Cd3 antigen binding fragments and compositions comprising the same

Abstract

The present disclosure relates to compositions having antibody binding fragments that specifically bind to CD3 or an epitope thereof. Some embodiments include compositions and antibody binding fragments having increased stability. Also disclosed are bispecific fusion proteins comprising such antibody binding fragments.

Description

[0001] sequence list

[0002] This application contains a sequence list, which has been electronically submitted in ASCII format and is hereby incorporated in its entirety by reference. The ASCII copy was created on June 24, 2020, is named 32808-775_601_SL.txt, and is 3,106,733 bytes in size.

[0003] Cross-reference declaration

[0004] This application claims the benefits 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, both of which are incorporated herein by reference in their entirety. Background of the Invention

[0006] Many approved cancer treatments are cytotoxic drugs that kill both normal and tumor cells. The therapeutic benefit of these cytotoxic drugs depends on the fact that tumor cells are more sensitive than normal cells, thus allowing for the use of doses that do not cause unacceptable side effects to achieve a clinical response. However, essentially all of these nonspecific drugs cause some (if not serious) damage to normal tissue, which often limits the suitability of treatment.

[0007] By directing immune effector cells to kill cancer cells, bispecific antibodies can provide different strategies for cytotoxic drugs. Bispecific antibodies combine the benefits of the different binding specificities derived from two monoclonal antibodies into a single composition, enabling methods or combinations of coverage that are impossible with monospecific antibodies. In one embodiment, the method 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 its 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 numerous bispecific antibody forms and compositions for redirecting effector cells to tumor targets, including bispecific antibodies that function by recruiting and activating polyclonal populations of T cells at tumor sites, without requiring co-stimulation or conventional MHC recognition. However, some patients still suffer from a dual problem, experiencing a severe side effect known as a “cytokine storm” or “cytokine release syndrome” (Lee DW et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014 124(2):188-195), mediated by the release of cytokines such as TNF-α and IFN-γ. Furthermore, some bispecific compositions have very short half-lives, requiring continuous infusion for four to eight weeks to maintain circulating concentrations sufficient for therapeutic effect or variable efficacy within the therapeutic window. Therefore, there is an unmet need for developing effective bispecific antibodies for cancer treatment. Invention Overview

[0009] This invention relates to an anti-differentiation cluster 3 (CD3) antigen-binding fragment incorporated into a chimeric fusion protein and its method of use.

[0010] In one aspect, this document discloses polypeptides comprising antigen-binding fragments, wherein the antigen-binding fragments comprise a light chain complementarity-determining region (CDR-L) and a heavy chain complementarity-determining region (CDR-H), and wherein the antigen-binding fragments a. specifically bind to the differentiation cluster 3 T cell receptor (CD3); and b. comprise CDR-H1, CDR-H2, and CDR-H3 having amino acid sequences having SEQ ID NO: 8, 9, and 10, respectively.

[0011] In another aspect, this document discloses a polypeptide comprising an anti-CD3 antigen-binding fragment, wherein the antigen-binding fragment comprises a light chain complementarity-determining region (CDR-L) and a heavy chain complementarity-determining region (CDR-H), and 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 high thermal stability, as demonstrated in in vitro assays, (i) a higher melting termperature (T) relative to the antigen-binding fragment comprising the sequence shown in SEQ ID NO:41. m (ii) after incorporating the anti-CD3 antigen-binding fragment into the anti-CD3 bispecific antibody, the bispecific antibody exhibits a higher Tm relative to the control bispecific antibody, wherein the anti-CD3 bispecific antibody comprises the anti-CD3 binding fragment and a reference antigen-binding fragment that binds antigens other than CD3, and wherein the control bispecific antigen-binding fragment consists of SEQ ID NO:41 and the reference antigen-binding fragment.

[0012] In some implementations, the T-cell antigen-binding fragment m T compared to the antigen-binding fragment composed 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.

