CD3 antigen-binding fragment and composition comprising same

Abstract

The present disclosure relates to compositions comprising antibody-binding fragments that specifically bind to CD3 or epitopes thereof. Some embodiments include compositions and antibody-binding fragments with increased stability. Bispecific fusion proteins comprising such antibody-binding fragments are also disclosed.

Description

[Technical Field]

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

[0002] Cross-reference statements This application claims the benefit of U.S. Provisional Application No. 62 / 866,746, filed June 26, 2019, entitled "CD3 ANTIGEN BINDING FRAGMENTS AND COMPOSITIONS COMPRISING SAME," and U.S. Provisional Application No. 63 / 041,059, filed June 18, 2020, entitled "CD3 ANTIGEN BINDING FRAGMENTS AND COMPOSITIONS COMPRISING SAME," both of which are incorporated herein in their entireties. [Background technology]

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

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

[0005] The present invention relates to anti-cluster of differentiation 3 (CD3) antigen-binding fragments incorporated into chimeric fusion proteins and methods of using 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 aspect, 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), wherein 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, wherein (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 is an antigen-binding fragment T 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 than

[0009] In yet another 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 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, each of which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or is identical to the amino acid sequences of SEQ ID NOs: 22, 23, 25, and 26, respectively. 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 an Fv, Fab, Fab', Fab'-SH, a linear antibody, and a single-chain variable fragment (scFv).

[0010] In some embodiments, CDR-H1 and CDR-H2 comprise the amino acid sequences of SEQ ID NOs: 8 and 9, respectively. In certain embodiments, the 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, the CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 1, CDR-L2 having the amino acid sequence of any one of SEQ ID NOs: 4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 or 7. In yet another embodiment, the CDR-L comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 2, CDR-L2 having the amino acid sequence of any one of SEQ ID NOs: 4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In one embodiment, the 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, the CDR-L comprises a CDR-L1 having the amino acid sequence of SEQ ID NO:2, a CDR-L2 having the amino acid sequence of SEQ ID NO:5, and a 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, each of which exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequences of SEQ ID NOs: 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 the amino acid sequence of any one 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 the amino acid sequence of any one 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 fragments include: 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 that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 31. In certain embodiments, the antigen-binding fragment comprises a variable light chain (VL) amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of any one of SEQ ID NOs: 27, 29, 30, 32, or 33. In other embodiments, the antigen-binding fragment comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, 99% sequence identity to or is identical to the amino acid sequence of any one of SEQ ID NOs: 36-40.

[0014] In some embodiments, the antigen-binding fragment specifically binds to human or cynomolgus monkey (cyno) CD3. In other embodiments, the antigen-binding fragment specifically binds to human and cynomolgus monkey (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 has a dissociation constant (K) of about 10 nM to about 400 nM as determined in an in vitro antigen binding assay involving human or cynomolgus CD3 antigen. d In other embodiments, the antigen-binding fragment specifically binds to human or cynomolgus monkey CD3 with a dissociation constant (K) 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. d In another embodiment, the antigen-binding fragment specifically binds to human or cynomolgus CD3 with a respective dissociation constant (K d ) 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 an antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 41.

[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 (inclusive). 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 units lower than the pI of a reference antigen-binding fragment consisting of the sequence set forth 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 a mammalian protease. 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 set forth in Table 5.

[0018] In some embodiments, the polypeptides disclosed herein further comprise a first extended recombinant polypeptide (XTEN1), wherein the XTEN1 is characterized in that: a. it 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 in the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P); and c. it has at least 4-6 different amino acids selected from G, A, S, T, E, and P. In some embodiments, the XTEN1 has at least about 36 to about 1000 amino acids or at least about 100-1000 amino acids. In certain embodiments, XTEN1 comprises an amino acid sequence selected from at least three 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 to 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 to a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7.

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

[0020] In certain embodiments, a polypeptide is provided comprising RS1, RS2, AF1, AF2, XTEN1, and XTEN2, wherein: a. RS1 and RS2 are each substrates for cleavage by a mammalian protease and 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; and b. AF1 exhibits binding to CD3. c. AF2 is an antigen-binding fragment comprising a VL and a 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 to a sequence selected from SEQ ID NOs: 665-718 and 922-926; and e. XTEN and f. the polypeptide comprises, from N-terminus to C-terminus, one of the following: XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN2-RS2-AF2-AF1- Disclosed herein are polypeptides having the structural arrangement of 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, and wherein the polypeptide exhibits increased thermal stability compared to an antibody fragment consisting of the sequence set forth in SEQ ID NO:41, as determined by an increased melting temperature (Tm) in an in vitro assay.

[0021] In some embodiments, AF1 comprises 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 has a melting temperature (T m ), compared to the Tm of an antigen-binding fragment consisting of the sequence set forth in SEQ ID NO: 41. In other embodiments, AF1 comprises a light chain complementarity-determining region (CDR-L) and a heavy chain complementarity-determining region (CDR-H), and AF1 comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10, and 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 to or is identical to the amino acid sequences of SEQ ID NOs: 20 or 21, 23, 24, and 26, respectively.

[0022] In certain embodiments, CDR-H1 and CDR-H2 comprise the amino acid sequences of SEQ ID NOs: 8 and 9, respectively. In some embodiments, the CDR-L comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 1 or 2, a CDR-L2 having the amino acid sequence of SEQ ID NO: 4 or 5, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In another embodiment, the CDR-L comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 1, a CDR-L2 having the amino acid sequence of any one of SEQ ID NOs: 4 or 5, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In other embodiments, the CDR-L comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 2, a CDR-L2 having the amino acid sequence of any one of SEQ ID NOs: 4 or 5, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In other embodiments, the CDR-L comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 1, a CDR-L2 having the amino acid sequence of any one of SEQ ID NOs: 4, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6. In other embodiments, the 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 comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), wherein AF1 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 one embodiment, AF1 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, AF1 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 yet another embodiment, AF1 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.

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

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

[0026] In certain embodiments, AF1 specifically binds to human or cynomolgus monkey (cyno) CD3. In some embodiments, AF1 specifically binds to human and cynomolgus monkey (cyno) CD3. In some embodiments, 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. In another embodiment, AF1 binds to CD3 epsilon. In another embodiment, AF1 has a dissociation constant (K) of about 10 nM to about 400 nM, as determined in an in vitro antigen binding assay. dIn certain embodiments, AF1 specifically binds to human or cynomolgus monkey CD3 with a dissociation constant (K) 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, as determined in an in vitro antigen binding assay. d In other embodiments, AF1 specifically binds to human or cynomolgus CD3 with a respective dissociation constant (K d ) 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 AF1 consisting of the amino acid sequence of SEQ ID NO: 41.

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

[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 (inclusive). 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 a reference antigen-binding fragment consisting of the sequence set forth 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 peptide 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: 719-918. In certain embodiments, AF2 comprises a VL and a 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: 819-918, and the VH is selected from the sequences of SEQ ID NOs: 719-818.

[0031] In some embodiments, the target cell marker is a tumor antigen. In some embodiments, the target cell marker is 1-40-beta-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, adrenoceptor beta 3 (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, B-cell activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), siblings of imprinted 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 IL-2 receptor), CD27, CD274, CD28, 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, 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), CXC chemokine receptor type 4, cyclin B1, cytochrome P4501B1 (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 (ART) 4), 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 type A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), episialin, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protein serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 (ETV6-AML) located on chromosome 12p, respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibulin 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 mutant (mutated) hsp70-2), hemagglutinin, hepatitis A virus cellular receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, globoH glycoceramide (GloboH) hexaglycoprotein, 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-gamma-inducible protein, interleukin-11 receptor alpha (IL-11Rα), 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 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 inhibitor of apoptosis melanoma (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, cell surface-associated mucin 1 (MUC1), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, mucin CanAg, myelin-associated glycoprotein, myostatin, N-acetylglucosaminyltransferase V (NA17), NCA-90 (granulocyte antigen), nectin-4, nerve growth factor (NGF), neuronal apoptosis-regulating proteinase 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitors (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), oncofetal antigen (h5T4), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), rabbit, 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), pre Protein convertase subtilisin / kexin type 9 (PCSK9), prostase, prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), T cell-recognized melanoma antigen 1 (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostate cancer cells, prostein, protease serine 21 (testisin or PRSS21), proteasome (prosome, 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-recognized squamous cell carcinoma antigens 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 Γ 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 signaling receptor 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 factors (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), estrogen-regulated LIV-1 protein (LIV1, also known as SLC39A6), neurotrophin 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 white Hemocyte 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-associated 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, MIC A, 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 Inhibitor of T-cell activation containing 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 (S ILTRK6), 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), teratoma-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 SEQ ID NOs: 719-918. In certain embodiments, AF2 comprises a VL and a 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 has a K of about 0.1 nM to about 100 nM as determined in an in vitro antigen binding assay that includes a target cell marker. d AF2 specifically binds to a target cell marker at a specific affinity, as measured in an in vitro antigen binding assay, that is at least 10-fold higher, at least 100-fold higher, or at least 1000-fold higher than the binding affinity of AF1 to CD3. In certain embodiments, AF2 comprises the CDRs of a monoclonal antibody that has binding affinity for the 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 a mammalian protease. 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%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 42-660. In another embodiment, the sequences of RS1 and RS2 are identical. In yet another embodiment, the sequences of RS1 and 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), which is characterized in that: a. it 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 in the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P); and c. it has at least 4-6 different amino acids selected from G, A, S, T, E, and P. In other embodiments, XTEN2 comprises an amino acid sequence, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of which comprise non-overlapping sequences selected from at least three 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 set forth in Table 7.

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

[0037] In some other embodiments, AF1 is fused to AF2 by a flexible peptide linker, wherein a. AF2 specifically binds to a second reference antigen other than CD3, resulting in a bispecific antigen-binding fragment in which the polypeptide is capable of binding to both CD3 and the second reference antigen; and b. the bispecific antigen-binding fragment exhibits a melting temperature (T m ) compared to a control bispecific antigen-binding fragment, wherein the control bispecific antigen-binding fragment comprises SEQ ID NO: 41 and AF2.

[0038] In certain embodiments, the second reference antigen is 1-40-beta-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, adrenoceptor beta 3 (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, B-cell activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), siblings of imprinted 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 IL-2 receptor), CD27, CD274, CD28, 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, 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), CXC chemokine receptor type 4, cyclin B1, cytochrome P4501B1 (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 (ART) 4), 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 type A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), episialin, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protein serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 (ETV6-AML) located on chromosome 12p, respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibulin 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 mutant (mutated) hsp70-2), hemagglutinin, hepatitis A virus cellular receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, globoH glycoceramide (GloboH) hexaglycoprotein, 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-gamma-inducible protein, interleukin-11 receptor alpha (IL-11Rα), 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 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 inhibitor of apoptosis melanoma (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, cell surface-associated 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 (NG) F), neuronal apoptosis-regulating proteinase 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitors (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), oncofetal antigen (h5T4), breakpoint cluster region (BCR), and Abelson mouse Oncogene fusion protein consisting of leukemia viral oncogene homolog 1 (Abl) (bcr-abl), European rabbit, 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-recognized melanoma antigen 1 (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostate cancer cells, prostein, protease serine 21 (testisin or PRSS21), proteasome (prosome, macropain) subunit β 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-recognized squamous cell carcinoma antigens 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 Γ 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 signaling receptor 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 endothelium Growth factors (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), estrogen-regulated LIV-1 protein (LIV1, also known as LIV-1) SLC39A6), neurotrophin 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), and interleukin-1 (IL-1). -Leukin-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-associated 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), CanAg glycoform of MUC1, integrin subunit alpha V (ITGAV), teratoma-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 fragments disclosed herein are 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 fragments disclosed herein are configured as (Fab')2 or single chain diabodies.

[0040] In certain embodiments, the binding affinity of AF2 to a 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 AF1 to 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 other embodiments, AF1 and AF2 each exhibit a pI of 5.5 to 6.6 (inclusive). 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, 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 epsilon subunit of CD3, and b. comprises a VH amino acid sequence comprising SEQ ID NO: 920. 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, disclosed herein is a pharmaceutical composition, comprising the polypeptide disclosed herein and one or more pharmaceutically suitable excipients.In some embodiments, the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intraperitoneal, intrathecal or intramuscular administration.In another embodiment, the pharmaceutical composition is in liquid form.In another embodiment, the pharmaceutical composition is in a pre-filled syringe for single injection.In yet another embodiment, the pharmaceutical composition is formulated as a lyophilized powder that is reconstituted before administration.

[0044] In another aspect, disclosed herein is the use of a polypeptide disclosed herein in the preparation of a medicament for treating a disease in a subject. In some embodiments, the disease is selected from the group consisting of carcinoma, 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 tract cancer, ovarian cancer, ovarian cancer with malignant ascites, vaginal cancer, vulvar cancer, Ewing's sarcoma, peritoneal carcinomatosis, uterine serous carcinoma, parathyroid cancer, endometrial cancer, cervical cancer, colorectal cancer, epithelial intraperitoneal malignant neoplasm 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, and the like. cancer), small intestine cancer, liver cancer, hepatocellular carcinoma, retinoblastoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, testicular cancer, bile duct cancer, bone cancer, salivary gland cancer, thyroid cancer, craniopharyngioma, carcinoid tumor, epithelial carcinoma, androgenic tumor, adenocarcinoma, sarcoma of any origin, primary hematologic malignancy (including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, chronic lymphocytic leukemia of B-cell origin, hairy cell leukemia, myeloproliferative neoplastic disorder, or myelodysplastic disorder), myasthenia gravis, Graves' disease, Kaposi's sarcoma, neuroblastoma, Hashimoto's thyroiditis, Wilms' tumor, or Goodpasture's syndrome.

[0045] In yet another aspect, disclosed herein is 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 a pharmaceutical composition disclosed herein. In certain embodiments, the subject is selected from the group consisting of a mouse, a rat, a monkey, and a human. 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, genitourinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous carcinoma, endometrial cancer, cervical cancer, colorectal cancer, epithelial intraperitoneal malignant tumor with malignant ascites, uterine cancer, peritoneal mesothelioma, kidney cancer, lung cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, and the like. cancer), small intestine cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, salivary gland cancer, thyroid cancer, epithelial carcinoma, adenocarcinoma, sarcoma of any origin, primary hematologic malignancy (including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, myeloproliferative neoplastic disorder, or myelodysplastic disorder), myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpasture's syndrome.

[0046] In other embodiments, the pharmaceutical composition is administered to a subject as one or more therapeutically effective doses 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 a subject as one or more therapeutically effective doses for a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. In some embodiments, the dose is administered intradermally, subcutaneously, intravenously, intraarterially, intraperitoneally, intraperitoneally, intrathecally, or intramuscularly.

