-EGFR binding domains. PCR products encoding the anti-HER2 light and heavy chains were amplified with primers containing an 18 bp 5’ homology region to the backbone DNA at the N-terminus and primers containing a 21 bp 3’ homology region to the second PCR product. A second PCR product encoding the anti-CD3 scFv sequence variant (CD3.23) was amplified using pJB0205 as a template with primers containing an 18 bp 5’ homology region to the first PCR product at the N-terminus and primers containing a 23 bp 3’ homology region to the vector backbone. These two PCR products were cloned into the digested backbone using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to construct pAH0013.

[0208] To generate pJB0244 and pJB0245, pAH0011 and pAH0013 were digested with BsaI and BsrDI to remove the sequences encoding the anti-Her2 (Her2.1) light and heavy chains. PCR products encoding the anti-Her2 (Her2.2) light and heavy chains were amplified with primers containing a 25 bp 5' homologous region to the 3' end of each N-terminal vector backbone and primers containing a 25 bp 3' homologous region to the 5' end of the vector backbone. The PCR product of pJB0244 was cloned into the digested pAH0011 backbone using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to construct pJB0244, which encodes AE868 XTEN868 with a 6xHIS affinity tag (SEQ ID NO: 1150), AE292 XTEN, RSR2295, an anti-HER2-anti-CD3 bispecific tandem scFv, RSR2295, and a C-tag affinity tag, under the control of the PhoA promoter and STII secretion leader. The DNA sequence and the encoded amino acid sequence are provided in Table 9. The PCR product of pJB0245 was cloned into the pAH0013 backbone to generate pJB0245, which encodes AE868 XTEN with a 6xHIS affinity tag (SEQ ID NO: 1150), AE292 XTEN, the release segment RSR3058, an anti-HER2-anti-CD3 bispecific tandem scFv, RSR3058, and a C-tag affinity tag, under the control of the PhoA promoter and STII secretion leader.

[0209] To introduce a new CD3 scFv with changes in 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. A DNA fragment encoding an anti-EGFR scFv variant paired with CD3.33 containing 40 bp homology to the digested vector at both the 5' and 3' ends was synthesized to facilitate Gibson DNA Assembly. Plasmids pJB0358 - pJB0372 with the structure of 6xHIS affinity tag (SEQ ID NO: 1150), AE292 XTEN, RSR2295, and individually, 15 anti-EGFR scFv variants paired with anti-CD3 scFv, RSR2295, and AE868 XTEN with C-tag affinity tag were constructed.

[0210] pAH0025 and pAH0026 were prepared by first digesting pJB0368 and pJB0373 with btsI to remove the anti-CD3 scFv. DNA fragments encoding anti-CD3.32 scFv adjacent to a 40 bp homologous region to the digested backbone were ordered. These fragments were introduced into pJB0368 and pJB0373 by Gibson Assembly to construct plasmids encoding 6xHIS affinity tag (SEQ ID NO: 1150), AE292 XTEN, RSR2295, anti-EGFR - anti-CD3 bispecific tandem scFv, RSR2295, and AE868 XTEN with C-tag affinity tag, constructed in pAH0025 construct and pAH0026 construct with two different anti-EGFR binding domains EGFR.23 and EGFR.2. The DNA sequences and the encoded amino acid sequences are provided in Table 9.

[0211] To generate bispecific antigen-binding polypeptide constructs with truncated C-terminal XTEN, pJB0244 was digested with BtsI and EcoRI to remove the C-terminal XTEN and C-tag. A PCR fragment encoding the AE584 XTEN sequence and C-tag was amplified from pJB0244. A vector backbone encoding a second fragment with 40 bp homology beyond the EcoRI site where the 34-base tail overlaps with the first fragment was synthesized. These two fragments were cloned into the digested pJB0244 backbone using the Gibson Assembly Kit to generate plasmid pJB0354, which encodes a 6xHIS affinity tag (SEQ ID NO: 1150), AE292, RSR2295, anti-HER2-anti-CD3 bispecific tandem scFv, RSR2295, AE584 XTEN, and a C-tag affinity tag. To generate pJB0355, a PCR fragment encoding the AE293 XTEN sequence and C-tag was amplified from pJB0244. This was cloned, together with the second fragment described above, into the digested pJB0244 backbone using the Gibson Assembly Kit to generate plasmid pJB0355, which encodes a 6xHIS affinity tag (SEQ ID NO: 1150), XTEN292, RSR2295, anti-Her2-anti-CD3 bispecific tandem scFv, RSR2295, AE300 XTEN, and a C-tag affinity tag (DNA and amino acid sequences in Table 9). Non-cleavable variants of pJB0354 and pJB0355 (pJB0377 and pJB0378, respectively) were also constructed by replacing RSR2295 with the sequence EAGRSANHTPAGLTGP (SEQ ID NO: 88).

[0212] To generate a protein with truncated N- and C-terminal XTENs, three PCR products were amplified. The first PCR product consisted of an N-terminal His tag and AE144_7A XTEN amplified from pCW1199. The second PCR product consisted of an N-terminal cleavage site 2295, an anti-HER2-anti-CD3 bispecific tandem scFv, and a C-terminal cleavage site 2295, as well as 286 amino acids of the XTEN sequence. These two fragments were cloned by Gibson Assembly into a backbone generated by PCR amplification to form pJB0380, whose backbone contained, at its 5' end, the last 17 XTEN amino acids homologous to the second PCR product and 30 bp homologous to the STII signal peptide, a 6xHis tag (SEQ ID NO: 1150), and, at its 3' end, five XTEN residues containing 39 bp homologous to the 5' end of the first PCR product. pJB0380 encodes a 6xHIS affinity tag (SEQ ID NO: 1150), AE144_7A XTEN, RSR2295, an anti-HER2-anti-CD3 bispecific tandem scFv, RSR2295, AE293 XTEN, and a C-tag affinity tag (DNA and amino acid sequences in Table 9). A non-cleavable variant of pJB0380 (pJB0379) was also constructed by replacing RSR2295 with the sequence EAGRSANHTPAGLTGP (SEQ ID NO: 88). The same methodology would be used to generate constructs having CD3.24, CD3.30, CD3.31, CD3.33 scFv, and scFv of antigen-binding fragments to the target cell markers described herein in any combination or orientation (i.e., AF1-AF2 or AF2-AF1 from the N-terminus to the C-terminus).