[0013] In another aspect, a polypeptide is disclosed herein comprising an antigen-binding fragment comprising 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 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with, or is identical to, the amino acid sequences of SEQ ID NO:22, 23, 25, and 26. In some embodiments, the antigen-binding fragments disclosed herein are chimeric or humanized antigen-binding fragments. In other embodiments, the antigen-binding fragments are selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibodies, and single-chain variable fragments (scFv).

[0014] In some embodiments, CDR-H1 and CDR-H2 comprise the amino acid sequences of SEQ ID NO:8 and 9, respectively. In some embodiments, CDR-L comprises: CDR-L1 having the amino acid sequence of SEQ ID NO:1 or 2, CDR-L2 having the amino acid sequence of SEQ ID NO:4 or 5, and CDR-L3 having the amino acid sequence of SEQ ID NO:6. In another embodiment, CDR-L comprises: CDR-L1 having the amino acid sequence of SEQ ID NO:1; CDR-L2 having the amino acid sequence of any one of SEQ ID NO:4 or 5; and CDR-L3 having the amino acid sequence of SEQ ID NO:6 or 7. In yet another embodiment, CDR-L comprises: CDR-L1 having the amino acid sequence of SEQ ID NO:2; CDR-L2 having the amino acid sequence of any one of SEQ ID NO:4 or 5; and CDR-L3 having the amino acid sequence of SEQ ID NO:6. In one embodiment, CDR-L comprises: CDR-L1 having the amino acid sequence of SEQ ID NO:1; CDR-L2 having the amino acid sequence of SEQ ID NO:4; and CDR-L3 having the amino acid sequence of SEQ ID NO:6. In some embodiments, CDR-L comprises: CDR-L1 having the amino acid sequence of SEQ ID NO:2; CDR-L2 having the amino acid sequence of SEQ ID NO:5; and CDR-L3 having the amino acid sequence of SEQ ID NO:6.

[0015] In some embodiments, the antigen-binding fragment further comprises FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with or identical to the amino acid sequences of SEQ ID NO: 12, 13, 18, and 19.

[0016] In other embodiments, the antigen-binding fragment further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and said 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 NO: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 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 some embodiments, the antigen-binding fragment comprises: a. FR-L1 having the amino acid sequence of SEQ ID NO:12; b. FR-L2 having the amino acid sequence of SEQ ID NO:13; c. FR-L3 having the amino acid sequence of SEQ ID NO:17; d. FR-L4 having the amino acid sequence of SEQ ID NO:19; e. FR-H1 having the amino acid sequence of SEQ ID NO:21; f. FR-H2 having the amino acid sequence of SEQ ID NO:23; g. FR-H3 having the amino acid sequence of SEQ ID NO:24; and h. FR-H4 having the amino acid sequence of SEQ ID NO:26.

[0017] In some embodiments, the antigen-binding fragment comprises a variable-heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:31. In some embodiments, the antigen-binding fragment comprises a variable-light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NO:27, 29, 30, 32, or 33. In other embodiments, the antigen-binding fragment comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NO:36-40, or the same amino acid sequence as any one of SEQ ID NO:36-40.

[0018] In some embodiments, the antigen-binding fragment specifically binds to human or cynomolgus monkey CD3. In other embodiments, the antigen-binding fragment specifically binds to human and cynomolgus monkey CD3. In some embodiments, the antigen-binding fragment binds to a CD3 complex subunit selected from the CD3ε, CD3δ, CD3γ, CD3δ, CD3α, and CD3βε units of CD3. In other embodiments, the antigen-binding fragment binds to the CD3ε fragment of CD3.

[0019] In some embodiments, the antigen-binding fragment has a dissociation constant (K0) between about 10 nM and about 400 nM. d The antigen-binding fragment specifically binds to human or cynomolgus monkey CD3, as determined in an in vitro antigen-binding assay containing human or cynomolgus monkey CD3 antigen. In other embodiments, the antigen-binding fragment has a dissociation constant (K0) 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. d Specifically binds to human or cynomolgus monkey CD3, as determined in an in vitro antigen binding assay. In another embodiment, the antigen-binding fragment exhibits at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times weaker binding affinity to CD3 relative to the antigen-binding fragment comprising the amino acid sequence of SEQ ID NO:41, as determined by their respective dissociation constants (K0) in an in vitro antigen binding assay. d As determined by ).