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

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

[0049] In another aspect, disclosed herein is an isolated host cell comprising 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 in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0051] Various features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and accompanying drawings that set forth illustrative embodiments, in which the principles of the invention are utilized. [Brief explanation of the drawings]

[0052] [Figure 1] Figure 1 shows the individual components of the bispecific antigen-binding fragment composition. Figure 1A shows an antigen-binding fragment with affinity for a target cell marker. Figure 1B shows an antigen-binding fragment with affinity for an effector cell. Figure 1C and Figure 1D show XTEN polypeptides of different lengths. Figure 1E shows a cleavable release segment. [Figure 2]

[0023] Figure 2A shows two different forms of the polypeptide compositions described herein. Figure 2A shows, on the left, a release segment and an antigen-binding fragment for an effector cell fused to an XTEN; the arrow indicates the action of a protease to cleave the release segment, which results in the release of the XTEN from the antigen-binding fragment of the polypeptide on the right, allowing the antigen-binding fragment to regain its maximum binding affinity potential because it is no longer shielded by the XTEN. Figure 2B shows, on the left, a bispecific composition having an antigen-binding fragment for an effector cell fused to an antigen-binding fragment with binding affinity for a target cell marker. The release segment and XTEN are also fused to an antigen-binding fragment with affinity for the effector cell; the arrow indicates the action of a protease to cleave the release segment, which results in the release of the XTEN and the fused antigen-binding fragment from the polypeptide on the right, allowing them to regain their maximum binding affinity potential because they are no longer shielded by the XTEN. [Figure 3] Two different forms of bispecific antigen-binding polypeptides are shown: on the left, a bispecific composition having an antigen-binding fragment for an effector cell is fused to an antigen-binding fragment with binding affinity for a target cell marker with a release segment (the scissors indicate susceptibility to protease cleavage) and an XTEN is fused to the antigen-binding fragment with binding affinity for the effector cell, while on the right, a bispecific composition having an antigen-binding fragment for an effector cell is fused to an antigen-binding fragment with binding affinity for a target cell marker and a release segment and an XTEN are fused to the antigen-binding fragment with binding affinity for the target cell marker. [Figure 4]

[0039] Figure 4A shows three different forms of bispecific antigen-binding polypeptides. Figure 4A shows a bispecific composition having an scFv antigen-binding fragment for an effector cell fused to an scFv antigen-binding fragment with binding affinity for a target cell marker having a release segment (the scissors indicate susceptibility to protease cleavage), and an XTEN fused to each antigen-binding fragment. Figures 4B and 4C are variations of Figure 4A in which the antigen-binding fragments are in a diabody configuration, and the release segment (the scissors indicate susceptibility to protease cleavage) and XTEN are fused to the antigen-binding fragment for the effector cell marker or target cell marker, respectively. [Figure 5] Schematic diagram of a bispecific antigen-binding polypeptide near tumor tissue (top) and normal tissue (bottom). The bispecific antigen-binding polypeptide is preferentially cleaved in tumor tissue, releasing one or more XTEN moieties compared to normal tissue. The cleaved bispecific antigen-binding polypeptide can bind to T cells and tumor cells expressing tumor-specific markers. [Figure 6] 1 shows the amino acid sequence of controlled release segment RSR-1517 (SEQ ID NO: 42) with the peptide cleavage sites of the listed proteases indicated. [Figure 7] Figure 1 shows the in vitro cytotoxic activity of N- and C-terminally Xtenylated anti-HER2-anti-CD3 XPAT constructs ("HER2-XPAT") compared to the same constructs, non-Xtenylated ("HER2-PAT"), in PBMC / SK-OV-3 cell (A) or PBMC / BT-474 cell (B) cytotoxicity assays. Cytotoxicity was assessed by caspase 3 / 7 assay or luminescent ATP assay, respectively. [Figure 8]Figure 8A shows the in vitro toxicity of non-Xtenylated HER2-CD3 constructs correlates with HER2 expression. Figure 8A shows the dose response of non-Xtenylated HER2-CD3 constructs ("HER2-PAT") in cell lines with varying HER2 expression in the presence of PBMCs. Figure 8B shows the dose response comparing non-Xtenylated HER2-CD3 constructs ("HER2-PAT") and Xtenylated HER2-CD3 constructs ("HER2-XPAT") in selected cell lines with varying HER2 expression in the presence of PBMCs. [Figure 9A] We illustrate a proposed model of how HER2-PAT molecules form an immune synapse between T cells and HER2-positive cells. [Figure 9B] Figure 9B shows that HER2-PAT and HER2-XPAT constructs can induce conventional markers of T cell activation in "naive" T cells (sourced as PBMCs). Figure 9B shows the dose response of a non-Xtenylated HER2-CD3 construct ("HER2-PAT") compared to an Xtenylated HER2-CD3 construct ("HER2-XPAT") in upregulating CD69+ T cells (an early marker of T cell activation) in PBMCs in the presence of HER2+ cells (SK-OV-3 cells), as assessed by flow cytometry. [Figure 10A] 1 shows that HER2-XPAT exhibits significantly reduced cytotoxicity compared to non-Xtenylated HER2-PAT in an in vitro PBMC / cardiomyocyte model. [Figure 11] Figure 11A shows T cell activation by HER2-PAT and HER2-XPAT constructs is dependent on HER2-positive cell binding. Figure 11A shows T cell activation by HER2-PAT or HER2-XPAT in the presence or absence of BT-474 HER2+ cells in an in vitro Jurkat T cell / BT-474 model, as measured by luciferase activity driven by the NFAT response element in Jurkat T cells. Figure 11B shows similar data showing T cell activation by HER2-PAT or HER2-XPAT in the presence or absence of SK-OV-3 cells in an in vitro Jurkat T cell / SK-OV-3 model. [Figure 12] Figure 12A shows the dose response of Jurkat cell activation by a non-Xtenylated HER2-CD3 construct, a single-terminal Xtenylated HER2-CD3 construct, or an N- and C-terminal Xtenylated HER2-CD3 molecule in the presence of BT-474 cells. Figure 12B shows the dose response of Jurkat cell activation by a non-Xtenylated HER2-CD3 construct, a single-terminal Xtenylated HER2-CD3 construct, or an N- and C-terminal Xtenylated HER2-CD3 molecule in the presence of SK-OV-3 cells. [Figure 13] Figure 13A shows tumor volume after 25 days of treatment with vehicle + / - PBMC, Xtenylated HER2-CD3 molecule ("XPAT"), and non-Xtenylated HER2-CD3 molecule ("PAT"). Figure 13B shows tumor volume after 25 days of treatment with vehicle + PBMC, Xtenylated HER2-CD3 molecule ("XPAT"), and Xtenylated HER2-CD3 molecule with non-cleavable XTEN. [Figure 14]Figure 14 shows that N- and C-terminally Xtenylated HER2-CD3 molecules ("HER2-XPAT") and non-Xtenylated HER2-CD3 molecules ("HER2-PAT") are effective in increasing activated CD4+ and CD8+ tumor-infiltrating lymphocyte populations in tumor tissue harvested from a BT-474 / human PBMC xenograft model after treatment. Figure 14A shows the percentage of "activated" (e.g., CD25+) CD4+ cells in tumors assessed by flow cytometry after treatment with vehicle, HER2-XPAT, or HER2-PAT. Figure 14B shows the percentage of "activated" (e.g., CD25+) CD8+ cells in tumors assessed by flow cytometry after treatment with vehicle, HER2-XPAT, or HER2-PAT. [Figure 14C] We show that alternative dosing schedules of HER2-XPAT are effective in reducing tumor burden in humanized BT-474 xenograft mice. [Figure 15] Figure 15A shows that Xtenylation of HER2-CD3 molecules reduces the toxicity of HER2-CD3 constructs in cynomolgus monkeys. Figure 15A shows a scheme of maximum tolerated dose testing of Xtenylation ("HER2-XPAT") or non-Xtenylation ("HER2-PAT") in monkeys. Figure 15B shows the plasma levels of Xtenylation or non-Xtenylation molecules after administration in monkeys, showing that the maximum tolerated dose of the Xtenylation construct is more than 1000 times that of the non-Xtenylation molecule. [Figure 16] Figure 16 shows that in cynomolgus monkeys, the HER2-XTEN construct induces T cell margination at doses above 2.5 mpk, but fails to activate peripheral T cells at doses as high as 50 mpk. Figure 16A shows the effect on total lymphocyte counts, demonstrating that the XTENized construct induces a decrease in total blood lymphocyte counts at doses above 2.5 mpk. Figure 16B shows the effect of HER2-XPAT 2275 on activated CD4+ and CD8+ T cell populations, demonstrating that the post-dose effect on systemic T cell activation is within the pre-dose range. [Figure 17]Figure 17A, Figure 17B, and Figure 17C show the maximum serum IL-6, TNF-alpha, or IFN-gamma concentrations induced by a dose series of HER2-PAT or HER2-XPAT in cynomolgus monkeys, demonstrating that HER2-XPAT does not induce appreciable cytokine release at the doses tested. DETAILED DESCRIPTION OF THE INVENTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0070] "Target cell marker" refers to a molecule expressed by a target cell, including, but not limited to, a cell surface receptor, antigen, glycoprotein, oligonucleotide, enzyme substrate, antigenic determinant, or binding site that may be present within or on the surface of a target tissue or cell that can serve as a ligand for an antibody.

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

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

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

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

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

[0076] "Framework" or "FR" residues are those variable domain residues in an antigen-binding fragment other than the hypervariable region residues as defined herein, which generally are located between or adjacent to the CDRs. Several FR delineations are used and encompassed herein, e.g., FR-L1 refers to the first FR region of the light chain, FR-H2 refers to the second FR region of the heavy chain, and so on.

[0077] "Isoelectric point" or "pI" are used interchangeably herein and refer to the pH at which a particular molecule has no net charge or is electrically neutral on statistical average. The formal term for expressing isoelectric point is pH, which results in units of pH. For example, an antigen-binding fragment with a pI of 6.3 would have a neutral charge in a solution at pH 6.3. Isoelectric point can be determined mathematically, and several algorithms for estimating the isoelectric point of peptides and proteins, including the Henderson-Hasselbalch equation, have different pK values. Isoelectric point can also be determined experimentally by in vitro assays such as capillary isoelectric focusing.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0104] The terms "stringent conditions" or "stringent hybridization conditions" include reference to conditions under which a polynucleotide hybridizes to its target sequence to a detectably greater extent than other sequences (e.g., at least twice background). Generally, the stringency of hybridization is expressed, in part, in terms of the temperature and salt concentration under which the wash step is carried out. Typically, stringent conditions are those in which the salt concentration is less than about 1.5 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short polynucleotides (e.g., 10 to 50 nucleotides) and at least about 60°C for long polynucleotides (e.g., more than 50 nucleotides). For example, "stringent conditions" may include hybridization in 50% formamide, 1 M NaCl, and 1% SDS at 37°C, followed by three washes (15 minutes each) in 0.1×SSC / 1% SDS at 60°C to 65°C. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentrations may vary from about 0.1 to 2×SSC, with SDS present at about 0.1%. Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point of the specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Equations for calculating Tm and conditions for nucleic acid hybridization are well known and are set forth in Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual," 3 rdThese washing conditions can be found in the "Blocking Agents for RNA-DNA Hybridization," Cold Spring Harbor Laboratory Press, 2001 edition. Typically, a blocking reagent is used to block nonspecific hybridization. Such a blocking reagent contains, for example, about 100-200 μg / ml of sheared and denatured salmon sperm DNA. Organic solvents such as formamide at a concentration of about 35-50% v / v may 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," "percent sequence identity," and "% identity" as applied to polynucleotide sequences refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such algorithms insert gaps in a standardized, reproducible manner within the sequences being compared to optimize the alignment between the two sequences, thus achieving a more meaningful comparison of the two sequences. Percent identity can be measured over the length of the entire defined polynucleotide sequence, or over a shorter length, e.g., over the length of a fragment taken from a larger, defined polynucleotide sequence, e.g., a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210, or at least 450 contiguous residues. It is understood that such lengths are exemplary only, and that any fragment length supported by the sequences set forth herein in the tables, figures, or sequence listing can be used to describe the length over which percent identity can be measured. The sequence identity percentage is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of matched positions (where identical residues occur in both polypeptide sequences), dividing the number of matched positions by the total number of positions within the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the sequence identity percentage. When sequences of different lengths are compared, the shortest sequence defines the length of the comparison window. Conservative substitutions are not taken into account when calculating sequence identity.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0126] I) General Techniques The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. Sambrook J et al., "Molecular Cloning: A Laboratory Manual," 3 rd edition, Cold Spring Harbor Laboratory Press, 2001, “Current protocols in molecular biology”, FMAusubel, et al. eds., 1987, the series “Methods in Enzymology,” Academic Press, San Diego, CA., “PCR 2: a practical approach”, MJ MacPherson, BD Hames and GRTaylor eds., Oxford University Press, 1995, “Antibodies, a laboratory manual” Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988, “Goodman & Gilman's The Pharmacological Basis of Therapeutics,” 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, the contents of which are incorporated herein by reference in their entireties.

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

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

[0129] II).CD3 antigen-binding fragment composition In a first aspect, the present disclosure provides polypeptides comprising an antigen-binding fragment (AF1) that has specific binding affinity for an effector cell antigen expressed on the surface of an effector cell selected from plasma cells, T cells, B cells, cytokine-induced killer cells (CIK cells), mast cells, dendritic cells, regulatory T cells (RegT cells), helper T cells, myeloid cells, and NK cells. 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 polypeptides comprising an antigen-binding fragment that has 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, e.g., CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta, either individually or in combination.

[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 a diseased cell or tissue in a compositional manner that results in cell killing of the diseased cell or tissue. Binding specificity can be determined by the complementarity-determining region, or CDR, such as the light chain CDR or the heavy chain CDR. In many cases, binding specificity is determined by the light chain CDR and the heavy chain CDR. A given combination of heavy chain CDR and light chain CDR provides a given binding pocket that confers higher affinity and / or specificity to CD3 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 can be produced using the hybridoma method described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (U.S. Patent No. 4,816,567). The structure of antibodies and their fragments, including antibody heavy and light chain variable regions (VH and VL), single-chain variable regions (scFv), complementarity-determining regions (CDRs), and domain antibodies (dAbs), are well understood. Methods for generating polypeptides having desired antigen-binding fragments of target cell markers are well known in the art.

[0132] Certain CD3-binding antigen-binding fragments of the present disclosure have been specifically modified to enhance their stability in the polypeptide embodiments described herein compared to CD3 antibodies and antigen-binding fragments known in the art. Protein aggregation of monoclonal and other antibodies continues to pose a significant challenge to their developability and remains a major area of ​​focus in antibody production. Antibody aggregation is triggered by partial unfolding of their domains, potentially resulting in monomer-monomer association followed by nucleation and aggregate growth. While the aggregation propensity of antibodies and antibody-based proteins can be influenced by external experimental conditions, it is highly dependent on intrinsic antibody properties determined by their sequence and structure. While it is well known that proteins are marginally stable in their folded state, it is less understood that most proteins are inherently prone to aggregation in their unfolded or partially unfolded state, and that the resulting aggregates may be highly stable and long-lived. Reduced aggregation propensity has also been shown to be accompanied by increased expression titers, indicating that reduced protein aggregation may be beneficial throughout the development process and lead to a more efficient path to clinical trials. For therapeutic proteins, aggregates are a significant risk factor for adverse immune responses in patients and can occur through a variety of mechanisms. Controlling aggregation can improve protein stability, manufacturability, wear rate, safety, formulation, potency, immunogenicity, and solubility. Intrinsic protein properties, such as size, hydrophobicity, electrostatics, and charge distribution, play an important role in protein solubility. It has been shown that poor solubility of therapeutic proteins due to surface hydrophobicity can make formulation development more challenging and lead to poor in vivo biodistribution, undesirable pharmacokinetic behavior, and immunogenicity. Reducing the overall surface hydrophobicity of candidate monoclonal antibodies can also provide benefits and cost savings related to purification and administration regimens. Individual amino acids can be identified through structural analysis as contributing to an antibody's aggregation potential and can be located in both CDRs and framework regions.In particular, residues may be predicted to have a high risk of causing hydrophobicity problems for a given antibody. In one embodiment, the present disclosure provides an AF1 having the ability to specifically bind to CD3, relative to a parent antibody or antibody fragment, wherein the AF1 has at least one amino acid substitution in a framework region with a hydrophobic amino acid selected from isoleucine, leucine, or methionine. In another embodiment, the CD3 AF1 has at least two amino acid substitutions in one or more framework regions with a hydrophobic amino acid selected from isoleucine, leucine, or methionine.