Table 9-1

Table 9-2

Table 9-3

Table 9-4

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Table 9-7

Table 9-8

Table 9-9

Table 9-10

Table 9-11

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Table 9-45

Table 9-46

Table 9-47

Table 9-48

Table 9-49

Table 9-50

Table 9-51

Table 9-52

Table 9-53

Table 9-54

Table 9-55

Table 9-56

Table 9-57

Table 9-58

Table 9-59

Table 9-60

Table 9-61

Table 9-62

Table 9-63

Table 9-64

Table 9-65

Table 9-66

Table 9-67

Table 9-68

Table 9-69

Table 9-70

Table 9-71

Table 9-72

Table 9-73

Table 9-74

Table 9-75

Table 9-76

Table 9-77

Table 9-78

Table 9-79

Table 9-80

Table 9-81

Table 9-82

Table 9-83

Table 9-84

Table 9-85

Table 9-86

Table 9-87

Table 9-88

Table 9-89

Table 9-90

Table 9-91

Table 9-92

Table 9-93

Table 9-94

Table 9-95

Table 9-96

Table 9-97

Table 9-98

Table 9-99

Table 9-100

Table 9-101

Table 9-102

Table 9-103

Table 9-104

Table 9-105

Table 9-106

Table 9-107

Table 9-108

Table 9-109

Table 9-110

Table 9-111

Table 9-112

Table 9-113

Table 9-114

Table 9-115

Table 9-116

Table 9-117

Table 9-118

Table 9-119

Table 9-120

Table 9-121

Table 9-122

Table 9-123

Table 9-124

Table 9-125

Table 9-126

Table 9-127

Table 9-128

Table 9-129

Table 9-130

Table 9-131

Table 9-132

Table 9-133

Table 9-134

Table 9-135

Table 9-136

Table 9-137

Table 9-138

Table 9-139

Table 9-140

Table 9-141

Table 9-142

Table 9-143

Table 9-144

Table 9-145

Table 9-146

Table 9-147

Table 9-148

Table 9-149

Table 9-150

Table 9-151

Table 9-152

Table 9-153

Table 9-154

Table 9-155

Table 9-156

Table 9-157

Table 9-158

Table 9-159

Table 9-160

Table 9-161

Table 9-162

Table 9-163

Table 9-164

Table 9-165

Table 9-166

Table 9-167

Table 9-168

Table 9-169

Table 9-170

Table 9-171

Table 9-172

Table 9-173

Table 9-174

Table 9-175

Table 9-176

Table 9-177

Table 9-178

Table 9-179

Table 9-180

Table 9-181

Table 9-182

Table 9-183

Table 9-184

Table 9-185

Table 9-186

Table 9-187

Table 9-188

Table 9-189

Table 9-190

Table 9-191

Table 10-1

Table 10-2

Table 10-3

Table 10-4

Table 10-5

Table 10-6

Table 10-7

Table 10-8

Table 10-9

Table 10-10

Table 10-11

Table 10-12

Table 10-13

Table 10-14

Table 10-15

Table 10-16

Table 10-17

Table 10-18

Table 10-19

Table 10-20

Table 10-21

Table 10-22

Table 10-23

Table 10-24

Table 10-25

Table 10-26

Table 10-27

Table 10-28

Table 10-29

Table 10-30

Table 10-31

Table 10-32

Table 10-33

Table 10-34

Table 10-35

Table 10-36

Table 10-37

Table 10-38

Table 10-39

Table 10-40

Table 10-41

Table 10-42

Table 10-43

Table 10-44

Table 10-45

Table 10-46

Table 10-47

Table 10-48

Table 10-49

Table 10-50

Table 10-51

Table 10-52

Table 10-53

Table 10-54

Table 10-55

Table 10-56

Table 10-57

[0213] Example 2: Evaluation of CD3 scFv Array Variants Compared with Parental CD3 scFv The purpose of this experiment was to evaluate four CD3 array variants and determine whether those variants had enhanced properties compared to the CD3.9 parental scFv.

[0214] 1. Determination of Melting Temperature (T m ) The melting temperature of each scFv variant was measured to determine its thermal stability. Briefly, a uniform amount of scFv in 200 μL of 1% BSA-PBST was aliquoted 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 for CD3 Added to ELISA plates coated with 79 μ target antigen (Creative Biomart) or BSA (reference for addressing adhesiveness). The wells of the ELISA plates were pre-filled with 1% BSA-PBST (50 μl / well). The plates were incubated at room temperature for 1 hour. The plates were washed three times with water and 0.05% TWEEN to remove unbound scFv. The bound scFv was detected by adding an anti-YOL antibody (Thermo Scientific, number MA180189) (diluted 1:500 in 1% BSA-PBST (0.05%)) that detects the porcine alpha-tubulin motif in the linker between the heavy and light chains. The samples were incubated at room temperature for 1 hour. The plates were washed three times with water and 0.05% TWEEN to remove unbound scFv. The anti-YOL antibody was detected by adding an anti-rat-HRP antibody (Thermo Scientific, number 31470) (diluted 1:7500 in 1% BSA-PBST (0.05%)) [100 μl / well] and incubating at room temperature for 1 hour. The plates were washed three times with water and 0.05% TWEEN to remove unbound antibody. The plates were developed using a 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). The relative activity was measured as the absorbance at 450 nM. The absorbance at each temperature was graphed. The melting temperature was determined to be the EC50 of each sample where the binding of the scFv decreased to 50% of the maximum signal. The results are presented in Table 11.

[0215] Results: The assay results showed that CD3 scFv 3.23 and 3.24 had a Tm 5 °C higher than the parental CD3.9, while CD3.25 and CD3.26 (sequences shown in Table 12) scFv had a T m equivalent to that of the parental CD3.9.

[0216] 2. Determination of binding affinity for 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) starting from 1000 nM in 1:1 dilution steps down to 62.5 nM for CD3.24 - 26 and from 400 nM in 1:1 dilution steps down to 25 nM for CD3.23. Biotinylated CD3 TIFF2025106277000313.tif77 μ antigen (Creative Biomart) was diluted in PBS to a final concentration of 30 μg / ml. Streptavidin biosensors (ForteBio) were activated in PBS for 10 minutes. To perform the measurement, the streptavidin biosensors were applied to the BLItz instrument. Tubes containing 300 μL of PBS were transferred to the BLItz instrument over 30 seconds. Biotinylated CD3 TIFF2025106277000314.tifTubes containing 79 μ (30 μg / ml, 300 μL / tube) were transferred to the BLItz instrument and the capture of the antigen to the sensor was measured over 120 seconds. Tubes containing 300 μL of PBS were transferred to the BLItz instrument over 30 seconds to measure the baseline signal. Tubes containing the test scFv (30 μg / ml, 300 μL / tube) were transferred to the BLItz instrument and the association of the scFv to the antigen-loaded biosensor was measured over 120 seconds. Tubes containing 300 μL of PBS were transferred to the BLItz instrument over 120 seconds to measure the dissociation of the scFv from the antigen-loaded biosensor. This protocol was repeated for each scFv dilution. The KD of each antibody was determined using BLI software (ForteBio). Results showing the melting temperature and binding affinity of the CD3-binding variants are presented in Table 11, indicating that variants such as CD3.23 have a decreased binding affinity for CD3.

[0217] The binding affinity of the bivalent anti-HER2, anti-CD3 XTENylated binder AC2275 (see Example 24) was measured against the targets (HER2 and CD3) using a ForteBio Octet Red instrument. This assay was performed in PBSTB buffer (10 mM sodium phosphate dibasic, 1.8 mM calcium phosphate monobasic, 137 mM sodium chloride, 2.7 mM potassium chloride, 0.5% BSA, 0.005% Tween-20). For binding to human HER2 or cynomolgus HERa2, a dilution series of each analyte was prepared in PBSTB buffer (500 μL / tube) starting from 64 nM in 1:1 dilution steps down to 1 nM. For binding to human CD3 or cynomolgus CD3, a dilution series of each analyte was prepared in PBSTB buffer (500 μL / tube) starting from 1010 nM in 1:1 dilution steps down to 16 nM. The targets were diluted in PBSTB to a final concentration of 33 μg / ml. Anti-human Fc biosensors (ForteBio) were activated in PBSTB buffer for 10 minutes. To perform the measurements, a series of anti-human Fc biosensors were placed on a sensor rack and transferred to the Octet Red instrument. A 96-well non-binding opaque plate containing 200 μL of PBSTB buffer, glycine buffer, the targets, and the analytes was transferred to the Octet Red instrument. The biosensors were transferred to PBSTB buffer for 600 seconds for equilibration. For activation, the biosensors were transferred to 10 mM glycine buffer (pH 1.5) for 10 seconds and then transferred to PBSTB buffer for 10 seconds. The activation step was repeated two more times. The biosensors were transferred to the target wells for 100 seconds for the loading step. The biosensors were transferred to PBSTB buffer for 600 seconds for the baseline measurement. The biosensors were transferred to the analyte wells for 200 - 400 seconds for the association step. The biosensors were transferred to the analyte wells for 300 - 400 seconds for the dissociation step. This protocol was repeated for each target. The binding affinity of each antibody was determined using Octet Data Analysis software (ForteBio). The results are presented in Table 11A.

[0218] Results: The assay results indicate that all CD3 sequence variants had a decreased binding affinity for CD3 compared to the parental CD3.9. [Table 11] [Table 12] [Table 13]

[0219] Conclusion: Two new anti-CD3 scFvs with improved thermal stability have been identified. The new scFvs each have 8 - 9 mutations relative to CD3.9, mainly present in the CDRs. These mutations result in a decreased affinity of the scFvs for their target (CD3) compared to parental CD3.9, but the bispecific T cell engager utilizing CD3.23 remains effective in cell killing assays and in vivo.