[0020] In some embodiments, the antigen-binding fragment exhibits an isoelectric point (pI) of less than or equal to 6.6. In other embodiments, the antigen-binding fragment exhibits a pI between 6.0 and 6.6, including the endpoints. In some 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 composed of the sequence shown in SEQ ID NO:41.

[0021] In other embodiments, the polypeptides disclosed herein further comprise a first releasing peptide (RS1), wherein said RS1 is a substrate for cleavage by a mammalian protease. In some embodiments, said RS1 is a substrate of a protease selected from the group consisting of asparagine endopeptidase (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 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO:42-660. In some embodiments, RS1 comprises an amino acid sequence selected from the following sequences: 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 shown in Table 5.

[0022] In some embodiments, the polypeptide disclosed herein further comprises a first extended recombinant polypeptide (XTEN1), wherein 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 of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (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, XTEN1 has at least about 36 to about 1000 amino acids or at least about 100 to 1000 amino acids. In some embodiments, XTEN1 comprises an amino acid sequence selected from at least three of SEQ ID NO: 661-664. In other embodiments, XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with sequences selected from SEQ ID NO:665-718 and 922-926. In another embodiment, XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the following: 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 as shown in Table 7.

[0023] In some embodiments, the peptides disclosed herein are expressed as fusion proteins, wherein the fusion protein in the uncleaved state has an N-terminal to C-terminal structural arrangement of AF1-RS1-XTEN1 or XTEN1-RS1-AF1, wherein AF1 is the first antigen-binding fragment.

[0024] In some respects, this document discloses a polypeptide comprising RS1, RS2, AF1, AF2, XTEN1, and XTEN2, wherein: a. RS1 and RS2 are each substrates for cleavage by mammalian proteases and each comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO:42-660; b. AF1 is an antigen-binding fragment of a monoclonal antibody with binding specificity to CD3; c. AF2 is an antigen-binding fragment comprising VL and VH of a monoclonal antibody with binding affinity to a target cell marker; d. XTEN1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO:42-660; The sequences NO:665-718 and 922-926 have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; e.XTEN2 contains amino acid sequences selected from SEQ ID NO:665-718 and 922-926. The sequences of NO:665-718 and 922-926 have amino acid sequences with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; f. the polypeptide has the following N-terminal to C-terminal 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, or XTEN2-RS2-dimer-RS1-XTEN1, wherein the dimer comprises VL and VH of AF1 and AF2; and g. relative to those in SEQ ID The antibody fragment consisting of the sequence shown in NO:41, the polypeptide exhibits high thermal stability, as determined by increasing the melting temperature (Tm) in in vitro assays.

[0025] 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 exhibits higher thermal stability relative to the antigen-binding fragment composed of the sequence shown in SEQ ID NO:41, such as by increasing the melting temperature (T) in in vitro assays. mAs determined by [reference needed]. In other embodiments, AF1 comprises a light chain complementarity-determining region (CDR-L) and a heavy chain complementarity-determining region (CDR-H), wherein AF1 comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO:10; and comprises FR-H1, FR-H2, FR-H3, and FR-H4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with or identical to the amino acid sequences of SEQ ID NO:20 or 21, 23, 24, and 26.