[0133] In the context of a subject antigen-binding fragment, the isoelectric point (pI) is the pH at which the antibody fragment has no net charge. If the pH is below the pI of an antibody fragment, it has a net positive charge. A greater positive charge tends to correlate with increased blood clearance and tissue retention, and generally a shorter half-life. If the pH is above the pI of an antibody fragment, it has a negative charge. A negative charge generally results in decreased tissue uptake and a longer half-life. This charge on framework residues can be manipulated through mutation. These considerations informed the design of the antigen-binding fragment sequences of the embodiments described herein, in which individual amino acid substitutions were made to the parent antibody used as a starting point. The isoelectric point of a polypeptide can be determined mathematically or experimentally in in vitro assays. The isoelectric point (pI) is the pH at which a protein has a net charge of zero and can be calculated using the charges of specific amino acids in the protein sequence. An estimate of the charge, called the acid dissociation constant or pKa value, is used to calculate the pI. pI can be determined in vitro by methods such as capillary isoelectric focusing (see Datta-Mannan, A., et al. The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies. mAbs 7:1084 (2015); Li, B., et al. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecular charge. mAbs 6, 1255-1264 (2014)) or other methods known in the art.

[0134] In some aspects of any of the embodiments disclosed herein, a subject polypeptide comprising AF1 comprises a light chain complementarity determining region (CDR-L) and a heavy chain complementarity determining region (CDR-H), wherein AF1 (a) specifically binds to the cluster of differentiation 3 (CD3) T-cell receptor, which may include all known CD3 subunits, e.g., CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta, either individually or in combination. In one embodiment, the antigen-binding fragment of any of the subject composition embodiments described herein is a chimeric or humanized antigen-binding fragment. In another embodiment, the antigen-binding fragment of any of the subject composition embodiments described herein is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, linear antibody, and single-chain variable fragment (scFv). An antigen-binding fragment having a CDR-H and a CDR-L can be configured in the (CDR-H)-(CDR-L) or (CDR-H)-(CDR-L) orientation from N-terminus to C-terminus.

[0135] In one embodiment, the disclosure provides a polypeptide comprising AF1 comprising a CDR-L and a CDR-H, wherein AF1 (a) specifically binds to the 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 disclosure, AF1 comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs: 8, 9, and 10, respectively. In another embodiment, a polypeptide according to any of the subject composition embodiments described herein can comprise AF1, wherein AF1 comprises a CDR-L and a CDR-H, and wherein 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 to or is identical to SEQ ID NOs: 22, 23, 25, and 26, respectively. In another embodiment, the disclosure provides a polypeptide comprising AF1, wherein AF1 comprises a CDR-L and a CDR-H, wherein AF1 (a) specifically binds 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) heavy chain framework regions (FR-H) FR-H1, FR-H2, FR-H3, FR-H4, which each have at least 86%, 87%, 88%, 89%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220, 230, 250, and 260, respectively. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to or identical to SEQ ID NOs: 12, 13, 18, and 19, respectively, and further comprising 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 to or identical to SEQ ID NOs: 12, 13, 18, and 19, respectively.

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

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

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

[0139] In some aspects of any of the embodiments disclosed herein, the subject polypeptide comprises AF1 that binds to CD3, wherein AF1 is configured as an scFv capable of specifically binding to CD3. In one embodiment, AF1 comprises an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with, or is identical to, the amino acid sequence of any one of SEQ ID NOs: 36-40.

[0140] In some cases, CD3 AF1 of polypeptide embodiments described herein specifically binds to human or cynomolgus monkey (cyno) CD3. In other cases, CD3 AF1 of polypeptide embodiments described herein specifically binds to human and cynomolgus monkey (cyno) CD3. In one embodiment, CD3 AF1 of polypeptide embodiments described herein 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 one embodiment, AF1 of polypeptide embodiments described herein binds to the CD3 epsilon fragment of CD3.

[0141] In another aspect, the present disclosure provides polypeptides comprising AF1s that bind to the CD3 protein complex and have enhanced stability compared to CD3-binding antibodies or AF1s known in the art. Additionally, certain CD3 AF1s of the present disclosure are designed to confer greater stability to chimeric bispecific antigen-binding compositions into which they are incorporated, which may result in improved expression and recovery, increased shelf life, and enhanced stability of the fusion protein when administered to a subject. In one approach, certain CD3 AF1s of the present disclosure are designed to have greater thermal stability compared to certain CD3-binding antibodies and antigen-binding fragments known in the art. As a result, CD3 AF1s utilized as components of chimeric bispecific antigen-binding fragment compositions into which they are incorporated exhibit favorable pharmaceutical properties, including high thermal stability and low aggregation tendency, resulting in improved expression and recovery during manufacturing and storage, and promoting a long serum half-life. Biophysical properties such as thermal stability are often limited by antibody variable domains, whose intrinsic properties vary widely. High thermal stability is often associated with high expression levels and other desirable properties, such as low susceptibility to aggregation (Buchanan A, et al. Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression. MAbs 2013;5:255). Thermal stability is measured by the "melting temperature" (T), defined as the temperature at which half of the molecule is denatured. m The melting temperature of each heterodimer indicates its thermal stability. mIn vitro assays for determining the melting point of heterodimers are known in the art. The melting point of heterodimers can be measured using techniques such as differential scanning calorimetry (Chen et al. (2003) Pharm Res 20:1952-60, Ghirlando et al. (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of heterodimers can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

[0142] Thermal denaturation curves of CD3-binding fragments of the present disclosure and anti-CD3 bispecific antibodies comprising the anti-CD3 binding fragments and a reference binding fragment show that various constructs of the present disclosure are more resistant to thermal denaturation than an antigen-binding fragment consisting of the sequence set forth in SEQ ID NO: 41 or a control bispecific antibody, where the control bispecific antigen-binding fragment comprises SEQ ID NO: 41 and a reference antigen-binding fragment that binds to an antigen other than CD3. In one embodiment, a polypeptide of the embodiments described herein comprises anti-CD3 AF1, where AF1 comprises a CDR-L and a CDR-H, where AF1 specifically binds CD3 and 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 in an in vitro assay, such that (i) the polypeptide has a melting temperature (T m ), or (ii) incorporation of the anti-CD3 AF1 into an anti-CD3 bispecific antibody results in the bispecific antibody having a higher Tm than a control bispecific antibody. mwherein 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, the control bispecific antigen-binding fragment consisting of SEQ ID NO: 41 and the reference antigen-binding fragment. For example, in some situations, the control bispecific antibody is identical to the subject 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 anti-CD3 AF1, wherein the T m T of an antigen-binding fragment consisting of the sequence of SEQ ID NO: 41 m In another embodiment, the disclosure provides a polypeptide comprising anti-CD3 AF1, wherein the T of AF1 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. m T of an antigen-binding fragment consisting of the sequence of SEQ ID NO: 41 m In yet another embodiment, a bispecific antigen-binding polypeptide is provided that has a melting temperature (T) of 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 than that of a control bispecific antibody control comprising the sequence set forth in SEQ ID NO: 41 and a reference antigen-binding fragment that binds to an antigen other than CD3. m ) is provided.

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

[0144] In some aspects of any of the embodiments disclosed herein, the subject polypeptide has a dissociation constant (K) of about 10 nM to about 400 nM, or about 50 nM to about 350 nM, or about 100 nM to 300 nM, as determined in an in vitro antigen binding assay involving human or cynomolgus monkey CD3 antigen. d In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF1 that specifically binds to human or cynomolgus monkey CD3 with a dissociation constant (K) weaker than about 10 nM, or about 50 nM, or about 100 nM, or about 150 nM, or about 200 nM, or about 250 nM, or about 300 nM, or about 350 nM, or about 400 nM, as determined in an in vitro antigen binding assay. d ) and may contain AF1 that specifically binds to human or cynomolgus monkey CD3. For clarity, d An antigen-binding fragment having a K d binds to its ligand more weakly than one with

[0145] In another embodiment, the present disclosure provides methods for determining the dissociation constant (K d 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 than the binding affinity of an antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 41, as determined by a dissociation constant (K d), is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold weaker than the binding affinity of a second antigen-binding fragment incorporated into the polypeptide that specifically binds to an antigen other than CD3. In the foregoing embodiments, the antigen other than CD3 is selected from any of the target cell marker embodiments disclosed herein, including, but not limited to, HER2, EGFR, EpCAM, or CD19, or the target cell markers in Table 8. The binding affinity of the subject compositions for the target ligand can be assayed using a binding or competitive binding assay, such as a Biacore assay using a chip-bound receptor or binding protein, or an ELISA assay as described in U.S. Pat. No. 5,534,617, the assays described in the Examples herein, a radioreceptor assay, or other assays known in the art. The binding affinity constant can then be determined using standard methods, such as Scatchard analysis as described by van Zoelen, et al., Trends Pharmacol Sciences (1998) 19)12):487, or other methods known in the art. The same methodology will be used to generate bispecific antigen-binding fragment constructs having antigen-binding fragments against CD3 and target cell markers described herein in any combination or orientation (i.e., AF1-AF2 or AF2-AF1 in 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 release segment (RS) peptides suitable for inclusion in the subject compositions described herein that are substrates for one or more mammalian proteases associated with or produced by cells found in or near diseased tissue. Such proteases include, but are not limited to, classes of proteases such as metalloproteinases, cysteine ​​proteases, aspartic acid proteases, and serine proteases. RSs are useful, among other things, to impart a prodrug format to the subject compositions that can be activated by cleavage of the RS by a mammalian protease. As described herein, RSs are incorporated into the subject composition embodiments described herein to link the incorporated antigen-binding fragments to XTENs (the configuration of which is described more fully below), such that upon cleavage of the RS by the action of one or more proteases for which the RS is a substrate, the antigen-binding fragments and XTENs are released from the composition, allowing the antigen-binding fragments, no longer shielded by the XTEN, to regain full binding capacity to their respective ligands. In a particular aspect, the RS serves as a substrate for a protease found in close association with or co-localized with diseased tissues or cells, such as, but not limited to, tumors, cancer cells, and inflamed tissues; upon cleavage of the RS, antigen-binding fragments that would otherwise be shielded by the XTEN of the subject compositions (and therefore have lower binding affinity for their respective ligands) are released from the composition and fully regain the ability to bind to target and / or effector cell ligands. In another embodiment, the RS of the subject polypeptide compositions comprises an amino acid sequence that is a substrate for a cellular protease located within the target cell. In another particular aspect of the subject compositions described herein, RSs that are substrates for two or three classes of proteases are designed with sequences that can be cleaved at different positions in the RS sequence by different proteases; a representative example is shown in FIG. 6.Thus, an RS that is a substrate for two, three, or more classes of proteases will have two, three, or multiple distinct cleavage sites within the RS sequence, but cleavage by a single protease will nevertheless result in the release of an antigen-binding fragment and an XTEN from a composition comprising the RS.

[0147] In one embodiment, the disclosure provides an activatable polypeptide comprising one or more release segments, wherein the release segments are substrates for cleavage by one or more mammalian proteases. In another embodiment, the disclosure provides a polypeptide comprising a first release segment (RS1) sequence, wherein RS1 is a substrate for cleavage by a mammalian protease, wherein RS1 is a substrate for a protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other instances, the polypeptide of any of the subject composition embodiments described herein comprises a first release segment (RS1) sequence, wherein RS1 is selected from the group consisting of meprin, neprilysin (CD10), PSMA, BMP-1, A disintegrin and metalloproteinase (ADAM), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM with thrombospondin motifs (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3 (MMP-3, stromelysin 1), matrix metalloproteinase-4 (MMP-4, stromelysin 1), matrix metalloproteinase-5 (MMP-5, stromelysin 1), matrix metalloproteinase-6 (MMP-6, stromelysin 1), matrix metalloproteinase-7 (MMP-7, stromelysin 1), matrix metalloproteinase-8 (MMP-8, stromelysin 1), matrix metalloproteinase-9 (MMP-8, stromelysin 1), matrix metalloproteinase-10 (MMP-8, stromelysin 1), matrix metalloproteinase-11 (MMP-8, stromelysin 1), matrix metalloproteinase-12 (MMP-8, stromelysin 1), matrix metalloproteinase-13 (MMP-8, stromelysin 1), matrix metalloproteinase-14 (MMP-8, stromelysin 1), matrix metalloproteinase-15 (MMP-8, stromelysin 1), matrix metalloproteinase-16 (MMP-8, stromelysin 1), matrix metalloproteinase-17 (MMP-8, strome matrix metalloproteinase-7 (MMP-7, matrilysin 1), matrix metalloproteinase-8 (MMP-8, collagenase 2), matrix metalloproteinase-9 (MMP-9, gelatinase B), matrix metalloproteinase-10 (MMP-10, stromelysin 2), matrix metalloproteinase-11 (MMP-11, stromelysin 3), matrix metalloproteinase-12 (MMP- 12, macrophage elastase), matrix metalloproteinase-13 (MMP-13, collagenase 3), matrix metalloproteinase-14 (MMP-14, MT1-MMP), matrix metalloproteinase-15 (MMP-15, MT2-MMP), matrix metalloproteinase-19 (MMP-19), matrix metalloproteinase-23 (MMP-23, CA-MMP),Matrix metalloproteinase-24 (MMP-24, MT5-MMP), matrix metalloproteinase-26 (MMP-26, matrilysin 2), matrix metalloproteinase-27 (MMP-27, CMMP), legumain, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X, cathepsin D, cathepsin E, secretase, urokinase (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, prostate-specific antigen (PSA, KLK3), human neutrophil elastase or a substrate for cleavage by one or more mammalian proteases selected from the group consisting of: tase (HNE), elastase, tryptase, type II transmembrane serine protease (TTSP), DESC1, hepsin (HPN), matriptase, matriptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast activation protein (FAP), kallikrein-related peptidase (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14.

[0148] In another embodiment, the disclosure provides a polypeptide comprising a first release segment (RS1) sequence for incorporation into a subject polypeptide composition described herein, wherein RS1 is a substrate for cleavage by one or more mammalian proteases, and RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 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 set forth in Table 5. As described in more detail below in the description of the composition and properties of the subject polypeptide compositions, the release segment is fused between the antigen-binding fragment and the XTEN polypeptide such that upon cleavage of the release segment, the XTEN is released from the composition.

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

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

[0150] In another aspect, the release segments (either RS1 and / or RS2) for incorporation into the polypeptides of any of the subject composition embodiments described herein can be designed to be selectively susceptible to different cleavage rates and cleavage efficiencies for the various proteases for which they are substrates. Because a given protease may be found at different concentrations in diseased tissues, including, but not limited to, tumors, blood cancers, or inflamed tissues or sites, compared to healthy tissues or circulating blood, the present disclosure provides RSs in which the individual amino acid sequences are engineered to have higher or lower cleavage efficiencies for a given protease compared to the cleavage rate of the release segment in healthy tissues or circulating blood, such that the released antigen-binding fragments have a higher ability to bind to ligands in diseased tissues compared to the prodrug form that remains in the circulating blood, ensuring that the polypeptide is preferentially converted from the prodrug form to the active form (i.e., by separation and release of the antigen-binding fragment and XTEN from the polypeptide after cleavage of the release segment) when in the vicinity of target cells or tissues and their co-localized proteases. Such selective design can improve the therapeutic index of the resulting composition and reduce side effects compared to conventional therapeutic agents that do not incorporate such site-specific activation.