[0220] Example 3: Fermentation and Purification of Stable and Unstable Chimeric Fusion Polypeptides Comprising Bispecific Antigen-Binding Fragments, Release Segments, and XTEN The following example describes the production of two highly similar chimeric bispecific antigen-binding fragment compositions that differ only in the anti-CD3 antigen-binding fragment utilized, the observed discrepancies in the tendency to aggregate between these two constructs, and the finding that the sequence of the anti-CD3 antigen-binding fragment had a significant impact on the production, recovery, and purification of a stable soluble product.

[0221] Construct ID pJB0169 is a molecule with eight distinct domains. From the N-terminus to the C-terminus, this molecule consists of an N-terminal polyhistidine tag (His6 (SEQ ID NO: 1150)), 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 glutamic acid, proline, glutamic acid, alanine (C-tag) (XTEN_AE868).

[0222] Construct ID pJB0231 is a sim...

Claims

**Claim 1** A polypeptide comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a light chain complementarity-determining region (CDR-L) and a heavy chain complementarity-determining region (CDR-H), and the antigen-binding fragment a. specifically binds to the cluster of differentiation 3 (CD3) T cell receptor, b. comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively. **Claim 2** A polypeptide comprising an anti-CD3 antigen-binding fragment, wherein the antigen-binding fragment comprises a light chain complementarity-determining region (CDR-L) and a heavy chain complementarity-determining region (CDR-H), and the antigen-binding fragment a. specifically binds to CD3, b. comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10, and c. exhibits higher thermal stability as demonstrated by in vitro assays, (i) A melting temperature (T m ) that is higher than that of the antigen-binding fragment consisting of the sequence shown in SEQ ID NO: 41, or (ii) When the anti-CD3 antigen-binding fragment is incorporated into an anti-CD3 bispecific antibody, the bispecific antibody exhibits a higher Tm compared to a control bispecific antibody, the anti-CD3 bispecific antibody comprises the anti-CD3 binding fragment and a reference antigen-binding fragment that binds to an antigen other than CD3, and the control bispecific antigen-binding fragment comprises SEQ ID NO: 41 and the reference antigen-binding fragment. A polypeptide. **Claim 3** The T of the antigen-binding fragment m of the antigen-binding fragment consisting of the sequence of SEQ ID NO: 41 m is at least 2°C higher, or at least 3°C higher, or at least 4°C higher, or at least 5°C higher than that of the polypeptide according to claim 2. **Claim 4** A polypeptide comprising an antigen-binding fragment, wherein the antigen-binding fragment comprises a light chain complementarity-determining region (CDR-L) and a heavy chain complementarity-determining region (CDR-H), and the antigen-binding fragment a. specifically binds to CD3, b. comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10, and c. comprises FR-H1, FR-H2, FR-H3, FR-H4, which each exhibit at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the amino acids of SEQ ID NOs: 22, 23, 25, and 26, respectively, or is identical thereto. A polypeptide. **Claim 5** The antigen-binding fragment further comprises FR-L1, FR-L2, FR-L3, and FR-L4, and each of them has 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 sequences of SEQ ID NOs: 12, 13, 18, and 19, respectively. The polypeptide according to claim 4.

6. The polypeptide according to any one of claims 2 to 5, wherein the CDR-H1 and the CDR-H2 each comprise the amino acid sequences of SEQ ID NOs: 8 and 9.

7. The CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 1 or 2, b. CDR-L2 having the amino acid sequence of SEQ ID NO: 4 or 5, and c. CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The polypeptide according to any one of the preceding claims.

8. The CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 1, b. CDR-L2 having the amino acid sequence of either one of SEQ ID NOs: 4 or 5, c. CDR-L3 having the amino acid sequence of SEQ ID NO: 6 or 7. The polypeptide according to any one of the preceding claims.

9. The CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 2, b. CDR-L2 having the amino acid sequence of either one of SEQ ID NOs: 4 or 5, c. CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The polypeptide according to any one of claims 1 to 7.

10. The CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 1, b. CDR-L2 having the amino acid sequence of SEQ ID NO: 4, c. CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The polypeptide according to claim 8.

11. The CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 2, b. CDR-L2 having the amino acid sequence of SEQ ID NO: 5, c. CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The polypeptide according to any one of claims 1 to 7 and 9.

12. The antigen-binding fragment further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen-binding fragment is a. FR-L1 having the amino acid sequence of SEQ ID NO: 12, b. FR-L2 having the amino acid sequence of SEQ ID NO: 13, c. FR-L3 having any one amino acid sequence of SEQ ID NOs: 14 to 17, d. FR-L4 having the amino acid sequence of SEQ ID NO: 19, e. FR-H1 having the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21, f. FR-H2 having the amino acid sequence of SEQ ID NO: 23, g. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and h. FR-H4 having any one amino acid sequence of SEQ ID NO: 26, the polypeptide according to any one of the preceding claims.

13. The antigen-binding fragment further comprises a light-chain framework region (FR-L) and a heavy-chain framework region (FR-H), and the antigen-binding fragment a. FR-L1 having the amino acid sequence of SEQ ID NO: 12, b. FR-L2 having the amino acid sequence of SEQ ID NO: 13, c. FR-L3 having the amino acid sequence of SEQ ID NO: 14, d. FR-L4 having the amino acid sequence of SEQ ID NO: 19, e. FR-H1 having the amino acid sequence of SEQ ID NO: 20, f. FR-H2 having the amino acid sequence of SEQ ID NO: 23, g. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26, the polypeptide according to any one of the preceding claims.

14. The antigen-binding fragment further comprises a light-chain framework region (FR-L) and a heavy-chain framework region (FR-H), and the antigen-binding fragment a. FR-L1 having the amino acid sequence of SEQ ID NO: 12, b. FR-L2 having the amino acid sequence of SEQ ID NO: 13, c. FR-L3 having the amino acid sequence of SEQ ID NO: 15, d. FR-L4 having the amino acid sequence of SEQ ID NO: 19, e. FR-H1 having the amino acid sequence of SEQ ID NO: 21, f. FR-H2 having the amino acid sequence of SEQ ID NO: 23, g. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26, the polypeptide according to any one of claims 1 to 12.

15. The antigen-binding fragment further comprises a light-chain framework region (FR-L) and a heavy-chain framework region (FR-H), and the antigen-binding fragment a. FR-L1 having the amino acid sequence of SEQ ID NO: 12, b. FR-L2 having the amino acid sequence of SEQ ID NO: 13, c. FR-L3 having the amino acid sequence of SEQ ID NO: 16, d. FR-L4 having the amino acid sequence of SEQ ID NO: 19, e. FR-H1 having the amino acid sequence of SEQ ID NO: 21, f. FR-H2 having the amino acid sequence of SEQ ID NO: 23, g. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26, the polypeptide according to any one of claims 1 to 12.

16. The antigen-binding fragment further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the antigen-binding fragment a. FR-L1 having the amino acid sequence of SEQ ID NO: 12, b. FR-L2 having the amino acid sequence of SEQ ID NO: 13, c. FR-L3 having the amino acid sequence of SEQ ID NO: 17, d. FR-L4 having the amino acid sequence of SEQ ID NO: 19, e. FR-H1 having the amino acid sequence of SEQ ID NO: 21, f. FR-H2 having the amino acid sequence of SEQ ID NO: 23, g. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and h. FR-H4 having the amino acid sequence of SEQ ID NO: 26, the polypeptide according to any one of claims 1 to 12.

17. The antigen-binding fragment comprises a variable heavy chain (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 31, or is identical thereto, the polypeptide according to any one of the preceding claims.

18. The antigen-binding fragment comprises a variable light chain (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with any one of the amino acid sequences of SEQ ID NO: 27, 29, 30, 32, or 33, or is identical thereto, the polypeptide according to any one of the preceding claims.

19. The antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity with any one of the amino acid sequences of SEQ ID NO: 36 to 40, or is identical thereto, the polypeptide according to any one of the preceding claims.

20. The antigen-binding fragment specifically binds to human or cynomolgus CD3, the polypeptide according to any one of the preceding claims.

21. The polypeptide according to any one of the preceding claims, wherein the antigen-binding fragment specifically binds to human and cynomolgus CD3.

22. The polypeptide according to any one of the preceding claims, wherein the antigen-binding fragment binds to a CD3 complex subunit selected from CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta epsilon units of CD3.

23. The polypeptide according to any one of the preceding claims, wherein the antigen-binding fragment binds to the CD3 epsilon fragment of CD3.

24. The polypeptide according to any one of the preceding claims, wherein the antigen-binding fragment exhibits an isoelectric point (pI) of 6.6 or less.

25. The polypeptide according to any one of the preceding claims, wherein the antigen-binding fragment exhibits a pI of 6.0 to 6.6 (including the boundary values).

26. The polypeptide according to any one of the preceding claims, wherein the antigen-binding fragment exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lower than the pI of a reference antigen-binding fragment consisting of the sequence shown in SEQ ID NO:

41.

27. When the antigen-binding fragment is determined in an in vitro antigen-binding assay comprising a human or cynomolgus CD3 antigen, it specifically binds to human or cynomolgus CD3 with a dissociation constant (K d ) constant of about 10 nM to about 400 nM, the polypeptide according to any one of the preceding claims.

28. When the antigen-binding fragment binds specifically to human or cynomolgus CD3 with a dissociation constant (K d d) of less than about 10 nM, or less than about 50 nM, or less than about 100 nM, or less than about 150 nM, or less than about 200 nM, or less than about 250 nM, or less than about 300 nM, or less than about 350 nM, or less than about 400 nM as determined by an in vitro antigen-binding assay, the polypeptide according to any one of the preceding claims.

29. When the antigen-binding fragment has a dissociation constant (K d ) determined by an in vitro antigen-binding assay, the polypeptide according to any one of the preceding claims, which exhibits a binding affinity for CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker than the binding affinity of the antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO:

41.

30. The polypeptide according to any one of the preceding claims, further comprising a first release segment peptide (RS1), wherein the RS1 is a substrate for cleavage by a mammalian protease.

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

32. The polypeptide according to any one of the preceding claims, wherein the RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 42 to 660.

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

34. Further comprising a first extended recombinant polypeptide (Xten1), wherein the Xten1 a. It has at least about 100 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, the polypeptide according to any one of the preceding claims.

35. The polypeptide according to claim 34, wherein the Xten1 has at least about 100 to about 1000 amino acids.

36. The polypeptide according to any one of the preceding claims, wherein the Xten1 comprises an amino acid sequence selected from at least three of SEQ ID NOs: 661 to 664.

37. The polypeptide according to any one of the preceding claims, wherein the Xten1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 665 to 718 and 922 to 926.

38. The Xten1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is described in Table 7, the polypeptide according to any one of the preceding claims. **Claim 39** The polypeptide according to any one of the preceding claims, wherein the antigen-binding fragment is a chimeric or humanized antigen-binding fragment. **Claim 40** The polypeptide according to any one of the preceding claims, wherein the antigen-binding fragment is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, single domain antibody (sdAb), and single chain variable fragment (scFv). **Claim 41** Expressed as a fusion protein, the fusion protein having a structural arrangement of AF1-RS1-Xten1 or Xten1-RS1-AF1 from the N-terminus to the C-terminus in a non-cleaved state, where AF1 is a first antigen-binding fragment, the polypeptide according to any one of the preceding claims. **Claim 42** The polypeptide according to any one of the preceding claims, further comprising a second antigen-binding fragment (AF2) that specifically binds to a target cell marker other than CD3. **Claim 43** The polypeptide according to claim 42, wherein the AF2 is fused to the AF1 by a flexible peptide linker. **Claim 44** The polypeptide according to claim 43, wherein the flexible linker comprises two or three types of amino acids selected from the group consisting of glycine, serine, and proline. **Claim 45** The polypeptide according to any one of claims 42 to 44, wherein (1) the AF2 fragment is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, single domain antibody, and single chain variable fragment (scFv), or (2) the AF1 and the AF2 are configured as (Fab′)2 or a single chain diabody.