[0151] As used herein, cleavage efficiency is defined as the log2 value of the ratio of the percentage of test substrate containing a cleaved release segment to the percentage of cleaved control substrate AC1611 when subjected to a protease enzyme in a biochemical assay (further detailed in the Examples) in which the reaction is performed, 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 product and uncleaved substrate are analyzed using non-reducing SDS-PAGE to establish the ratio of the percentage of cleaved release segment. Cleavage efficiency is calculated as follows:

number

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

[0153] IV). XTEN Polypeptides In another aspect, the present disclosure relates to polypeptides comprising at least a first extended recombinant polypeptide (XTEN) incorporated into the subject composition embodiments described herein, thereby increasing the mass and size of the construct and also serving to significantly reduce the ability of the antigen-binding fragment to bind to a ligand when the molecule is in its intact, uncleaved state, as described more fully below. In some embodiments, the present disclosure provides polypeptides comprising a single XTEN fused to the end of an RS located between the antigen-binding fragment and the XTEN. In other embodiments, the present disclosure provides polypeptides comprising a first XTEN and a second XTEN (XTEN1 and XTEN2) fused to the N- and C-termini of RS1 and RS2, respectively, located between each antigen-binding fragment and the XTEN.

[0154] Without being bound by theory, the incorporation of XTEN can be incorporated into the design of the subject compositions to impart certain properties: 1) providing the polypeptide composition with XTEN that shields the antigen-binding fragments and reduces their binding affinity for target cell markers and effector cell antigens when the composition is in its intact prodrug form; ii) providing the polypeptide composition with XTEN that provides an enhanced half-life when administered to a subject; iii) contributing to the solubility and stability of the intact composition, thereby enhancing the pharmaceutical properties of the subject compositions; and iv) providing the polypeptide composition with XTEN that reduces extravasation in normal tissues and organs, but allows some extravasation in diseased tissues (e.g., tumors) that have larger pore sizes in the vasculature and may nevertheless be released from the tissue by the action of certain mammalian proteases, thereby allowing the antigen-binding fragments of the composition to more easily penetrate diseased tissues, e.g., tumors, and bind to and link together target cell markers on effector cells and tumor cells. To meet these needs, the present disclosure provides compositions comprising one or more XTENs, wherein the XTENs provide increased mass and hydrodynamic radius to the resulting compositions. The XTEN polypeptides of the embodiments not only provide increased mass and hydrodynamic radius to the compositions, but their flexible, unstructured nature can also provide a shielding effect for the antigen-binding fragments of the compositions, thereby providing a particular advantage in the design of the subject compositions in that they reduce binding to antigens in normal tissues or the vasculature of normal tissues that do not express, or express at reduced levels of, target cell markers and / or effector cell antigens. Additionally, the incorporation of XTENs into the subject compositions can enhance the solubility and proper folding of single-chain antibody-binding fragments during expression and recovery.

[0155] XTENs are polypeptides having non-naturally occurring, substantially non-repetitive sequences that have some degree of secondary or tertiary structure under physiological conditions, as well as one or more additional properties described in the following paragraphs. In some embodiments, the present disclosure provides polypeptides comprising one or more XTENs having at least about 36, 72, 96, 100, 144, 200, 288, 292, 293, 300, 576, 584, 800, 864, 867, 868, 900, or at least about 1000 or more amino acids. In one embodiment, the disclosure provides a polypeptide comprising XTEN1, wherein the XTEN1 has at least about 36 or 100 amino acid residues, and wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues in the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), and wherein the polypeptide has at least 4-6 different amino acids selected from G, A, S, T, E, and P. In some embodiments, the disclosure provides a polypeptide comprising XTEN1 having at least about 36 to about 1000, 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 instances, the disclosure provides polypeptides 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 four to six types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P).In other instances, the disclosure provides a polypeptide comprising XTEN1, wherein the XTEN1 is characterized by having at least about 36 to about 1000 amino acid residues or at least about 100 to about 1000 amino acid residues, and wherein 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues in the XTEN1 sequence are selected from six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P).

[0156] In another embodiment, the disclosure provides a polypeptide according to any of the embodiments described herein comprising XTEN1, wherein the 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 wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues in the XTEN1 sequence are selected from at least three of the sequences set forth in 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 in 12 amino acid increments, such that any length of at least 36 amino acids or greater (e.g., 36, 48, 60, 72, 84, 96 amino acids, etc.) can be achieved. In other cases, a polypeptide of any of the subject composition embodiments described herein can comprise XTEN1, wherein the XTEN1 is characterized by having 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 in the XTEN1 sequence are selected from the sequences of SEQ ID NOs: 665-718 and 922-926. In another embodiment, the XTEN 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 the sequence EPEA (SEQ ID NO: 1149) added to the N- or C-terminus of the XTEN in the composition to facilitate purification of the composition to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% purity by chromatographic methods known in the art, such as IMAC chromatography or C-tagXL chromatography, or the methods described in the Examples below.

[0157] In another embodiment, the disclosure provides a polypeptide comprising XTEN1, wherein XTEN1 is selected from the group consisting of AE36 (comprising a sequence selected from any three of the sequences of SEQ ID NOs: 661-664), or 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_3A, AE288_4B, AE288_5A, AE288_6B, AE288_7A, AE288_8A, AE288_9A, AE288_10A, AE288_11A, AE288_12A, AE288_13A, AE288_14A, AE288_15A, AE288_16A, AE288_17A, AE288_18A, AE288_19A, AE288_20A, AE288_21A, AE288_22A, AE288_23A, AE288_24A, AE288_25A, AE288_26A, AE288_27A, AE288_28A, AE288_29A, AE288_30A, AE288_31A, AE288_32A, AE288_33A, AE288_34A, AE288_35A, AE288_36A, AE288 AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7.

[0158] In some aspects of any of the embodiments disclosed herein, the subject polypeptide comprises XTEN1 and XTEN2. The composition of polypeptides comprising XTEN1 and XTEN2, among other components, is described herein below. In one embodiment, the disclosure provides a polypeptide comprising XTEN1 and XTEN2, wherein XTEN1 and XTEN2 each have at least about 36 to about 1000 amino acid residues or at least about 100 to about 1000 amino acid residues, and wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of XTEN1 and XTEN2 are selected from at least three of the sequences set forth in SEQ ID NOs: 661-664. In another embodiment, the present disclosure provides a polypeptide comprising XTEN1 and XTEN2, wherein XTEN1 and XTEN2 each have at least about 36 to about 1,000 amino acid residues or at least about 100 to about 1,000 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: 665-718 and 922-926. In another embodiment, the polypeptide of any of the subject composition embodiments described herein can comprise XTEN1 and XTEN2, wherein XTEN1 and XTEN2 each comprise at least about 90% of the amino acid sequence of: AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, and AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is set forth in Table 7.In some cases of the preceding embodiments of this paragraph, XTEN1 and XTEN2 are identical. In other cases of the preceding embodiments of this paragraph, XTEN1 and XTEN2 of the preceding embodiments of this paragraph have different amino acid sequences. In some cases, XTEN1 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 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, in any of the polypeptide composition embodiments having two XTENs, XTEN1 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 compositions according to any of the embodiments described herein, including XTENs of intermediate lengths relative to those in Table 7, as well as XTENs of lengths longer than those in Table 7, e.g., those in which the 12 amino acid motif of Table 6 is added to the N- or C-terminus of an XTEN of Table 7.

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

[0161] Additional examples of XTEN sequences that can be used in accordance with the present disclosure are described in U.S. Patent Publication Nos. 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, or 2011 / 0172146A1, or International Patent Publication Nos. W and 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 a target cell marker antigen other than CD3, which can be incorporated into any of the subject composition embodiments described herein. A first antigen-binding fragment (AF1) having binding affinity for CD3 is linked to a second antigen-binding fragment (AF2) having binding affinity for a second, non-CD3 antigen using a short, flexible peptide linker, resulting in a bispecific composition in which each antigen-binding fragment has specific binding affinity for its respective ligand. It will be understood that in such compositions, an antigen-binding fragment directed against a target cell marker of a diseased tissue is used in combination with a second antigen-binding fragment directed against an effector cell marker to bring effector cells into close proximity with the cells of the diseased tissue, resulting in cytolysis of the cells of the diseased tissue. Additionally, AF1 and AF2 can be incorporated into specifically designed polypeptides comprising a cleavable release segment and XTEN to impart prodrug properties to the composition that are activated by release of the fused AF1 and AF2 upon cleavage of the release segment when in the vicinity of diseased tissue that has a protease capable of cleaving the release segment at one or more positions in the release segment sequence.

[0163] In one embodiment, the polypeptide of any of the subject composition embodiments described herein may comprise AF2 having specific binding affinity for a target cell marker expressed on the cell surface, in the plasma membrane, or within target cells associated with cancer, autoimmune disease, inflammatory disease, and other conditions in which local activation of the polypeptide is desirable. In one embodiment, the antigen to which AF2 has specific binding affinity is 1-40-beta-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, adrenoceptor beta 3 (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, B-cell activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), siblings of imprinted 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 IL-2 receptor), CD27, CD274, CD28, 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), CXC chemokine receptor type 4, cyclin B1, cytochrome P4501B1 (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 (ART) 4), 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 type A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), episialin, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protein serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 (ETV6-AML) located on chromosome 12p, respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II beta chain, fibroblast activation protein alpha (FAP), fibronectin extracellular domain-B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), folate binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor alpha, folate receptor beta, 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 mutant (mut hsp70-2), hemagglutinin, hepatitis A virus cellular receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, hexaglycoprotein of globoH glycoceramide (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, 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 gamma-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 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 (M-ACA) L-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, cell surface-associated 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), neuronal apoptosis-regulating proteinase 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitory factor (e.g., For example, NOGO-A, NOGO-B, NOGO-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), oncofetal antigen (h5T4), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), European rabbit, OX-40, oxLDL, p53 mutant, Pediatrics Paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), P-cadherin, sodium phosphate symporter, 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-recognized melanoma antigen 1 (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostate cancer cells, prostein, protease serine 21 (testisin or PRSS21), proteasome (prosome, 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-recognized squamous cell carcinoma antigens 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 Γ 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 signaling receptor 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 (UPK) 2), vascular endothelial growth factors (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), L Estrogen-regulated IV-1 protein (LIV1, also known as SLC39A6), neurotrophin 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), cell surface-associated 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 proteoglycans, Can 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), teratoma-derived growth factor 1 (TDGF1, also known as Crypto The antigen may be selected from, but is not limited to, antigens including, but not limited to, SLAM family member 6 (SLAMF6, also known as CD352), and Notch receptor 3 (NOTCH3).

[0164] Therapeutic monoclonal antibodies from which AF2 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 to treat many lymphomas, leukemias, and some autoimmune disorders; ofatumumab, an anti-CD20 antibody approved for use in chronic lymphocytic leukemia and in development for follicular non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing-remitting multiple sclerosis; lucatumumab (HCD122), an anti-CD40 antibody for non-Hodgkin's or Hodgkin's 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's lymphoma; veltuzumab (hA20), an antibody that binds to cells expressing CD20 for treating immune thrombocytopenic purpura; Antibodies that bind to cells expressing CD20 for the treatment of rheumatoid arthritis include HumaLYM, an antibody 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, breast, and ovarian cancer (see, e.g., U.S. Patent No. 4,753,894); cetuximab; an anti-EGFR antibody used to treat epidermal growth factor receptor (EGFR)-expressing, KRAS wild-type metastatic colorectal cancer and head and neck cancer (see, e.g., U.S. Patent No. 4,943,533; PCT International Publication No. WO 2014 / 023111); WO 96 / 40210), panitumumab, a fully human monoclonal antibody specific for the epidermal growth factor receptor (also known as EGF receptor, EGFR, ErbB-1, and HER1), currently marketed for the treatment of metastatic colorectal cancer (U.S. Patent No. 6,235,883), zalutumumab, a fully human IgG1 monoclonal antibody directed against the epidermal growth factor receptor (EGFR) for the treatment of head and neck squamous cell carcinoma (see, e.g., U.S. Pat. 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 (see, e.g., U.S. Pat. No. 5,891,996, U.S. Pat. No. 6,506,883), matuzumab, a soluble IgG1 antibody directed against the epidermal growth factor receptor (EGFR) ... Humanized monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been developed for the treatment of colorectal, lung, esophageal, and gastric cancers (see, e.g., U.S. Patent Application No. 2009 / 0175858A1), cetuximab, a chimeric (murine / human) monoclonal antibody directed against the epidermal growth factor receptor (EGFR) that has been used to treat metastatic colorectal cancer, metastatic non-small cell lung cancer, and head and neck cancer (see, e.g., U.S. Patent No. 6,217,(See, e.g., 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 some forms of B-cell non-Hodgkin's lymphoma; gemtuzumab ozogamicin, an anti-CD33 (p67 protein) antibody linked to the radioisotope-conjugated cytotoxic chelator tiuxetan used in the treatment of acute myeloid leukemia; ABX-CBL, an anti CD147 antibody, ABX-IL8, anti-IL8 antibody, ABX-MA1, anti-MUC18 antibody, pemtumomab (R1549, 90Y-muHMFG1), anti-MUC1 in development, Therex (R1550), anti-MUC1 antibody, AngioMab (AS1405) developed by Antisoma, HuBC-1 developed by Antisoma, Thioplatin (AS1407) developed by Antisoma, ANTEGREN (natalizumab), anti-alpha-4-beta-1 (VLA4) and alpha-4-beta-7 antibodies, VLA-1 mAb, anti-VLA-1 integrin antibody, LTBR mAb, anti-lymphotoxin beta receptor (LTBR) antibody, CAT-152, anti-TGF-β2 antibody, J695, anti-IL-12 antibody, CAT-192, developed anti-TGF-β1 antibody, CAT-213, developed anti-eotaxin 1 antibody, LYMPHOSTAT-B, anti-Blys antibody, TRAIL-R1 mAb, anti-TRAIL-R1 antibody, Herceptin, anti-HER receptor family antibody, anti-tissue factor (ATF), anti-tissue factor antibody, Xolair (omalizumab), anti-IgE antibody, MLN-02 antibody (formerly LDP-02), HuMax CD4®, anti-CD4 antibody, tocilizumab, and anti-IL6R antibody, HuMax-IL15, anti-IL15 antibody, HuMax-inflammation, HuMax-cancer, anti-heparanase I antibody, HuMax-lymphoma,, HuMax-TAC, IDEC-131, anti-CD40, IDEC-151 (clenoliximab), 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 (labetuzumab), anti-carcinoembryonic antigen (CEA) antibody developed by Immunomedics, Yervoy (ipilimumab), anti-CTLA4 antibody used to treat melanoma, Lumphocide® (epratuzumab), anti-CD22 antibody, AFP- Cide, 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®-CD4, anti-CD4 antibody, HuMax-IL15, anti-IL15 antibody, anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibody, MOR201, tremelimumab, anti-CTLA-4 antibody, Protein Antibody references may include, but are not limited to, anti-α5β1 integrin developed by Protein Design Labs, anti-IL-12, ING-1, an anti-Ep-CAM antibody developed by Xoma, and MLN01, an anti-Beta2 integrin antibody, all of which are expressly incorporated herein by reference. The sequences of the above antibodies may be obtained from publicly available databases, patents, or literature references. Additionally, non-limiting examples of monoclonal antibodies against cancer, tumor, or target cell markers suitable for incorporation into the subject compositions of the present disclosure, as well as VH and VL sequences (and in some cases, the indicated CDR sequences that can be incorporated into AF2) are provided in Table 8.

[0165] According to the above-described antigen-binding fragment embodiments, it may be advantageous if the binding site recognizing the target cell marker antigen has a high binding affinity to capture the target cells for destruction with high efficiency. In a preferred embodiment, the AF2 target cell antigen-binding fragment has a binding affinity of 10 or more, as determined in an in vitro binding assay. -7 ~10 -10 K in the M range d Because the subject polypeptides described in any of the embodiments of the present disclosure have an advantage that they can be used multiple times to kill tumor cells, because they have affinities at low affinity. If the affinity of a bispecific antigen-binding fragment for binding to a target cell marker is too high, the composition will bind to the expressing target cell and remain on its surface, unable to be released to bind to other cells. In one embodiment, a polypeptide of any of the subject composition embodiments described herein comprises AF2, which 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, as determined in an in vitro antigen binding assay involving a target cell marker. d In another embodiment, AF2 specifically binds to a target cell marker with a binding affinity (K in an in vitro binding assay) of less than about 0.1 nM, or less than about 0.5 nM, or less than about 1.0 nM, or less than about 10 nM, or less than about 50 nM, or less than about 100 nM. d In another embodiment, the present disclosure provides a polypeptide comprising AF2, wherein the binding affinity of AF2 for the target cell marker is at least 10-fold, or at least 100-fold, or at least 1000-fold greater than the binding affinity of AF1 for CD3, as measured in an in vitro antigen binding assay. In another embodiment, the AF1 antigen-binding fragment of any of the subject embodiments of the present disclosure specifically binds to a target cell marker at a K d When determined as a constant, AF2 has a lower binding affinity for the CD3 antigen that is at least one order of magnitude, at least two orders of magnitude, or at least three orders of magnitude lower than the higher binding affinity of AF2 for the target cell marker antigen. dIt is understood to mean a value, e.g., 1 x 10 -9 M is 1 x 10 -8 It 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 binding affinity for a target cell marker antigen. In another embodiment, the polypeptide of any of the subject composition embodiments described herein comprises AF2, wherein AF2 comprises a CDR derived from a monoclonal antibody having 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 of any of the embodiments disclosed herein, a subject polypeptide comprises AF2, wherein AF2 comprises a VL and a VH of a monoclonal antibody having binding affinity for a target cell marker antigen. In some cases, a polypeptide of any of the subject composition embodiments described herein can comprise AF2, wherein AF2 comprises a VL and a VH of a monoclonal antibody having binding affinity for a target cell marker antigen, wherein the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to or 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%, or 99% sequence identity to or 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 composition embodiments disclosed herein is intended to include portions or fragments of antibodies that retain the ability to bind to an antigen that is the ligand of the corresponding intact antibody. In such embodiments, the antigen-binding fragment may be, but is not limited to, CDRs and intervening framework regions, variable or hypervariable regions of the antibody light and / or heavy chains (VL, VH), variable fragments (Fv), Fab' fragments, F(ab')2 fragments, Fab fragments, single-chain antibodies (scAbs), VHH camelid antibodies, single-chain variable fragments (scFv), linear antibodies, single-domain antibodies, complementarity-determining regions (CDRs), domain antibodies (dAbs), BHH-type or BNAR-type single-domain heavy chain immunoglobulins, single-domain light chain immunoglobulins, or other polypeptides known in the art that include fragments of antibodies capable of binding to antigens. The VL and VH of the two antigen-binding fragments can also be configured in a single-chain diabody configuration, i.e., the VL and VH of AF1 and AF2 are configured with a linker of appropriate length to allow for configuration as a diabody.

[0169] In certain embodiments, the VL and VH of the antigen-binding fragment are fused together by a relatively long linker of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids that has flexible properties when linked together. In one embodiment, the VL and VH of any of the scFv embodiments described herein are linked together by a hydrophilic amino acid linker selected from the sequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1142), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 1143), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 1144), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 1145). In other cases, AF1 and AF2 of the subject compositions are linked together by a short hydrophilic amino acid linker 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 in which, after folding, the first domain (VL or VH) pairs with the last domain (VH or VL) to form one scFv, and these two domains pair in the middle to form the other scFv, where the first and second domains and the third and last domains are fused together by one of the aforementioned short linkers, and the second and third variable domains are fused by one of the aforementioned long linkers. The selection of short and long linkers can prevent incorrect pairing of adjacent variable domains, thereby facilitating the formation of a single-chain diabody configuration comprising the VL and VH of the first 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

[0170] VI. Bispecific antigen-binding compositions—construction and functional properties In another aspect, the present disclosure relates to novel chimeric bispecific antigen-binding compositions that bind to an antigen or epitope of the CD3 protein complex of effector cells (e.g., T cells) and a second target cell marker associated with diseased cells or tissues. They may therefore be referred to as T cell engagers. As described in more detail below, bispecific antigen-binding compositions that offer advantages over bispecific T cell engagers and related compounds known in the art are comprised of activatable prodrug forms. Various compositions of the present disclosure possess properties including enhanced stability during their manufacture and purification, enhanced stability and increased half-life in the circulation when administered to a subject, the ability to be activated at the intended site of therapy but not in normal, healthy tissues, and, upon activation by proteolytic cleavage of the release segment and release of the fused AF1 and AF2, exhibit binding affinities for target and effector cells at least comparable to those of corresponding conventional bispecific IgG antibodies. Upon binding of effector and target cells by AF1 and AF2, an immunological synapse is formed that leads to activation of the effector cell and promotes the subsequent destruction of the target cell by apoptosis or cytolysis.

[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 fragments, reducing their ability to bind to their ligands until they are released from the composition by protease cleavage at one of the protease cleavage sites located within the release segment. Proteases known to be associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine ​​proteases, aspartic acid proteases, and metalloproteases, including, but not limited to, the specific proteases described herein. This prodrug property of the bispecific antigen-binding compositions improves the specificity of the compositions for diseased tissues or cells compared to bispecific T cell engager therapeutics that are not in prodrug form. In contrast, by activating the bispecific antigen-binding composition specifically in the microenvironment of a target cell or diseased tissue where a target cell marker and a protease capable of cleaving the release segment are highly expressed, the bispecific antigen-binding fragment and its construct XTEN are released upon cleavage of the release segment, and the fused antigen-binding fragment can crosslink cytotoxic effector cells with cells expressing the target cell marker in a highly specific manner, thereby directing the cytotoxic potential of T cells toward 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 bearing the target cell marker and effector cells, such as cytotoxic T cells, by binding to the CD3 antigen, thereby forming part of a T cell receptor complex and triggering T cell activation that mediates the subsequent lysis of target cells expressing the specific target cell marker. Thus, it is contemplated that the bispecific antigen-binding composition of the present disclosure exhibits potent, specific, and efficient target cell killing. In such cases, cells are selectively eliminated, 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 an effector cell and a second antigen-binding fragment that targets a cell marker associated with a diseased tissue or cell, both of which have specific binding affinity for their respective ligands. The design of the subject compositions having first and second antigen-binding fragments (AF1 and AF2, respectively) was informed by consideration of at least three properties: 1) the composition has a bispecific antigen-binding fragment capable of binding to and linking effector and target cells 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 their ability to bind to target and effector cell ligands when the composition is in an intact prodrug form; ii) provides an enhanced half-life when administered to a subject; iii) reduces extravasation of the intact composition from the blood circulation in normal tissues and organs compared to diseased tissues (e.g., tumors); and iv) confers an increased safety profile compared to conventional bispecific cytotoxic antibody therapeutics; and 3) the RS is activated when cleaved by one or more mammalian proteases near the diseased tissue, thereby releasing the fused bispecific antigen-binding fragments, allowing them to fully regain their binding affinity for the target ligands. The design of the subject compositions utilizes the properties of the XTEN and release segment (RS) components and their positioning relative to the bispecific antigen-binding fragment to achieve the aforementioned properties, as evidenced by the results of the illustrative examples below.

[0173] In one embodiment, a polypeptide of 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, when uncleaved, can have, from N-terminus to C-terminus, the structural arrangement 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 other aspects, 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 consideration that the addition of a second XTEN further reduces the binding affinity of the uncleaved composition to the ligands of each of the AF1 and AF2 antibody fragments, further reducing unintended binding of the composition to healthy tissues or cells when administered to a subject, thereby further improving the therapeutic index of the subject compositions compared to compositions having only one RS and one XTEN. The addition of a second RS and a second XTEN resulted in a surprising reduction in the binding affinity of the uncleaved intact polypeptide to the ligands of each of the AF1 and AF2 antibody fragments when assayed in vitro compared to compositions having a single RS and XTEN, and also resulted in reduced toxicity in animal models of disease when administered at therapeutically effective doses, as described in the Examples below. In embodiments of the subject compositions having two antigen-binding fragments, two RSs, and two XTENs, the compositions, in their uncleaved state, can have, from N-terminus to C-terminus, the structural arrangement: XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-diabody-RS1-XTEN1 (wherein the diabody comprises the VL and VH of AF1 and AF2), or XTEN1-RS1-diabody-RS2-XTEN2 (wherein the diabody comprises the VL and VH of AF1 and AF2).

[0175] It is a feature of various designed compositions that, when the RS of the bispecific antigen-binding composition is cleaved by a mammalian protease in the environment of a target cell and converted from a prodrug form to an activated or apoprotein form, upon cleavage and release of the bispecific antigen-binding fragment and XTEN from the composition, the fused AF1 and AF2 bind to and link together effector cells (e.g., CD3-bearing T cells) and diseased cells (e.g., tumor or cancer cells) bearing a target cell marker antigen that can bind to AF2, whereupon the effector cells are activated. In one embodiment, the RS of the bispecific antigen-binding composition is cleaved, the antigen-binding fragments are released, and subsequent simultaneous binding to the effector and target cells can result in at least 3-fold, 10-fold, 30-fold, 100-fold, 300-fold, or 1000-fold activation of the effector cells, as assessed by the production of cytokines, cytolytic proteins, or lysis of target cells, as assessed in an in vitro cell-based assay. In another embodiment, simultaneous binding of the released fusion AF1 and AF2 to a T cell bearing the CD3 antigen and a diseased cell bearing the target cell marker antigen forms an immunological synapse, which results in the release of T cell-derived effector molecules that can lyse the diseased cell.Non-limiting examples of in vitro assays for measuring effector cell activation and / or cell lysis include cell membrane integrity assays, mixed cell culture assays, FACS-based propidium iodide assays, trypan blue influx assays, photometric enzyme release assays, ELISA, radiometric 51Cr release assays, fluorometric europium release assays, Calcein AM release assays, photometric MTT assays, XTT assays, WST-1 assays, Alamar Blue assays, radiometric 3H-Thd incorporation assays, clonogenic assays measuring mitotic activity, fluorometric Rhodamine 123 assays measuring mitochondrial transmembrane gradients, FACS-based phosphatidylserine exposure-monitored apoptosis assays, ELISA-based TUNEL assays, caspase activity assays, and cell morphology assays, or other assays known in the art for assaying cytokines, cytolytic proteins, or cell lysis, or methods described in the Examples below.

[0176] Without being bound by any particular theory, it is believed that by using the above-mentioned bispecific antigen-binding composition format, upon cleavage of RS, the released fusion AF1 and AF2 can kill target cells by recruiting cytotoxic effector cells without the need for pre-stimulation and / or costimulation. Furthermore, the independence of effector cells from pre-stimulation and / or costimulation may substantially contribute to the extremely high cytotoxicity mediated by the released fusion AF1 and AF2 antigen-binding fragments. In some embodiments, the released AF1 and AF2, in which AF1 remains fused to AF2 by a linker peptide, are engineered with binding specificity such that they have the ability to bind to a pre-selected target cell marker and link them together in close proximity, with AF2 having binding specificity for a target cell marker associated with cytotoxic effector cells (e.g., T cells, NK cells, cytokine-induced killer cells (CIK cells), tumor cells, cancer cells, or cells associated with diseased tissue), thereby creating an immunological synapse and selective, directed, and localized effect of the released cytokine and effector molecule on the target tumor or cancer cell, resulting in damage or destruction of the tumor or cancer cell and providing a therapeutic benefit to the subject. The released AF1 that binds to the effector cell antigen binds to the effector cell antigen. AF2 can regulate one or more functions of a cell, causing or contributing to the cytolytic effect on target cells, such as tumor cells, to which it binds. Effector cell antigens can be expressed by effector cells or other cells. In one embodiment, effector cell antigens are expressed on the cell surface of effector cells. Non-limiting examples of effector cell antigens include CD3, CD4, CD8, CD16, CD25, CD38, CD45RO, CD56, CD57, CD69, CD95, CD107, and CD154. Therefore, those skilled in the art will understand that the configuration of the subject composition is intended to selectively or disproportionately deliver the active form of the composition to target tumor tissues or cancer cells compared to healthy tissues or healthy cells in the subject to which the composition is administered, resulting in a therapeutic benefit.As is apparent from the foregoing, the present disclosure provides a large family of polypeptides in configurations designed to provide desired properties.

[0177] It is an object of the present disclosure that the design of subject bispecific antigen binding compositions in which the shielding effect is conferred by the XTEN in the intact circulating composition, and which concomitantly reduces the likelihood of binding to effector cells and target tissues, will result in reduced production of Th1 T cell-associated cytokines or other pro-inflammatory mediators during systemic exposure when administered to a subject, such that the overall side effect and safety profile (e.g., therapeutic index) is improved compared to bispecific antigen binding compositions that are not linked to a shielding moiety, such as XTEN. As key components of cellular immunity, the production of IL-2, TNF-alpha, and IFN-gamma is a hallmark of Th1 responses (Romagnani ST-cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol. 2000. 85(1):9-18), particularly in anti-CD3 stimulated T cells (Yoon, SH. Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro. Exp Mol Med. 2009. 41(3):161-170). IL-4, IL-6, and IL-10 are also pro-inflammatory cytokines important in the cytotoxic response to bispecific antibody compositions (Zimmerman, Z., et al. Unleashing the clinical power of T cells: CD19 / CD3 bi-specific T cell engager (BiTE®) antibody composition blinatumomab as a potential therapy. Int. Immunol. (2015) 27(1):31-37).In one embodiment, an uncleaved, intact bispecific antigen-binding composition of the embodiments described herein may exhibit at least a 3-fold, or at least a 4-fold, or at least a 5-fold, or at least a 6-fold, or at least a 7-fold, or at least a 8-fold, or at least a 9-fold, or at least a 10-fold, or at least a 20-fold, or at least a 30-fold, or at least a 50-fold, or at least a 100-fold, or at least a 1000-fold decrease in the likelihood of resulting in the production of Th1 and / or pro-inflammatory cytokines when the uncleaved, intact polypeptide is contacted with effector and target cells in an in vitro cell-based cytokine stimulation assay compared to the Th1 and / or cytokine levels stimulated by the corresponding released AF1 and AF2 (which remain fused together after release by RS proteolysis) of the corresponding protease-treated composition in an in vitro cell-based cytokine stimulation assay performed under comparable conditions, e.g., at an equivalent molar concentration. Non-limiting examples of Th1 and / or pro-inflammatory cytokines are IL-2, IL-4, IL-6, IL-10, TNF-alpha, and IFN-gamma. In one aforementioned embodiment, Th1 cytokine production is assayed in an in vitro assay including effector cells, such as PBMCs or CD3+ T cells, and target cells bearing a target cell marker antigen as disclosed herein. In another embodiment, cytokines can be assessed from blood, body fluid, or tissue samples taken from a subject to which the polypeptide composition has been administered. In the aforementioned embodiments, the subject can be a mouse, rat, monkey, or human. However, an advantage of the subject bispecific antigen-binding compositions of the embodiments described herein is that the cytolytic properties of the compositions do not require cytokine pre-stimulation; it has been found that 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 the target cells.Nevertheless, the production of pro-inflammatory cytokines is a useful marker for assessing the efficacy or effectiveness of a subject polypeptide composition, whether by in vitro assay or by monitoring the treatment of a subject having an affected tissue (e.g., a tumor, etc.) following administration of a subject bispecific antigen-binding composition.