46. The polypeptide according to any one of claims 42 to 45, wherein the target cell marker is a tumor antigen.

47. The target cell marker is 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A kinase anchor protein 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenergic receptor β3 (ADRB3), AGS-22M6, alpha folate receptor, alpha-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoietin-binding cell surface receptor 2 (Tie 2), anthrax toxin, AOC3 (VAP-1), B cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthracis anthrax, B cell activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), brother of regulator of imprinted sites (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCTC-binding factor (CTCF), CCL11 (eotaxin-1), CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD171, CD179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (alpha chain of IL-2 receptor), CD27, CD274, CD28, CD3, CD3 ε, CD30, CD300 molecular-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-related antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), claudin 18.2 (CLDN18.2), claudin 6 (CLDN6), Clostridium difficile,Clamping factor A, CLCA2, colony stimulating factor 1 receptor (CSF1R), CSF2, CTLA-4, C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-X-C chemokine receptor type 4, cyclin B1, cytochrome P450 1B1 (CYP1B1), cyp-B, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, E. coli Shiga toxin type 1, E. coli Shiga toxin type 2, ecto ADP-ribosyltransferase 4 (ART4), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), EGF-like domain multiple 7 (EGFL7), elongation factor 2 variant (ELF2M), endotoxin, ephrin A2, ephrin B2, ephrin A receptor type 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epicyanin, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 (ETV6-AML) located on chromosome 12p, F protein of respiratory syncytial virus, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II β chain, fibroblast activation protein α (FAP), fibronectin extracellular domain-B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor α, folate receptor β, Fos-related antigen 1, frizzled receptor, fucosyl GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5 member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), glypican-3 (GPC3), GMCSF receptor α chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, heat shock protein 70-2 variant (mut hsp70-2), hemagglutinin,Hepatitis A virus cellular receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, globoH glycosphingolipid (GloboH) hexasaccharide protein, HGF, HHGF receptor, high molecular weight melanoma-associated antigen (HMW-MAA), histone complex, HIV-1, HLA-DR, HNGF, Hsp90, HST-2 (FGF6), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), human TNF, ICAM-1 (CD54), iCE, IFN-α, IFN-β, IFN-γ, IgE, IgE Fc region, IGF-1, IGF-1 receptor, IGHE, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-1β, IL-20, IL-22, IL-23, IL-31, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin α4β7, integrin β2, integrin α2, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor, interferon γ-induced protein, interleukin 11 receptor α (IL-11Rα), interleukin 13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, kinase domain region (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), lymphocyte antigen 6 (Ly-6), Lewis Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex locus K 9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-α (LT-α) or tumor necrosis factor-β (TNF-β),Lysosome-associated membrane protein 1 (LAMP1), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, mammary differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma apoptosis inhibitory factor melanoma (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, cell surface-bound mucin 1 (MUC1), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, mucin CanAg, myelin-associated glycoprotein, myostatin, N-acetylglucosaminyl-transferase V (NA17), NCA-90 (granulocyte antigen), nectin-4, nerve growth factor (NGF), nerve apoptosis regulatory protease 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitor (e.g., Nogo-A, Nogo-B, Nogo-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), tumor fetal antigen (h5T4), breakpoint cluster region (BCR) and Abelson murine leukemia virus cancer gene homolog 1 (Abl) (bcr-abl) oncogene fusion protein, anasarca, OX-40, oxLDL, p53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), P-cadherin, sodium phosphate cotransporter, phosphatidylserine placenta-specific 1 (PLAC1), platelet-derived growth factor receptor α (PDGF-Rα), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), T cell recognition melanoma antigen 1 (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP),Prostate cancer cells, prostain, protease serine 21 (testisin or PRSS21), proteasome (prososome, macropain) subunit beta type 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), receptor activator of nuclear factor kappa-B ligand (RANKL), receptor for advanced glycation end products (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), respiratory syncytial virus, Rh blood group D antigen, Rh factor, sarcoma translocation breakpoint, sclerostin (SOST), selectin P, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), sphingosine-1-phosphate, T cell recognition squamous cell carcinoma antigen 1, 2, and 3 (SART1, SART2, and SART3), stage-specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1, syndecan 1 (SDC1)+A314, SOX10, survivin, survivin-2B, synovial sarcoma, X breakpoint 2 (SSX2), T cell receptor, TCR gamma alternative reading frame protein (TARP), telomerase, TEM1, tenascin C, TGF-beta (e.g., TGF-beta1, TGF-beta2, TGF-beta3), thyroid stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen ((Tn Ag) or (GalNAc alpha-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), TNF-alpha, TRAIL-R1, TRAIL-R2, TRG, transglutaminase 5 (TGS5), tumor antigen CTAAl6.88, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tumor protein p53 (p53), tumor-specific glycosylation of MUC1, tumor-related calcium signaling molecule 2 (TROP-2), tumor-associated glycoprotein 72 (TAG72), tumor-associated glycoprotein 72 (TAG-72)+A327, TWEAK receptor, tyrosine kinase, tyrosine kinase-related protein 1 (TYRP1 or glycoprotein 75), tyrosine kinase-related protein 2 (TYRP2), uroplakin 2 (UPK2), vascular endothelial growth factor (e.g., VEGF-A,VEGF-B, VEGF-C, VEGF-D, PlGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), vimentin, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), von Willebrand factor (VWF), Wilms tumor protein (WT1), X antigen family member, -1A (XAGE1), β-amyloid, κ-light chain, fibroblast growth factor receptor 2 (FGFR2), LIV-1 protein estrogen-regulated (LIV1, also known as SLC39A6), neurotrophic tyrosine kinase receptor type 1 (NTRK1, also known as TRK), Ret proto-oncogene (RET), B-cell maturation antigen (BCMA, also known as TNFRSF17), transferrin receptor (TFRC, also known as CD71), activated leukocyte cell adhesion molecule (ALCAM, also known as CD166), somatostatin receptor 2 (SSTR2), KIT proto-oncogene receptor tyrosine kinase (cKIT), V-Set immunoregulatory receptor (VSIR, also known as VISTA), glycoprotein Nmb (GPNMB), delta-like canonical Notch ligand 3 (DLL3), interleukin-3 receptor subunit alpha (IL3RA, also known as CD123), lysosome-associated membrane protein 1 (LAMP1), cadherin 3, type 1, P-cadherin (CDH3), ephrin A4 (EFNA4), protein tyrosine kinase 7 (PTK7), solute carrier family 34 member 2 (SLC34A2, also known as NaPi-2b), GCC, PLAUR domain-containing 3 (LYPD3, also known as LY6 or C4.4a), cell surface-bound mucin 17 (MUC17), Fms-related receptor tyrosine kinase 3 (FLT3), NKG2D ligands (e.g., ULBP1, ULBP2, ULBP3, H60, Rae-1α, Rae-1β, Rae-1δ, Rae-1γ, MICA, MICB, hHLA-A), SLAM family member 7 (SLAMF7), interleukin-13 receptor subunit alpha 2 (IL13RA2), C-type lectin domain family 12 member A (CLEC12A, also known as CLL-1), CEA cell adhesion molecule 5 (CEACAM, also known as CD66e), interleukin-3 receptor subunit alpha (IL3RA), CD5 molecule (CD5), UL16-binding protein 1 (ILBP1), V-Set domain-containing T cell activation inhibitor 1 (VTCN1, also known as B7-H4), chondroitin sulfate proteoglycan 4 (CSPG4), syndecan 1 (SDC1, also known as CD138), interleukin-1 receptor accessory protein (IL1RAP), baculoviral IAP repeat-containing 5 (BIRC5, also known as survivin), CD74 molecule (CD74),The polypeptide according to claim 46, selected from hepatitis A virus cellular receptor 1 (HAVCR1, also known as TIM1), SLIT and NTRK-like family member 6 (SILTRK6), CD37 molecule (CD37), coagulation factor III, tissue factor (CD142, also known as F3), AXL receptor tyrosine kinase (AXL), endothelin receptor type B (EDNRB, also known as ETBR), cadherin 6 (CDH6), fibroblast growth factor receptor 3 (FGFR3), carbonic anhydrase 6 (CA6), the CanAg glycoform of MUC1, integrin subunit alpha V (ITGAV), teratocarcinoma-derived growth factor 1 (TDGF1, also known as Crypto 1), SLAM family member 6 (SLAMF6, also known as CD352), and Notch receptor 3 (NOTCH3).

48. When the AF2 is determined in an in vitro antigen-binding assay comprising the target cell marker, a K of about 0.1 nM to about 100 nM d The polypeptide according to any one of claims 42 to 47, which specifically binds to the target cell marker with.

49. The polypeptide according to any one of claims 42 to 48, wherein the binding affinity of the AF2 for the target cell marker is at least 10 times higher, or at least 100 times higher, or at least 1000 times higher than the binding affinity of the AF1 for CD3 when measured by an in vitro antigen binding assay.

50. The polypeptide according to any one of claims 42 to 49, wherein the AF2 comprises the CDRs of a monoclonal antibody having a binding affinity for the target cell marker.

51. The polypeptide according to claim 49, wherein the CDR of the AF2 is selected from the sequences of SEQ ID NOs: 719 to 918.

52. The polypeptide according to any one of claims 42 to 51, wherein the AF2 comprises the VL and VH of a monoclonal antibody having a binding affinity for the target cell marker.

53. The polypeptide according to claim 52, wherein the VL of the AF2 is selected from the sequences of SEQ ID NOs: 819 to 918, and the VH is selected from the sequences of SEQ ID NOs: 719 to 818.

54. The polypeptide according to any one of claims 30 to 53, further comprising a second release segment (RS2), wherein the RS2 is a substrate for cleavage by a mammalian protease.

55. The polypeptide according to claim 54, wherein the RS2 is a substrate for a protease selected from legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase.

56. The polypeptide according to claim 54 or claim 55, wherein the RS2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a sequence selected from SEQ ID NOs: 42 to 660.

57. The polypeptide according to any one of claims 54 to 56, wherein the sequence of the RS1 is the same as the sequence of the RS2.

58. The polypeptide according to any one of claims 54 to 56, wherein the sequence of the RS1 is not the same as the sequence of the RS2.

59. The polypeptide according to any one of claims 54 to 58, wherein the RS1 and the RS2 are each a substrate for cleavage by a plurality of proteases at one, two, or three cleavage sites within each release segment array.

60. Further comprising a second extended recombinant polypeptide (Xten2), wherein the Xten2 a. has at least about 100 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. has at least 4 to 6 different amino acids selected from G, A, S, T, E, and P, the polypeptide according to any one of claims 54 to 59.

61. The polypeptide according to claim 60, wherein the Xten2 comprises an amino acid sequence, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence comprises non-overlapping sequences selected from at least three of SEQ ID NOs: 661 to 664.

62. The polypeptide according to claim 60 or claim 61, wherein the Xten2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 665 to 718 and 922 to 926.

63. The Xten2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is described in Table 7, the polypeptide according to any one of claims 60-62.

64. The polypeptide has, from the N-terminus to the C-terminus, the following structural arrangements: 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, or Xten1-RS1-diabody-RS2-Xten2, wherein the diabody comprises the VL and VH of the AF1 and the AF2, the AF1 specifically binds to CD3, the AF2 specifically binds to a target cell marker, and Xten1 and Xten2 are of different amino acid lengths or sequences, the polypeptide according to any one of claims 60-63.