[0178] In the context of use of bispecific antigen-binding compositions in subjects, for purposes of this disclosure, the subject bispecific antigen-binding compositions are designed to take advantage of the difference in pore size of the vasculature in tumor or inflamed tissue compared to healthy vasculature through the addition of XTEN, such that extravasation of the intact bispecific antigen-binding composition in normal tissue is reduced, but in the leaky environment of tumor vasculature or other inflammatory areas in diseased tissue, the intact construct leaks proteases in the diseased tissue environment and can be activated thereby, releasing antigen-binding fragments to effector and target cells (see, e.g., Figure 5). In the case of the RS of the bispecific antigen-binding composition, this design takes advantage of the situation where, when the bispecific antigen-binding composition is near diseased tissue, e.g., a tumor, that produces one or more proteases, RS sequences that are sensitive to one or more proteases expressed by the tumor can be cleaved by the proteases (described more fully above). The action of protease cleaves the release segment (RS) of the composition, separating the antigen-binding fragment from XTEN, resulting in components with reduced molecular weight and hydrodynamic radius, particularly for the released fusion AF1 and AF2.As can be understood, the reduced molecular weight and hydrodynamic radius of the composition also gives the released fusion AF1 and AF2 the properties of moving more freely in solution, moving through smaller pore spaces in tissues and tumors, leaking more easily through the larger pores of tumor vasculature, penetrating more easily into tumors, and increasing the ability to bind to and link effector cells and tumor cells together.Such properties can be measured by different assays.Therefore, those skilled in the art will understand that in the context of treating a subject using the subject composition, the bispecific antigen-binding composition exists in a prodrug form, and is converted into a more active form when entering a certain cellular environment by the action of proteases co-localized with affected tissues or cells.Upon release from the composition by the action of proteases in the target tissue, AF1, which has binding specificity for an effector cell antigen, and the fused AF2, which has binding specificity for a target cell marker antigen of the diseased cell, fully regain the ability to bind to and link effector and target cells together, forming an immunological synapse. Formation of the immunological synapse activates the effector cell, and various signaling pathways activate new gene transcription and release the effector molecule contents of the vesicle by exocytosis. Depending on the type of effector cell, different cytokines and lymphokines are released; for example, type 1 helper T cells (Th1) release cytokines such as IFN-gamma, IL-2, and TNF-alpha, while type 2 helper T cells (Th2) release cytokines such as IL-4, IL-5, IL-10, and IL-13 that stimulate B cells, and cytotoxic T lymphocytes (CTLs) release cytotoxic molecules such as perforin and granzymes (collectively "effector molecules") that kill targets. It is specifically contemplated that simultaneous binding and linking of effector cells and target tumor cells by the released bispecific antigen-binding fragments of the bispecific antigen-binding composition, at very low effector:target (E:T) ratios, will result 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 with a disease such as a tumor, the prodrug form remains in the circulation in normal tissues but can leak into the more permeable vasculature of the tumor such that the prodrug form of the construct is activated by proteases co-localized with the tumor and the released antigen-binding fragment binds to and links together effector cells (e.g., T cells) and tumor cells expressing the target cell marker targeted by AF2 of the composition, whereupon the effector cells are activated, resulting in tumor cell lysis.In other words, in some cases, the more permeable vasculature in tumor tissue allows the bispecific antigen-binding polypeptide to leak into that tissue, where tumor-associated proteases can act on the release segment (RS), cleave it, releasing the binding moiety, and then bind to effector cells and tumor-associated cells, linking them together. In normal tissue, extravasation may be blocked by a tighter vasculature barrier, or if the bispecific antigen-binding polypeptide leaks to some extent, the bispecific antigen-binding polypeptide may remain primarily in the "pro" form because insufficient proteases may be present in healthy tissue to release the binding moiety, with the net effect being that no immunological synapse is formed. In some cases, the released fused AF1 and AF2 in the subject's tumor, bound to both tumor cells and effector cells, exhibit at least a 10-fold, or at least a 30-fold, or at least a 100-fold, or at least a 200-fold, or at least a 300-fold, or at least a 400-fold, or at least a 500-fold, or at least a 1000-fold increase in the ability to activate effector cells compared to the corresponding uncleaved intact bispecific antigen-binding composition. In other cases, the released linked AF1 and AF2 in the subject's tumor, bound to both tumor cells and effector cells, exhibit at least a 10-fold, or at least a 30-fold, or at least a 100-fold, or at least a 200-fold, or at least a 300-fold, or at least a 400-fold, or at least a 500-fold, or at least a 1000-fold increase in the ability to lyse tumor cells compared to the corresponding uncleaved intact bispecific antigen-binding composition in the tumor. In the foregoing embodiments, effector cell activation and / or cytotoxicity can be assayed by conventional methods known in the art, such as measuring cell counts of activated effector cells, assaying cytokines, measuring tumor size, or histopathology. In the foregoing embodiments, the subject can be a mouse, rat, dog, monkey, or human.Specifically, it is specifically contemplated that the subject compositions are designed such that, when administered to a subject with a disease having a target cell marker to which AF2 can bind, the bispecific antigen-binding composition exhibits an enhanced therapeutic index and reduced incidence of side effects compared to conventional bispecific antibodies known in the art, achieved by a combination of steric hindrance and the shielding effect of XTEN on the binding affinity for the antigen-binding fragment in a prodrug form, but is capable of releasing the bispecific AF1 and AF2 (achieved by inclusion of a cleavage sequence in the RS) near or within the target tissue (e.g., tumor) that produces the protease for which the RS is a substrate.

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

[0180] In particular, several therapeutic strategies have been used to design polypeptide compositions for use in methods for treating subjects with cancerous diseases, including regulating T cell responses by targeting TcR signaling using the VL and VH portions of anti-human CD3 monoclonal antibodies, which are widely used clinically in immunosuppressive regimens. The CD3-specific monoclonal OKT3 was the first such monoclonal approved for human use (Sgro, Toxicology 105 (1995), 23-29) and is widely used clinically as an immunosuppressant during transplantation (Chatenoud L: Immunologic monitoring during OKT3 therapy. Clin Transplant 7:422-430, 1993). Furthermore, anti-CD3 monoclonal antibodies can induce partial T cell signaling and clonal anergy (Smith, J. Exp. Med. 185 (1997), 1413-1422). OKT3 reacts with and blocks the function of the CD3 complex in the membrane of T cells, which associates with the antigen-recognition structure of T cells (TCR), essential for signal transduction. These and other such CD3-specific antibodies can induce various T cell responses, including cytokine production (Von Wussow, Human gamma interferon production by leukocytes induced with monoclonal antibodies recognizing T cells. J. Immunol. 127:1197-1200 (1981)), proliferation, and the induction of suppressor T cells. In cancer, attempts have been made to lyse cancer cells using cytotoxic T cells. Without being bound by theory, it is believed that cytotoxic T cells require direct cell-to-cell contact to bring about target cell lysis, and the TCR on the cytotoxic T cell must recognize and engage with the appropriate antigen on the target cell. This creates an immunological synapse that then initiates a signaling cascade within the cytotoxic T cell, leading to T cell activation and the production of a variety of cytotoxic cytokines and effector molecules.Perforin and granzymes are highly toxic molecules stored in preformed granules present in activated cytotoxic T cells. After target cell recognition, the cytoplasmic granules of the engaged cytotoxic T cells migrate toward the cytotoxic T cell membrane, eventually fusing with it and releasing their contents in a directed manner into the immunological synapse, forming pores in the target cell membrane and disrupting the tumor cell plasma membrane. The created pores act as entrances for granzymes, a family of serine proteases that induce apoptosis in tumor cells.

[0181] The subject bispecific antigen binding compositions described herein, in which AF1, which has specific binding affinity for CD3 on T cells, is tightly fused to AF2, which has specific binding affinity for a target cell marker, are T cell engagers that, upon release from the intact prodrug form of the composition by cleavage of the release segment, fully regain their ability to bind to T cells and target cells, form an immunological synapse that promotes T cell activation and the subsequent destruction of tumor cells by apoptosis or cytolysis.

[0182] The present disclosure contemplates the use of bispecific antigen-binding compositions engineered to target a wide range of malignant cells, such as tumors, in addition to effector cells to initiate target cell lysis and provide beneficial therapeutic outcomes in that bispecific antigen-binding compositions can be designed such that AF1 binds and associates with CD3 to activate cytotoxic T cells, while AF2 can be designed to target a variety of different target cell markers characteristic of a particular malignancy, bringing them together to create an immunological synapse. A particular advantage of this design is that the physical association of cytotoxic effector cells with cells bearing 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, those disclosed herein. Because of the range of such target cell markers (described more broadly above) that can be engineered into various embodiments of the subject bispecific antigen-binding compositions, it will be appreciated that the resulting compositions have utility for a variety of diseases, including hematological cancers and solid tumors. In one embodiment, the present disclosure provides a method of treating a subject with a tumor. The tumors to be treated may contain tumor cells derived from cells selected from the group consisting of stromal cells, fibroblasts, myofibroblasts, glial cells, epithelial cells, adipocytes, lymphocytes, vascular cells, smooth muscle cells, mesenchymal cells, breast tissue cells, prostate cells, kidney cells, brain cells, colon cells, ovarian cells, uterine cells, bladder cells, skin cells, stomach cells, urogenital tract cells, cervical cells, uterine cells, small intestine cells, liver cells, pancreatic cells, gallbladder cells, bile duct cells, esophageal cells, salivary gland cells, lung cells, and thyroid cells. A further advantage of the composition is that the cytotoxic effector cells are not consumed during the damage / destruction of the crosslinked target cancer cells; after causing lysis of one target cell, the activated effector cells can be released and migrate through the local tissue toward other target cancer cells, re-bind to AF1-AF2 and the target antigen, and initiate additional cell lysis.In addition, it is contemplated that in a localized environment such as a solid tumor, the release of effector cell molecules such as perforin and granzymes will result in damage to tumor cells adjacent to, but not bound by, a given molecule of the bispecific binding domain, resulting in stasis of tumor growth or regression.

[0183] Thus, one will appreciate the utility of the present disclosure that, after administering a therapeutically effective dose of a pharmaceutical composition comprising the bispecific antigen-binding composition described herein to a subject with a cancer or tumor bearing 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, thereby enabling an immunological synapse to be created by linkage between the target cell and an effector cell, resulting in the release of effector molecules from the effector cell that can lyse the target cell, resulting in apoptosis, cytolysis, or death of the target cancer or tumor cell. Furthermore, one skilled in the art will appreciate that the formation of an immunological synapse by binding of the released binding domains to effector cells and target cancer cells can result in the use of bispecific antigen-binding compositions to provide a beneficial therapeutic effect that is sustained and more systemic than "single killing."

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

[0185] The therapeutically effective dose of the bispecific antigen-binding compositions described herein generally provides therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the bispecific antigen-binding compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Using cell culture assays and animal experiments, LD 50 (lethal dose for 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects can be determined as the LD 50 / ED 50 The therapeutic index can be expressed as a ratio of ED to ED. Bispecific antigen-binding compositions that exhibit a large therapeutic index are preferred. In one aspect, the bispecific antigen-binding molecules of the present disclosure exhibit a high therapeutic index. Data obtained from cell culture assays and animal experiments can be used to formulate a suitable dose range for human use. Dosages are determined to be ED with little or no toxicity. 50 The blood concentration range is preferably within the range including the range of 0.1 to 1.0 mg / kg of the bispecific antigen-binding composition. The dosage can vary within this range depending on various factors, such as the dosage form used, the route of administration utilized, the condition of the subject, etc. The exact formulation, route of administration, and dosage can be chosen by the individual physician in consideration of the patient's condition (see, for example, Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Ch. 1, p. 1). Those skilled in the art will readily recognize that in many cases, a bispecific antigen-binding composition may provide only a partial benefit rather than a cure. In some embodiments, physiological changes that have some benefit are also considered therapeutically beneficial. Thus, in some embodiments, the amount of a bispecific antigen-binding composition that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount." The subject, patient, or individual in need of treatment is typically a mouse, rat, dog, monkey, or human.

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

[0187] In one embodiment of the method of treating a disease in a subject, the disease to be treated is carcinoma, 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 tract cancer, ovarian cancer, ovarian cancer with malignant ascites, vaginal cancer, vulvar cancer, Ewing's sarcoma, peritoneal carcinomatosis, uterine serous carcinoma, parathyroid cancer, endometrial cancer, cervical cancer, colorectal cancer, epithelial intraperitoneal malignant tumor with malignant ascites, uterine cancer, peritoneal mesothelioma, kidney cancer, lung cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, The therapeutically effective amount may be a cancer or tumor, such as small intestine cancer, liver cancer, hepatocellular carcinoma, retinoblastoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, testicular cancer, bile duct cancer, bone cancer, salivary gland cancer, thyroid cancer, craniopharyngioma, carcinoid tumor, epithelial cancer, androgenic tumor, adenocarcinoma, sarcoma of any origin, primary hematologic malignancy (including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, B-cell derived chronic lymphocytic leukemia, hairy cell leukemia, myeloproliferative neoplastic disorder, or myelodysplastic disorder), myasthenia gravis, Graves' disease, Kaposi's sarcoma, neuroblastoma, Hashimoto's thyroiditis, Wilms' tumor, or Goodpasture's syndrome. A therapeutically effective amount may provide a beneficial effect in helping to treat (e.g., cure or reduce the severity of) or prevent (e.g., reduce the likelihood of recurrence of) a cancer or tumor. In another embodiment of the method of treating a disease in a subject, the pharmaceutical composition is administered to the subject in two or more therapeutically effective doses administered twice weekly, once weekly, once every two weeks, once every three weeks, once every four weeks, or once monthly. In another embodiment of the method, the pharmaceutical composition is administered to the subject in two or more therapeutically effective doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months.In another embodiment of the method, a first low priming dose is administered to the subject, followed by one or more higher maintenance doses over a dosing schedule of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. The initial priming dose administered is selected from the group consisting of at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, and at least about 0.1 mg / kg, and the one or more subsequent maintenance doses administered are selected from the group consisting of at least about 0.02 mg / kg, at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.22 mg / kg, at least about 0.26 mg / kg, at least about 0.28 mg / kg, at least about 0.29 mg / kg, at least about 0.32 mg / kg, at least about 0.36 mg / kg, at least about 0.38 mg / kg, at least about 0.39 mg / kg, at least about 0.42 mg / kg, at least about 0.46 mg / kg, at least about 0.49 mg / kg, at least about 0.52 mg / kg, at least about 0.56 mg / kg, at least about 0.58 mg / kg, at least about 0.59 mg / kg, at least about 0.62 mg / kg, at least about 0.64 mg / kg, at least about 0.66 mg / kg, at least about 0.68 mg / kg, at least about 0.70 mg / kg, at least about 0.72 mg / kg, at least about 0.7 In another embodiment of the method, the pharmaceutical composition is administered to the subject intradermally, subcutaneously, intravenously, intraarterially, intraperitoneally, intraperitoneally, 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 over a period of 5 minutes to 96 hours, as tolerated for maximum safety and efficacy.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 over a period of 5 minutes to 96 hours, wherein 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 0.25 mg / kg, at least about 0.30 mg / kg, at least about 0.31 mg / kg, at least about 0.32 mg / kg, at least about 0.33 mg / kg, at least about 0.34 mg / kg, at least about 0.35 mg / kg, at least about 0.36 mg / kg, at least about 0.37 mg / kg, at least about 0.38 mg / kg, at least about 0.40 mg / kg, at least about 0.41 mg / kg, at least about 0.42 mg / kg, at least about 0.43 mg / kg, at least about 0.44 mg / kg, at least about 0.45 mg / kg, at least about 0.46 mg / kg, at least about 0.47 mg / kg, at least about 0.48 mg / kg, at least about 0.49 mg / kg, at least about 0.50 mg / kg, at least about 0.51 mg / kg, at least about 0.52 mg / kg, at least about 0.53 mg / kg, at least about 0.54 mg / kg, at least about 0.55 mg / kg, at least about 0.56 mg / kg, at least about 0.5 0.22 mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg, or at least about 5.0 mg / kg. In another embodiment of this method, the pharmaceutical composition is administered to the subject as one or more therapeutically effective bolus doses or by infusion over a period of from 5 minutes to 96 hours, and wherein administration to the subject results in a C plasma concentration of uncleaved intact bispecific antigen-binding composition in the subject of at least about 0.1 ng / mL to at least about 2 μg / mL or more that is maintained for at least about 3 days, at least about 7 days, at least about 10 days, at least about 14 days, or at least about 21 days.A therapeutically effective dose may be at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.14 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.22 mg / kg, at least about 0.2 4 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg. In one embodiment, the initial dose is selected from the group consisting of at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, and at least about 0.1 mg / kg, and subsequent doses are selected from the group consisting of at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.14 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.2 2 mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg.In the foregoing embodiments, administration to a subject results in a plasma concentration of the polypeptide in the subject of at least about 0.1 ng / mL to at least about 2 ng / mL or more for at least about 3 days, at least about 7 days, at least about 10 days, at least about 14 days, or at least about 21 days. In the foregoing embodiments of the method, the subject can be a mouse, rat, dog, monkey, or human.