65. A polypeptide comprising RS1, RS2, AF1, AF2, Xten1, and Xten2, a. The RS1 and the RS2 are each a substrate for cleavage by a mammalian protease, each comprising 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: 42-660, b. The AF1 is an antigen-binding fragment of a monoclonal antibody having binding specificity for CD3, c. The AF2 is an antigen-binding fragment comprising the VL and VH of a monoclonal antibody having binding affinity for a target cell marker. d. The Xten1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 665-718 and 922-926, e. The Xten2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NOs: 665-718 and 922-926, f. The polypeptide has a structural configuration from the N-terminus to the C-terminus as follows: Xten1-RS1-AF2-AF1-RS2-Xten2, Xten1-RS1-AF1-AF2-RS2-Xten2, Xten2-RS2-AF2-AF1-RS1-Xten1, Xten2-RS2-AF1-AF2-RS1-Xten1, or Xten2-RS2-diabody-RS1-Xten1, wherein the diabody comprises the VL and VH of the AF1 and the AF2, g. The polypeptide exhibits high thermal stability as determined by an increase in the melting temperature (Tm) in an in vitro assay, compared to an antibody fragment consisting of the sequence shown in SEQ ID NO:

41. **Claim 66** The AF1 is a. comprising heavy chain complementarity determining regions (CDR-H) CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10, and b. When determined by an increase in the melting temperature (T m ) in an in vitro assay, the polypeptide according to claim 65, which exhibits high thermal stability as compared to the Tm of the antigen-binding fragment consisting of the sequence set forth in SEQ ID NO:

41. **Claim 67** The AF1 comprises light chain complementarity determining regions (CDR-L) and heavy chain complementarity determining regions (CDR-H), and the AF1 is a. comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10, and b. comprising FR-H1, FR-H2, FR-H3, FR-H4, each of 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 acids of SEQ ID NOs: 20 or 21, 23, 24, and 26, respectively, the polypeptide according to claim 65. **Claim 68** The AF1 further includes FR-L1, FR-L2, FR-L3, and FR-L4, and each of them has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the amino acid sequences of SEQ ID NOs: 12, 13, 14-17, and 19, or is identical thereto. The polypeptide according to any one of claims 65 to 67.

69. The polypeptide according to claim 67 or claim 68, wherein the CDR-H1 and the CDR-H2 each include the amino acid sequences of SEQ ID NOs: 8 and 9.

70. The CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 1 or 2, b. CDR-L2 having the amino acid sequence of SEQ ID NO: 4 or 5, and c. CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The polypeptide according to any one of claims 67 to 69.

71. The CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 1, b. CDR-L2 having the amino acid sequence of either one of SEQ ID NOs: 4 or 5, and c. CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The polypeptide according to any one of claims 67 to 70.

72. The CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 2, b. CDR-L2 having the amino acid sequence of either one of SEQ ID NOs: 4 or 5, and c. CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The polypeptide according to any one of claims 67 to 70.

73. The CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 1, b. CDR-L2 having the amino acid sequence of any one of SEQ ID NO: 4, and c. CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The polypeptide according to any one of claims 67 to 70.

74. The CDR-L is a. CDR-L1 having the amino acid sequence of SEQ ID NO: 2, b. CDR-L2 having the amino acid sequence of any one of SEQ ID NO: 5, and c. CDR-L3 having the amino acid sequence of SEQ ID NO:

6. The polypeptide according to any one of claims 67 to 70.

75. The AF1 includes a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the AF1 is i. FR-L1 having the amino acid sequence of SEQ ID NO: 12, j. FR-L2 having the amino acid sequence of SEQ ID NO: 13, k. FR-L3 having the amino acid sequence of SEQ ID NO: 14, l. FR-L4 having the amino acid sequence of SEQ ID NO: 19, m. FR-H1 having the amino acid sequence of SEQ ID NO: 20, n. FR-H2 having the amino acid sequence of SEQ ID NO: 23, o. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and p. FR-H4 having the amino acid sequence of SEQ ID NO: 26, the polypeptide according to any one of claims 67 to 70.

76. wherein the AF1 includes a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the AF1 i. FR-L1 having the amino acid sequence of SEQ ID NO: 12, j. FR-L2 having the amino acid sequence of SEQ ID NO: 13, k. FR-L3 having the amino acid sequence of SEQ ID NO: 15, l. FR-L4 having the amino acid sequence of SEQ ID NO: 19, m. FR-H1 having the amino acid sequence of SEQ ID NO: 21, n. FR-H2 having the amino acid sequence of SEQ ID NO: 23, o. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and p. FR-H4 having the amino acid sequence of SEQ ID NO: 26, the polypeptide according to any one of claims 67 to 70.

77. wherein the AF1 includes a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the AF1 i. FR-L1 having the amino acid sequence of SEQ ID NO: 12, j. FR-L2 having the amino acid sequence of SEQ ID NO: 13, k. FR-L3 having the amino acid sequence of SEQ ID NO: 16, l. FR-L4 having the amino acid sequence of SEQ ID NO: 19, m. FR-H1 having the amino acid sequence of SEQ ID NO: 21, n. FR-H2 having the amino acid sequence of SEQ ID NO: 23, o. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and p. FR-H4 having the amino acid sequence of SEQ ID NO: 26, the polypeptide according to any one of claims 67 to 70.

78. wherein the AF1 includes a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and the AF1 i. FR-L1 having the amino acid sequence of SEQ ID NO: 12, j. FR-L2 having the amino acid sequence of SEQ ID NO: 13, k. FR-L3 having the amino acid sequence of SEQ ID NO: 17, l. FR-L4 having the amino acid sequence of SEQ ID NO: 19, m. FR-H1 having the amino acid sequence of SEQ ID NO: 21, n. FR-H2 having the amino acid sequence of SEQ ID NO: 23, o. FR-H3 having the amino acid sequence of SEQ ID NO: 24, and p. The polypeptide according to any one of claims 67 to 70, comprising FR-H4 having the amino acid sequence of SEQ ID NO:

26.

79. The polypeptide according to any one of claims 65 to 78, wherein the AF1 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 31, or comprises a variable heavy chain (VH) amino acid sequence identical thereto.

80. The polypeptide according to any one of claims 65 to 79, wherein the AF1 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 27, 29, 30, 32, or 33, or comprises a variable light chain (VL) amino acid sequence identical thereto.

81. The polypeptide according to any one of claims 65 to 80, wherein the AF1 has at least 95%, 96%, 97%, 98%, 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 36 to 40, or comprises an amino acid sequence identical thereto.

82. (1) The AF1 fragment and the AF2 fragment are each selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, single domain antibody, and single chain variable fragment (scFv), or (2) the AF1 and the AF2 are configured as (Fab′)2 or single chain diabody. The polypeptide according to any one of claims 65 to 81.

83. The polypeptide according to any one of claims 65 to 82, wherein the AF1 specifically binds to human or cynomolgus CD3.

84. The polypeptide according to any one of claims 65 to 82, wherein the AF1 specifically binds to human and cynomolgus CD3.

85. The polypeptide according to any one of claims 65 to 84, wherein the AF1 binds to a CD3 complex subunit selected from CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta epsilon fragments of CD3.

86. The polypeptide according to any one of claims 65 to 85, wherein the AF1 binds to CD3 epsilon.

87. The T of the AF1 m When determined by the increase in melting temperature in an in vitro assay, the T of the antigen-binding fragment consisting of the sequence of SEQ ID NO: 41 m is at least 2°C higher, or at least 3°C higher, or at least 4°C higher, or at least 5°C higher, or at least 6°C higher, or at least 7°C higher, or at least 8°C higher, or at least 9°C higher, or at least 10°C higher than that of the polypeptide according to any one of claims 66 to 86.

88. The polypeptide according to any one of claims 65 to 87, wherein the AF1 exhibits an isoelectric point (pI) of 6.6 or less.

89. The polypeptide according to any one of claims 65 to 88, wherein the AF1 exhibits a pI of 6.0 to 6.6 (including the boundary values).

90. The polypeptide according to any one of claims 65 to 87, wherein the AF1 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 lower than the pI of the reference antigen-binding fragment consisting of the sequence shown in SEQ ID NO:

41.

91. When the AF1 is determined in an in vitro antigen-binding assay, it specifically binds to human or cynomolgus CD3 with a dissociation constant (K d ) constant of about 10 nM to about 400 nM, the polypeptide according to any one of claims 65 to 90.

92. When the AF1 is determined by an in vitro antigen-binding assay, the dissociation constant (K d ) specifically binds to human or cynomolgus monkey CD3 with a dissociation constant of less than about 3 nM, or less than about 10 nM, or less than about 50 nM, or less than about 100 nM, or less than about 150 nM, or less than about 200 nM, or less than about 250 nM, or less than about 300 nM. The polypeptide according to any one of claims 65 to 90.