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

[0189] In some embodiments, the present 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 the complement of that polynucleotide sequence. In one embodiment, the present invention provides an isolated polynucleotide sequence encoding a polypeptide or bispecific antigen-binding composition according to any of the embodiments described herein, or the complement of that polynucleotide sequence. In one embodiment, the present invention provides an isolated polynucleotide sequence encoding a polypeptide or bispecific antigen-binding composition having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a polynucleotide sequence listed in Table 9.

[0190] In another aspect, the present disclosure relates to methods for generating polynucleotide sequences encoding a polypeptide or bispecific antigen-binding composition according to any of the embodiments described herein, or sequences complementary to the polynucleotide sequences (including homologous variants thereof), as well as methods for expressing proteins expressed by the polynucleotide sequences. Generally, the methods involve generating a polynucleotide sequence encoding a proteinaceous polypeptide or bispecific antigen-binding composition according to any of the embodiments described herein and incorporating the encoding gene into a suitable expression vector in a host cell. To produce the encoded polypeptide or bispecific antigen-binding composition according to any of the embodiments described herein, the method involves transforming a suitable host cell with the expression vector and culturing the host cell under conditions that cause or allow the resulting polypeptide or bispecific antigen-binding composition according to 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 according to any of the embodiments described herein is used to generate a recombinant DNA molecule that directs expression in a suitable host cell. Several cloning strategies are suitable for practicing the present disclosure, many of which are used to generate constructs comprising genes encoding the compositions of the present disclosure, or their complements. In one embodiment, a cloning strategy is used to generate a gene encoding a construct comprising nucleotides encoding the polypeptide or bispecific antigen-binding composition that is used to transform a host cell to express the composition. In the foregoing embodiments described in this paragraph, the gene may comprise nucleotides encoding the antigen-binding fragment, release segment, and XTEN in the configurations disclosed herein.

[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 an expression vector, transforming a host cell, and expressing and recovering XTEN are described in the Examples.

[0193] Genes encoding polypeptide or bispecific antigen-binding composition constructs can be generated in one or more steps, either entirely synthetically or synthetically, combined with enzymatic processes such as restriction enzyme-mediated cloning, PCR, and overlap extension, including methods more fully described in the Examples. The methods disclosed herein can be used, for example, to ligate polynucleotide sequences encoding various component (e.g., binding domain, linker, release segment, and XTEN) genes of desired length and sequence. Genes encoding polypeptide compositions are assembled from oligonucleotides using standard gene synthesis techniques. Gene design can be performed using algorithms that optimize codon usage and amino acid composition appropriate for the E. coli or mammalian host cell utilized for polypeptide or bispecific antigen-binding composition production. In one method of the present disclosure, a library of polynucleotides encoding the components of the construct is generated and then assembled, as described above. The resulting genes are then constructed, host cells are transformed with the resulting genes, and the polypeptide compositions are produced and recovered, and their properties evaluated, as described herein.

[0194] The resulting polynucleotides encoding the polypeptide or bispecific antigen-binding composition sequences can then be individually cloned into expression vectors. Nucleic acid sequences can be inserted into vectors by a variety of procedures. Generally, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components uses standard ligation techniques known to those skilled in the art. Such techniques are well known in the art and are fully described in the scientific and patent literature. Various vectors are publicly available. Vectors can be in the form of, for example, a plasmid, cosmid, viral particle, or phage that can be conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it will be introduced. Thus, vectors can be autonomously replicating vectors, i.e., vectors that exist as extrachromosomal entities and whose replication is independent of chromosomal replication, such as plasmids. Alternatively, the vector may be a vector that, when introduced into a host cell, is integrated into the host cell genome and replicates together with the chromosome into which it is integrated. Once introduced into a suitable host cell, the expression of the antigen-binding fragment or bispecific antigen-binding composition can be determined using any nucleic acid or protein assay known in the art. For example, the presence of transcribed mRNA of the light chain CDR or heavy chain CDR, antigen-binding fragment, or bispecific antigen-binding composition can be detected and / or quantified by conventional hybridization assays (e.g., Northern blot analysis), amplification procedures (e.g., RT-PCR), SAGE (U.S. Patent No. 5,695,937), and array-based technologies (see, e.g., U.S. Patent Nos. 5,405,783, 5,412,087, and 5,445,934) using probes complementary to any region of the antigen-binding unit polynucleotide.

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

[0196] Suitable vectors include, but are not limited to, SV40 and pcDNA and derivatives of known bacterial plasmids, e.g., col EI, pCR1, pBR322, pMal-C2, pET, pGEX (described in Smith, et al., Gene 57:31-40 (1988)), pMB9, and their derivatives, plasmids such as RP4, phage DNA, e.g., numerous derivatives of phage I, such as NM98 9, and other phage DNA, e.g., M13 and filamentous single-stranded phage DNA; yeast plasmids, e.g., 2 micron plasmid or derivatives of 2 micron plasmid, and centromeric and integrative yeast shuttle vectors; vectors useful in eukaryotic cells, e.g., vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, e.g., phage DNA or plasmids modified to use expression control sequences, and the like. Yeast expression systems that may also be used in the present disclosure include, but are not limited to, the non-fusion pYES2 vector (Invitrogen), the fusion pYESHisA, B, C (Invitrogen), the pRS vector, and the like. Control sequences in the vector include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.Suitable promoters for use in expression vectors using prokaryotic hosts include, for example, the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, tryptophan (trp) promoter systems [Goeddel, Nucleic Acids Res., 8:4057 (1980); 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 operably linked to DNA encoding the XTEN polypeptide. Promoters for use in bacterial systems may also contain a Shine-Dalgarno (SD) sequence operably linked to DNA encoding the polypeptide.

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

[0198] IX. Methods of Producing Polypeptides and Bispecific Antigen-Binding Compositions In another aspect, the present disclosure provides methods for producing a subject composition. In one embodiment, the method comprises culturing a host cell containing a nucleic acid construct encoding a polypeptide or bispecific antigen-binding composition according to any of the embodiments described herein under conditions that promote expression of the polypeptide or bispecific antigen-binding composition, and then recovering the polypeptide or bispecific antigen-binding composition using standard purification methods (e.g., column chromatography, HPLC, etc.) to recover the composition, wherein at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the binding fragments of the expressed polypeptide or bispecific antigen-binding composition are correctly folded. In another embodiment of the method of making, the expressed polypeptide or bispecific antigen-binding composition is recovered, wherein at least or at least 90%, or at least 95%, or at least 97%, or at least 99% of the polypeptide or bispecific antigen-binding composition is recovered in monomeric, soluble form.

[0199] In another aspect, the disclosure relates to methods for producing polypeptides and bispecific antigen-binding compositions at high fermentative expression levels of functional proteins using E. coli or mammalian host cells, and methods for providing expression vectors encoding constructs useful in the methods for generating cytotoxically active polypeptide construct compositions at high expression levels. In one embodiment, the method comprises: 1) preparing a polynucleotide encoding a polypeptide according to any of the embodiments disclosed herein; 2) cloning the polynucleotide into an expression vector, which may be a plasmid or other vector under the control of transcription and translation sequences suitable for high-level protein expression in a biological system; 3) transforming a suitable host cell with the expression vector; and 4) culturing the host cell in a conventional nutrient medium under conditions suitable for expression of the polypeptide composition. If desired, the host cell is E. coli. By this method, expression of the polypeptide results in a fermentation titer of at least 0.05 g / L, or at least 0.1 g / L, or at least 0.2 g / L, or at least 0.3 g / L, or at least 0.5 g / L, or at least 0.6 g / L, or at least 0.7 g / L, or at least 0.8 g / L, or at least 0.9 g / L, or at least 1 g / L of host cell expression product, and at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the expressed protein is correctly folded. As used herein, the term "correctly folded" means that the antigen-binding fragment component of the composition has the ability to specifically bind to its target ligand.In another embodiment, the disclosure provides a method for producing a polypeptide or bispecific antigen-binding composition, comprising culturing host cells comprising a vector encoding a polypeptide comprising a polypeptide or bispecific antigen-binding composition under conditions effective to express the polypeptide product at a concentration of greater than about 10 milligrams per gram (mg / g) of the polypeptide, or at least about 250 mg / g, or about 300 mg / g, or about 350 mg / g, or about 400 mg / g, or about 450 mg / g, or about 500 mg / g dry weight host cells in a fermentation reaction when the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, wherein the antigen-binding fragment of the expressed protein is correctly folded. In another embodiment, the disclosure provides a method for producing a polypeptide or bispecific antigen-binding composition, comprising culturing host cells comprising a vector encoding the composition under conditions effective to express the polypeptide product in a fermentation reaction at a concentration of greater than about 10 milligrams per gram (mg / g) of the polypeptide, or at least about 250 mg / g, or about 300 mg / g, or about 350 mg / g, or about 400 mg / g, or about 450 mg / g, or about 500 mg / g dry weight host cells when the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, wherein the expressed polypeptide product is soluble.

[0200] The following are examples of compositions and composition evaluations of the present disclosure. It will be understood that various other embodiments can be practiced in light of the summary provided above. [Example]

[0201] Example 1: Construction of a bispecific antigen-binding polypeptide with two release segments. To generate a plasmid from which individual scFvs could be removed by restriction digestion, pCW1700, encoding the anti-EpCAM-anti-CD3 (UCHT1) bispecific tandem scFv along with the release segment RSR2486, AE866 XTEN, and 6X His-tag affinity tag (SEQ ID NO: 1150), was digested with SacII and BstXI to remove the 3' end of the anti-EpCAM binding domain, the linker between the anti-EpCAM and anti-CD3 domains, and the 5' end of the anti-CD3 domain. A DNA fragment encoding the same region was synthesized with a silent point mutation at the junction between the anti-EpCAM binding domain and the linker to introduce a Bsu36I site. The synthetic DNA fragment was cloned into the digested backbone using an In-Fusion kit (New England Biolabs) to construct pJB0035. pJB0035 was then digested with NheI and BsaI to remove the BSRS1 release segment sequence. Overlapping single-stranded oligonucleotides encoding RSR2486 were synthesized with single-stranded tails that annealed to the NheI and BsaI overhangs. These oligonucleotides were annealed together and ligated into digested pJB0035 to yield pCW1880, which encodes the anti-EpCAM-anti-CD3 (UCHT1) bispecific tandem scFv, RSR2486, XTEN866, and a 6X His-tag affinity tag (SEQ ID NO: 1150).

[0202] To generate plasmids carrying 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 containing restriction sites at the 5' and 3' ends to serve as DNA overlaps for the Gibson DNA construct. The synthetic DNA fragments were cloned into the digested backbone using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to construct pJB0205, pJB0206, pJB0207, and pJB0208.

[0203] To generate bispecific antigen-binding polypeptides with both N- and C-terminal XTEN, AE292 XTEN was PCR-amplified from a plasmid using primers containing backbone DNA at the N-terminus and a 17- to 21-bp 5' region of homology to a non-cleavable release segment (RSR3058, amino acid sequence TTGEAGEAAGATSAGATGP (SEQ ID NO: 100)) at the C-terminus. A second PCR product encoding the light chain of anti-EpCAM antibody 4D5MOCB and a portion of its heavy chain was amplified using primers containing RSR3058 at the N-terminus and a 16- to 21-bp 5' region of homology to the heavy chain of 4D5MOCB at the C-terminus. These PCR fragments were cloned into a BsiWI-SacII-digested backbone vector encoding the remainder of the 4D5MOCB heavy chain / anti-CD3 tandem scFv, a second copy of the non-cleavable release segment RSR3058, and the AE837 XTEN with a 6xHIS (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 STII secretion leader, consisting of (N- to C-terminally) the AE292 XTEN, the non-cleavable release segment RSR3058, and the anti-EpCAM-anti-CD3 bispecific tandem scFv, with RSR3058 fused to the AE867 XTEN with a 6xHIS (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 was generated encoding the AE292 XTEN, cleavable release segment RSR2295, and anti-EpCAM-anti-CD3 bispecific tandem scFv, with RSR2295 fused to the AE868 XTEN. The plasmid utilized two PCR products using pJB0084 as a template: the first encodes the AE292 XTEN with a 6xHIS (SEQ ID NO: 1150) affinity tag and a 5' region of homology to the vector backbone and a 3' region encoding the first RSR2295, and the second encodes the anti-EpCAM-anti-CD3 bispecific tandem scFv with 5' and 3' regions of homology encoding the tandem scFv's 5' and 3' release segments RSR2295. The third fragment encoded the AE868 XTEN with a C-tag affinity tag (amino acid sequence EPEA (SEQ ID NO: 1149)) with a 5' homology region encoding the second RSR2295 and a 3' homology region to the backbone vector. These three PCR fragments were cloned into BsiWI-NotI-digested pJB0084 using the In-Fusion Plasmid Assembly Kit. The final vector, pJB0169, encodes a bispecific antigen-binding polypeptide molecule under the control of the PhoA promoter and STII secretion leader, consisting of (N- to C-terminus) a 6xHIS affinity tag (SEQ ID NO: 1150), the AE292 XTEN, the release segment RSR2295, the anti-EGFR-anti-CD3 bispecific tandem scFv, RSR2295, the AE868 XTEN with a C-tag affinity tag, and its DNA sequence.

[0205] To construct pJB0163 and pJB0179, pJB0169 was digested with DraIII and BtsI to remove the 5' RSR2295, anti-EGFR-anti-CD3 bispecific tandem scFv, RSR2295, and the first 72 amino acids of AE868 XTEN. For pJB0163, DNA fragments encoding RSR3058, anti-CD3 light chain, anti-EGFR light and heavy chains, anti-CD3 heavy chain, RSR3058, and the first 72 amino acids of AE868 XTEN were synthesized. For pJB0179, DNA fragments encoding RSR2295, anti-CD3 light chain, anti-EGFR light and heavy chains, 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 containing restriction sites at the 5' and 3' ends to serve 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 sequences encoding the anti-CD3 and anti-EGFR binding domains. PCR products encoding the anti-HER2 light and heavy chains were amplified with a primer containing an 18-bp 5' homology region to the backbone DNA on the N-terminus and a 21-bp 3' homology region to the second PCR product. A second PCR product encoding an anti-CD3 scFv sequence variant (CD3.23) was amplified with a primer containing an 18-bp 5' homology region to the first PCR product on the N-terminus and a 23-bp 3' homology region to the vector backbone, using pJB0205 as a template. 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 sequences encoding the anti-CD3 and anti-EGFR binding domains. PCR products encoding the anti-HER2 light and heavy chains were amplified with a primer containing an 18-bp 5' homology region to the backbone DNA on the N-terminus and a 21-bp 3' homology region to the second PCR product. A second PCR product encoding an anti-CD3 scFv sequence variant (CD3.23) was amplified with a primer containing an 18-bp 5' homology region to the first PCR product on the N-terminus and a 23-bp 3' homology region to the vector backbone, using pJB0205 as a template. 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 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 a primer containing a 25-bp 5' homology region to the 3' end of the N-terminal vector backbone and a primer containing a 25-bp 3' homology region to the 5' end of the vector backbone, respectively. The pJB0244 PCR product 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, anti-HER2-anti-CD3 bispecific tandem scFv, RSR2295, C-tag affinity tag, under the control of the PhoA promoter and STII secretion leader, the DNA sequence and encoded amino acid sequence of which are provided in Table 9. The pJB0245 PCR product was cloned into the pAH0013 backbone to generate pJB0245, which encodes the AE868 XTEN with a 6xHIS affinity tag (SEQ ID NO: 1150), release segment RSR3058, anti-HER2-anti-CD3 bispecific tandem scFv, RSR3058, and C-tag affinity tag under the control of the PhoA promoter and STII secretion leader.

[0209] To introduce new CD3 scFvs with altered isoelectric points and removal of potential aggregation sites within the amino acid sequence, pJB0244 was digested with BsaI and BbvCI to remove both HER2 and CD3 scFvs. DNA fragments encoding anti-EGFR scFv variants paired with CD3.33 were synthesized containing 40 bp of homology to the digested vector at both the 5' and 3' ends to facilitate Gibson DNA assembly. Plasmids pJB0358 to pJB0372 were constructed, each containing the constructs for 15 anti-EGFR scFv variants paired with a 6xHIS affinity tag (SEQ ID NO: 1150), AE292 XTEN, RSR2295, and, individually, AE868 XTEN with anti-CD3 scFv, RSR2295, and C-tag affinity tags.

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

[0211] To generate a bispecific antigen-binding polypeptide construct with a 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 was synthesized encoding a second fragment with 40 bp of homology across the EcoRI site, where the 34-base tail overlaps with the first fragment. 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, an anti-HER2-anti-CD3 bispecific tandem scFv, RSR2295, the 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, along with the second fragment described above, was cloned into the digested pJB0244 backbone using the Gibson Assembly Kit to create plasmid pJB0355, which encodes a 6xHIS affinity tag (SEQ ID NO: 1150), XTEN292, RSR2295, an anti-Her2-anti-CD3 bispecific tandem scFv, RSR2295, the 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 proteins with truncated N- and C-terminal XTEN, three PCR products were amplified. The first PCR product consisted of an N-terminal His tag and the AE144_7A XTEN amplified from pCW1199. The second PCR product consisted of an N-terminal release site 2295, an anti-HER2-anti-CD3 bispecific tandem scFv, and a C-terminal release site 2295, as well as 286 amino acids of XTEN sequence. These two fragments were cloned by Gibson Assembly into the backbone generated by PCR amplification to form pJB0380, which contains the last 17 XTEN amino acids from the second PCR product and 30 bp of homology to the STII signal peptide at its 5' end, a 6xHis tag (SEQ ID NO: 1150), and five XTEN residues at its 3' end that contain 39 bp of homology 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 will be used to generate constructs with antigen-binding fragments CD3.24, CD3.30, CD3.31, CD3.33 scFv, and scFvs against the target cell markers described herein in any combination or orientation (i.e., AF1-AF2 or AF2-AF1 in N- to C-terminal orientation). [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

Table 9-5

Table 9-6

Table 9-7

Table 9-8

Table 9-9

Table 9-10

Table 9-11

Table 9-12

Table 9-13

Table 9-14

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Table 9-16

Table 9-17

Table 9-18

Table 9-19

Table 9-20

Table 9-21

Table 9-22

Table 9-23

Table 9-24

Table 9-25

Table 9-26

Table 9-27

Table 9-28

Table 9-29

Table 9-30

Table 9-31

Table 9-32

Table 9-33

Table 9-34

Table 9-35

Table 9-36

Table 9-37

Table 9-38

Table 9-39

Table 9-40

Table 9-41

Table 9-42

Table 9-43

Table 9-44

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

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Table 9-105

Table 9-106

Table 9-107

Table 9-108

Table 9-109

Table 9-110

Table 9-111

Table 9-112

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Table 9-115

Table 9-116

Table 9-117

Table 9-118

Table 9-119

Table 9-120

Table 9-121

Table 9-122

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Table 9-125

Table 9-126

Table 9-127

Table 9-128

Table 9-129

Table 9-130

Table 9-131

Table 9-132

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Table 9-134

Table 9-135

Table 9-136

Table 9-137

Table 9-138

Table 9-139

Table 9-140

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Table 9-144

Table 9-145

Table 9-146

Table 9-147

Table 9-148

Table 9-149

Table 9-150

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Table 9-157

Table 9-158

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Table 9-172

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Table 10-1

Table 10-2

Table 10-3

Table 10-4

Table 10-5

Table 10-6

Table 10-7

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Table 10-16

Table 10-17

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Table 10-48

[0213] Example 2: Evaluation of CD3 scFv sequence variants compared to the parent CD3 scFv The purpose of this experiment was to evaluate four CD3 sequence variants to determine whether they had enhanced properties compared to the CD3.9 parent scFv.

[0214] 1. Melting temperature (T m ) determination The melting temperature of each scFv variant was measured to determine its thermal stability. Briefly, 200 μL of 1% BSA-PBST was aliquoted into PCR tubes. The tubes were incubated at several different temperatures (50°C, 51.4°C, 53.7°C, 57.3°C, 61.7°C, 65.5°C, and 68°C) for 1 hour. 50 μL of each sample was added to the CD3 TIFF0007651481000311.tif79μ target antigen (Creative Biomart) or BSA (see reference for sticky material) was added to an ELISA plate coated with 1% BSA-PBST. The wells of the ELISA plate were pre-filled with 1% BSA-PBST (50μl / well). The plate was incubated for 1 hour at room temperature. The plate was washed three times with water and 0.05% TWEEN to remove unbound scFv. Bound scFv was detected by adding anti-YOL antibody (Thermo Scientific, no. MA180189) (1:500 dilution (0.05%) in 1% BSA-PBST), which detects the porcine alpha-tubulin motif in the linker between the heavy and light chains. The sample was incubated for 1 hour at room temperature. The plate was washed three times with water and 0.05% TWEEN to remove unbound scFv. Anti-YOL antibodies were detected by adding anti-rat-HRP antibody (Thermo Scientific, No. 31470) (1:7500 dilution (0.05%) in 1% BSA-PBST) [100 μl / well] and incubating at room temperature for 1 hour. The plate was washed three times with water and 0.05% TWEEN to remove unbound antibody. The plate was developed using TMB (3,3',5,5'-tetramethylbenzidine) substrate (100 μL / well, 6 minutes at room temperature). The reaction was stopped with H2SO4 (0.5 M, 100 μL / well). Relative activity was measured as absorbance readings at 450 nM. Absorbance at each temperature was graphed. The melting temperature was determined to be the EC50 for each sample, the temperature at which scFv binding decreased to 50% of the maximum signal. The results are presented in Table 11.

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

[0216] 2. Determination of binding affinity to CD3 The binding affinity of each scFv was measured using a ForteBio BLItz instrument. A dilution series of each scFv was prepared in PBS (300 μL / tube) starting from 1000 nM to 62.5 nM in 1:1 dilution steps for CD3.24-26 and from 400 nM to 25 nM in 1:1 dilution steps for CD3.23. Biotinylated CD3 TIFF0007651481000312.tif 77μ antigen (Creative Biomart) was diluted in PBS to a final concentration of 30μg / ml. Streptavidin biosensors (ForteBio) were activated in PBS for 10 minutes. To perform the measurements, the streptavidin biosensors were applied to the BLItz instrument. A tube containing 300μL of PBS was transferred to the BLItz instrument for 30 seconds. Biotinylated CD3 Tubes containing 79µg (30µg / ml, 300µL / tube) were transferred to the BLItz instrument to measure antigen capture to the sensor for 120 seconds. Tubes containing 300µL of PBS were transferred to the BLItz instrument for 30 seconds to measure baseline signal. Tubes containing test scFvs (30µg / ml, 300µL / tube) were transferred to the BLItz instrument to measure association of the scFv to the antigen-loaded biosensor for 120 seconds. Tubes containing 300µL of PBS were transferred to the BLItz instrument for 120 seconds to measure 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 temperatures and binding affinities of the CD3 binding variants are presented in Table 11 and show that variants such as CD3.23 have reduced binding affinity for CD3.

[0217] The binding affinity of the bivalent anti-HER2, anti-CD3 Xtenated binder AC2275 (see Example 24) was measured for targets (HER2 and CD3) using a ForteBio Octet Red instrument. The assay was performed in PBSTB buffer (10 mM dibasic sodium phosphate, 1.8 mM monobasic calcium phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride, 0.5% BSA, 0.005% Tween-20). For binding to human HER2 or cynomolgus monkey HERa2, a dilution series of each analyte was prepared in PBSTB buffer (500 μL / tube) starting from 64 nM to 1 nM in 1:1 dilution steps. For binding to human CD3 or cynomolgus monkey CD3, a dilution series of each analyte was prepared in PBSTB buffer (500 μL / tube) starting from 1010 nM to 16 nM in 1:1 dilution steps. The target was 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 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, target, and analyte were 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 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 biosensor was transferred to the analyte well for a dissociation step of 300-400 seconds. 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 show that all CD3 sequence variants had reduced binding affinity to CD3 compared to the parent CD3.9. [Table 11] [Table 12] [Table 13]

[0219] Conclusions: Two new anti-CD3 scFvs with improved thermal stability have been identified. Each of the new scFvs contains eight to nine mutations to CD3.9, primarily located in the CDRs. Although these mutations result in reduced affinity of the scFvs for their target (CD3) compared to the parent CD3.9, bispecific T cell engagers utilizing CD3.23 remain effective in cell killing assays and in vivo.

[0220] Example 3: Fermentation and purification of stable and unstable chimeric fusion polypeptides containing 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 discrepancy in aggregation propensity between the two constructs, and the discovery 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 N- to C-terminus, it 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 the four C-terminal residues glutamic acid, proline, glutamic acid, alanine (C-tag) (XTEN_AE868).

[0222] Construct ID pJB0231 is a similarly constructed molecule consisting of, from N- to C-terminus, an N-terminal polyhistidine tag (His6 (SEQ ID NO: 1150)), an unstructured 292 amino acid chain (XTEN_AE292), a protease-cleavable release segment (RS), an anti-EGFR scFv (aEGFR.2), an anti-CD3 scFv (aCD3.23), another protease-cleavable release segment (RS), an unstructured 864 amino acid chain, and the four C-terminal residues glutamic acid, proline, glutamic acid, alanine (C-tag) (XTEN_AE868).

[0223] Expression: Both molecules (pJB0169 and pJB0231) were expressed in a proprietary E. coli AmE098 strain and distributed to the periplasm via an N-terminal secretion leader sequence (MKKNIAFLLASMFVFSIATNAYA- (SEQ ID NO: 1155)), which was cleaved during translocation. Fermentation cultures were grown in animal-free complex medium at 37°C and phosphate depleted after a temperature shift to 26°C. After phosphate depletion, fermentation was continued for 12 hours. During harvest, the fermentation whole broth was centrifuged to pellet the cells. At harvest, the total volume and wet cell weight (WCW, the ratio of pellet to supernatant) were recorded, and the pelleted cells were collected and frozen at -80°C.

[0224] Purification: Frozen cell pellets of each molecule (pJB0169 and pJB0231) were resuspended three times in lysis buffer (60 mM acetic acid, 350 mM NaCl) at pH 4.5, and cells were lysed by homogenization. The homogenate was allowed to flocculate overnight at pH 4.5 and 2-8°C. The flocculated homogenate was centrifuged, and the supernatant was retained. The supernatant was diluted approximately three times with water and then adjusted to 7 ± 1 mS / cm with NaCl. The supernatant was then adjusted to 0....

Claims

1. A polypeptide comprising an antigen-binding fragment, the antigen-binding fragment specifically binds to cluster of differentiation 3 (CD3); The antigen-binding fragment comprises a heavy chain variable domain (VH) having at least 90% identity to the amino acid sequence of SEQ ID NO:28, and comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:8, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:10; The antigen-binding fragment comprises a light chain variable domain (VL) having at least 90% identity with the amino acid sequence of SEQ ID NO:27 and comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO:1, CDR-L2 comprising the amino acid sequence of SEQ ID NO:4, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:6; Polypeptides.

2. A polypeptide comprising an antigen-binding fragment, the antigen-binding fragment specifically binds to cluster of differentiation 3 (CD3); the antigen-binding fragment comprises a heavy chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3; said CDR-H1 comprising the amino acid sequence of SEQ ID NO:8; said CDR-H2 comprising the amino acid sequence of SEQ ID NO:9; the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 10; the antigen-binding fragment comprises a light chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3; said CDR-L1 comprising the amino acid sequence of SEQ ID NO:1; said CDR-L2 comprising the amino acid sequence of SEQ ID NO:4; A polypeptide wherein the CDR-L3 comprises the amino acid sequence of SEQ ID NO:

6.

3. The polypeptide of claim 1 or 2, wherein the VH comprises the amino acid sequence of SEQ ID NO:

28.

4. The polypeptide of any one of claims 1 to 3, wherein the VL comprises the amino acid sequence of SEQ ID NO:

27.

5. The polypeptide of claim 4, wherein the VH comprises the amino acid sequence of SEQ ID NO:28 and the VL comprises the amino acid sequence of SEQ ID NO:

27.

6. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 5 and one or more excipients.

7. 1. An isolated nucleic acid comprising: (a) a polynucleotide encoding the polypeptide according to any one of claims 1 to 5; or (b) an isolated nucleic acid comprising the complement of the polynucleotide of (a).

8. An isolated host cell comprising the nucleic acid of claim 7.

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