93. When the AF1 is determined by the respective dissociation constants (K d ), a polypeptide according to any one of claims 65 to 90, which specifically binds to human or cynomolgus monkey CD3 with a binding affinity that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold lower than that of AF1 consisting of the amino acid sequence of SEQ ID NO:

41.

94. When the AF2 is determined by an in vitro antigen-binding assay, a K of about 0.1 nM to about 100 nM d The polypeptide according to any one of claims 65 to 93, which specifically binds to the target cell marker.

95. The polypeptide according to any one of claims 65 to 94, wherein the binding affinity of the AF2 for the target cell marker is at least 10-fold higher, or at least 100-fold higher, or at least 1000-fold higher than the binding affinity of the AF1 for CD3 when measured in an in vitro antigen-binding assay.

96. The polypeptide according to any one of claims 65 to 95, wherein the AF2 contains the CDR of a monoclonal antibody having a binding affinity for the target cell marker.

97. The polypeptide according to claim 96, wherein the CDR of the AF2 is selected from the CDR sequences of the sequences of SEQ ID NOs: 719 to 918.

98. The polypeptide according to any one of claims 65 to 95, wherein the AF2 contains the VL and VH of a monoclonal antibody having a binding affinity for the target cell marker.

99. The polypeptide according to claim 98, wherein the VL sequence is selected from the sequences of SEQ ID NOs: 719 to 818, and the VH sequence is selected from the sequences of SEQ ID NOs: 819 to 918.

100. A polypeptide comprising an antigen-binding fragment, wherein the antigen-binding fragment contains a light-chain complementarity-determining region (CDR-L) and a heavy-chain complementarity-determining region (CDR-H), and the antigen-binding fragment a. specifically binds to the epsilon subunit of CD3 and b. contains a VH amino acid sequence containing SEQ ID NO:

920.

101. The polypeptide according to claim 100, wherein the antigen-binding fragment contains a VL amino acid sequence containing SEQ ID NO:

919.

102. The polypeptide according to claim 100 or claim 101, wherein the antigen-binding fragment consists of SEQ ID NO:

921.

103. The AF1 is fused to the AF2 by a flexible polypeptide linker, a. The AF2 specifically binds to a second reference antigen other than CD3, whereby the polypeptide becomes a bispecific antigen-binding fragment capable of binding to both CD3 and the second reference antigen, b. When the dual - specificity antigen - binding fragment exhibits high thermal stability as determined by an increase in the melting temperature (T m m) in an in - vitro assay, compared to a control dual - specificity antigen - binding fragment that comprises SEQ ID NO: 41 and AF2, the polypeptide according to any one of claims 42 to 102.

104. The polypeptide according to claim 103, wherein the AF1 and the AF2 each exhibit an isoelectric point (pI) of 6.6 or less.

105. The polypeptide according to claim 103 or claim 104, wherein the AF1 and the AF2 each exhibit a pI of 5.5 to 6.6 (including the boundary values).

106. The polypeptide according to any one of claims 103 to 105, wherein the pI of the AF1 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 the AF2.

107. The second reference antigen is 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A kinase anchor protein 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenergic receptor β3 (ADRB3), AGS-22M6, α-folate receptor, α-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoietin-binding cell surface receptor 2 (Tie 2), anthrax toxin, AOC3 (VAP-1), B cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthracis anthrax, B cell activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), brother of imprint site regulator (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCTC-binding factor (CTCF), CCL11 (eotaxin-1), CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD171, CD179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (α-chain of IL-2 receptor), CD27, CD274, CD28, CD3, CD3 ε, CD30, CD300 molecule-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-related antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), claudin 18.2 (CLDN18.2), claudin 6 (CLDN6).Clostridium difficile, Clumping factor A, CLCA2, Colony stimulating factor 1 receptor (CSF1R), CSF2, CTLA-4, C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-X-C chemokine receptor type 4, Cyclin B1, Cytochrome P450 1B1 (CYP1B1), cyp-B, Cytomegalovirus, Cytomegalovirus glycoprotein B, Dabigatran, DLL4, DPP4, DR5, E. coli Shiga toxin type 1, E. coli Shiga toxin type 2, Ecto ADP ribosyltransferase 4 (ART4), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), EGF-like domain multiple 7 (EGFL7), Elongation factor 2 variant (ELF2M), Endotoxin, Ephrin A2, Ephrin B2, Ephrin A receptor 2, Epidermal growth factor receptor (EGFR), Epidermal growth factor receptor variant III (EGFRvIII), Epicyanin, Epithelial cell adhesion molecule (EpCAM), Epithelial glycoprotein 2 (EGP-2), Epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (Transmembrane serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), Respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), Fetal acetylcholine receptor, Fibrin II β chain, Fibroblast activation protein α (FAP), Fibronectin extracellular domain-B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), Folic acid binding protein (FBP), Folic acid hydrolase, Folic acid receptor 1, Folic acid receptor α, Folic acid receptor β, Fos-related antigen 1, Frizzled receptor, Fucosyl GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5 member D (GPRC5D), Ganglioside G2 (GD2), GD3 ganglioside, Glycoprotein 100 (gp100), Glypican-3 (GPC3), GMCSF receptor α chain, GPNMB, GnT-V, Growth differentiation factor 8, GUCY2C, Heat shock protein 70-2 variant (mut hsp70-2),Hemagglutinin, hepatitis A virus cell receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, globoH glycosphingolipid (GloboH) hexasaccharide protein, HGF, HHGFR, high molecular weight melanoma-associated antigen (HMW-MAA), histone complex, HIV-1, HLA-DR, HNGF, Hsp90, HST-2 (FGF6), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), human TNF, ICAM-1 (CD54), iCE, IFN-α, IFN-β, IFN-γ, IgE, IgE Fc region, IGF-1, IGF-1 receptor, IGHE, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-1β, IL-20, IL-22, IL-23, IL-31, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin α4β7, integrin β2, integrin α2, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor, interferon γ-induced protein, interleukin 11 receptor α (IL-11Rα), interleukin 13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, kinase domain region (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), lymphocyte antigen 6 (Ly-6), Lewis Y antigen, LFA-1 (CD11a), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex locus K 9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-α (LT-α) or tumor necrosis factor-β (TNF-β),Lysosome-associated membrane protein 1 (LAMP1), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, mammary differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma apoptosis inhibitory factor melanoma (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, cell surface-bound mucin 1 (MUC1), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, mucin CanAg, myelin-associated glycoprotein, myostatin, N-acetylglucosaminyl-transferase V (NA17), NCA-90 (granulocyte antigen), nectin-4, nerve growth factor (NGF), nerve apoptosis regulatory protease 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitor (e.g., Nogo-A, Nogo-B, Nogo-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), tumor fetal antigen (h5T4), breakpoint cluster region (BCR) and Abelson murine leukemia virus cancer gene homolog 1 (Abl) (bcr-abl) cancer gene fusion protein, anasarca, OX-40, oxLDL, p53 variant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), P-cadherin, sodium phosphate cotransporter, phosphatidylserine placenta-specific 1 (PLAC1), platelet-derived growth factor receptor α (PDGF-Rα), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), T cell recognition melanoma antigen 1 (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP)Prostate cancer cells, prostain, protease serine 21 (testisin or PRSS21), proteasome (prososome, macropain) subunit beta type 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), receptor activator of nuclear factor kappa-B ligand (RANKL), receptor for advanced glycation end products (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), respiratory syncytial virus, Rh blood group D antigen, Rh factor, sarcoma translocation breakpoint, sclerostin (SOST), selectin P, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), sphingosine-1-phosphate, T cell recognition squamous cell carcinoma antigen 1, 2, and 3 (SART1, SART2, and SART3), stage-specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1, syndecan 1 (SDC1)+A314, SOX10, survivin, survivin-2B, synovial sarcoma, X breakpoint 2 (SSX2), T cell receptor, TCR gamma alternative reading frame protein (TARP), telomerase, TEM1, tenascin C, TGF-beta (e.g., TGF-beta1, TGF-beta2, TGF-beta3), thyroid stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), TNF-alpha, TRAIL-R1, TRAIL-R2, TRG, transglutaminase 5 (TGS5), tumor antigen CTAAl6.88, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7 related (TEM7R), tumor protein p53 (p53), tumor-specific glycosylation of MUC1, tumor-associated calcium signal transducer 2 (TROP-2), tumor-associated glycoprotein 72 (TAG72), tumor-associated glycoprotein 72 (TAG-72)+A327, TWEAK receptor, tyrosine kinase, tyrosine kinase-related protein 1 (TYRP1 or glycoprotein 75), tyrosine kinase-related protein 2 (TYRP2), uroplakin 2 (UPK2), vascular endothelial growth factor (e.g., VEGF-A,VEGF-B, VEGF-C, VEGF-D, PlGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), vimentin, v-myc myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), von Willebrand factor (VWF), Wilms tumor protein (WT1), , X antigen family member 1A (XAGE1), β-amyloid, κ-light chain, fibroblast growth factor receptor 2 (FGFR2), LIV-1 protein estrogen-regulated (LIV1, also known as SLC39A6), neurotrophic tyrosine kinase receptor type 1 (NTRK1, also known as TRK), Ret proto-oncogene (RET), B-cell maturation antigen (BCMA, also known as TNFRSF17), transferrin receptor (TFRC, also known as CD71), activated leukocyte cell adhesion molecule (ALCAM, also known as CD166), somatostatin receptor 2 (SSTR2), KIT proto-oncogene receptor tyrosine kinase (cKIT), V-Set immunoregulatory receptor (VSIR, also known as VISTA), glycoprotein Nmb (GPNMB), delta-like canonical Notch ligand 3 (DLL3), interleukin-3 receptor subunit alpha (IL3RA, also known as CD123), lysosome-associated membrane protein 1 (LAMP1), cadherin 3, type 1, P-cadherin (CDH3), ephrin A4 (EFNA4), protein tyrosine kinase 7 (PTK7), solute carrier family 34 member 2 (SLC34A2, also known as NaPi-2b), GCC, PLAUR domain-containing 3 (LYPD3, also known as LY6 or C4.4a), cell surface-bound mucin 17 (MUC17), Fms-related receptor tyrosine kinase 3 (FLT3), NKG2D ligand (e.g., ULBP1, ULBP2, ULBP3, H60, Rae-1α, Rae-1β, Rae-1δ, Rae-1γ, MICA, MICB, hHLA-A), SLAM family member 7 (SLAMF7), interleukin-13 receptor subunit alpha 2 (IL13RA2), C-type lectin domain family 12 member A (CLEC12A, also known as CLL-1), CEA cell adhesion molecule 5 (CEACAM, also known as CD66e), interleukin-3 receptor subunit alpha (IL3RA), CD5 molecule (CD5), UL16-binding protein 1 (ILBP1), V-Set domain-containing T cell activation inhibitor 1 (VTCN1, also known as B7-H4), chondroitin sulfate proteoglycan 4 (CSPG4), syndecan 1 (SDC1, also known as CD138), interleukin-1 receptor accessory protein (IL1RAP), baculovirus IAP repeat-containing 5 (BIRC5, also known as survivin), CD74 molecule (CD74),The polypeptide according to any one of claims 103 to 106, which is a target cell marker selected from hepatitis A virus cellular receptor 1 (HAVCR1, also known as TIM1), SLIT and NTRK-like family member 6 (SILTRK6), CD37 molecule (CD37), coagulation factor III, tissue factor (CD142, also known as F3), AXL receptor tyrosine kinase (AXL), endothelin receptor type B (EDNRB, also known as ETBR), cadherin 6 (CDH6), fibroblast growth factor receptor 3 (FGFR3), carbonic anhydrase 6 (CA6), CanAg glycoform of MUC1, integrin subunit alpha V (ITGAV), teratocarcinoma-derived growth factor 1 (TDGF1, also known as Cryptol), SLAM family member 6 (SLAMF6, also known as CD352), and Notch receptor 3 (NOTCH3).

108. (1) The AF2 fragment is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibodies, single-domain antibodies, and single-chain variable fragments (scFv), or (2) the AF1 and the AF2 are configured as (Fab′)2 or single-chain diabodies. The polypeptide according to any one of claims 103 to 107.

109. The binding affinity of the AF2 for the target cell marker is at least 10-fold higher, or at least 100-fold higher, or at least 1000-fold higher than the binding affinity of the AF1 for CD3 when measured in an in vitro antigen-binding assay. The polypeptide according to any one of claims 103 to 108.

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

111. The pharmaceutical composition according to claim 110, wherein the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intracavitary, intrathecal, or intramuscular administration.

112. The pharmaceutical composition according to claim 111, wherein the pharmaceutical composition is in liquid form or frozen form.

113. The pharmaceutical composition according to any one of claims 110 to 112, wherein the pharmaceutical composition is in a pre-filled syringe for single injection.

114. The pharmaceutical composition according to claim 110, wherein the pharmaceutical composition is formulated as a lyophilized powder to be reconstituted before administration.

115. Use of the polypeptide according to any one of claims 1 to 109 in the preparation of a medicament for treating a disease in a subject.

116. The disease is selected from the group consisting of cancer tumor, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, diffuse large 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, vaginal cancer, vulvar cancer, Ewing sarcoma, peritoneal carcinomatosis, uterine serous cancer, parathyroid cancer, endometrial cancer, cervical cancer, colorectal cancer, epithelial intraperitoneal malignancy with malignant ascites, uterine cancer, peritoneal mesothelioma, kidney cancer, lung cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular cancer, retinoblastoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, testicular cancer, bile duct cancer, bone cancer, salivary gland cancer, thyroid cancer, craniopharyngioma, carcinoid tumor, epithelial cancer, masculinizing tumor, adenocarcinoma, sarcoma of any origin, primary hematological malignancy (including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, B-cell-derived chronic lymphocytic leukemia, hairy cell leukemia, myeloproliferative neoplasm disorder, or myelodysplastic disorder), myasthenia gravis, Graves' disease, Kaposi's sarcoma, neuroblastoma, Hashimoto's thyroiditis, Wilms' tumor, or Goodpasture syndrome.

117. A method for treating a disease in a subject, comprising administering to the subject in need thereof one or more therapeutically effective doses of the pharmaceutical composition according to any one of claims 110 to 114.

118. The method according to claim 117, wherein the disease is selected from the group consisting of carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, urogenital cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous cancer, endometrial cancer, cervical cancer, colorectal cancer, epithelial intraperitoneal malignancy with malignant ascites, uterine cancer, peritoneal mesothelioma, kidney cancer, lung cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular cancer, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, salivary gland cancer, thyroid cancer, epithelial cancer, adenocarcinoma, sarcoma of any origin, primary hematological malignancies (including acute or chronic lymphocytic leukemia, acute or chronic myelogenous leukemia, myeloproliferative neoplasm disorders, or myelodysplastic disorders), myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpasture's syndrome.

119. The method according to claim 117 or claim 118, wherein the pharmaceutical composition is administered to the subject as one or more therapeutically effective doses administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, or once a month.

120. The method according to any one of claims 117 to 119, wherein 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.

121. The method according to any one of claims 117 to 120, wherein the dose is administered intradermally, subcutaneously, intravenously, intraarterially, intraperitoneally, intra-abdominally, intrathecally, or intramuscularly.

122. The method according to any one of claims 117 to 121, wherein the subject is selected from the group consisting of mice, rats, monkeys, and humans.

123. An isolated nucleic acid comprising (a) a polynucleotide encoding the polypeptide according to any one of claims 1 to 109, or (b) a complement of the polynucleotide of (a).

124. An expression vector comprising the polynucleotide sequence according to claim 123 and a recombinant regulatory sequence operably linked to the polynucleotide sequence. **Claim 125** An isolated host cell comprising the expression vector according to claim 124. **Claim 126** The host cell according to claim 126, wherein the host cell is a prokaryote. **Claim 127** The host cell according to claim 125 or claim 126, wherein the host cell is E. coli.

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