[0026] In some embodiments, CDR-H1 and CDR-H2 comprise the amino acid sequences of SEQ ID NO:8 and 9, respectively. In some embodiments, CDR-L comprises: CDR-L1 having the amino acid sequence of SEQ ID NO:1 or 2; CDR-L2 having the amino acid sequence of SEQ ID NO:4 or 5; and CDR-L3 having the amino acid sequence of SEQ ID NO:6. In another embodiment, CDR-L comprises: CDR-L1 having the amino acid sequence of SEQ ID NO:1; CDR-L2 having the amino acid sequence of any one of SEQ ID NO:4 or 5; and CDR-L3 having the amino acid sequence of SEQ ID NO:6. In other embodiments, CDR-L comprises: CDR-L1 having the amino acid sequence of SEQ ID NO:2; CDR-L2 having the amino acid sequence of any one of SEQ ID NO:4 or 5; and CDR-L3 having the amino acid sequence of SEQ ID NO:6. In other embodiments, CDR-L comprises: CDR-L1 having the amino acid sequence of SEQ ID NO:1; 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 other embodiments, CDR-L comprises: CDR-L1 having the amino acid sequence of SEQ ID NO:2; CDR-L2 having the amino acid sequence of SEQ ID NO:5; and CDR-L3 having the amino acid sequence of SEQ ID NO:6.

[0027] In some embodiments, AF1 comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and wherein said 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.

[0028] In some other embodiments, AF1 further comprises FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with or identical to the amino acid sequences of SEQ ID NO: 12, 13, 14-17, and 19.

[0029] In some embodiments, AF1 comprises a variable weight (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:31, or the same as the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:31. In some embodiments, AF1 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NO:27, 29, 30, 32, or 33, or the same as the amino acid sequence of any one of SEQ ID NO:27, 29, 30, 32, or 33. In some embodiments, AF1 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with any of the amino acid sequences in SEQ ID NO:36-40, or the same amino acid sequence as any of the amino acid sequences in SEQ ID NO:36-40.

[0030] In some embodiments, AF1 specifically binds to human or cynomolgus monkey CD3. In other embodiments, AF1 binds to CD3 complex subunits selected from CD3ε, CD3δ, CD3γ, CD3δ, CD3α, and CD3βε fragments. In yet another embodiment, AF1 binds to CD3ε. In yet another embodiment, AF1 uses a dissociation constant (Ki) between about 10 nM and about 400 nM. d AF1 specifically binds to human or cynomolgus monkey CD3, as determined in an in vitro antigen binding assay. In some embodiments, AF1 has a dissociation constant (Ki) 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. d AF1 specifically binds to human or cynomolgus monkey CD3, as determined in an in vitro antigen binding assay. In other embodiments, AF1, relative to the amino acid sequence of SEQ ID NO:41, specifically binds to human or cynomolgus monkey CD3 with a binding affinity at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times smaller, as determined by their respective dissociation constants (K0) in an in vitro antigen binding assay. d As determined by ).

[0031] In some implementations, AF1's T m T compared to the antigen-binding fragment composed of the sequence of SEQ ID NO:41 m The temperature is at least 2°C higher, or at least 3°C ​​higher, or at least 4°C higher, or at least 5°C higher, or at least 6°C higher, or at least 7°C higher, or at least 8°C higher, or at least 9°C higher, or at least 10°C higher, as determined by increasing the melting temperature in an in vitro determination.

[0032] In other embodiments, AF1 exhibits an isoelectric point (pI) of less than or equal to 6.6. In some embodiments, AF1 exhibits a pI between 6.0 and 6.6, including the endpoints. In other embodiments, AF1 exhibits a pI that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 lower than the pI of the reference antigen-binding fragment composed of the sequence shown in SEQ ID NO:41.

[0033] In another embodiment, the polypeptide disclosed herein further comprises a second antigen-binding fragment (AF2) that specifically binds to target cell markers other than CD3. In some embodiments, AF2 is fused to AF1 via a flexible peptide linker. In other embodiments, the flexible linker comprises two or three amino acids selected from the group consisting of glycine, serine, and proline. In some embodiments, (1) the AF2 fragment is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibodies, single-domain antibodies, and single-chain variable fragments (scFv), or (2) AF1 and AF2 are constructed as (Fab')2 or a single-chain dimeric compound.

[0034] In some embodiments, the CDR of AF2 is selected from the sequence of SEQ ID NO:719-918. In some embodiments, AF2 comprises VL and VH of a monoclonal antibody having binding affinity for a target cell marker. In other embodiments, VL is selected from the sequence of SEQ ID NO:819-918, and VH of AF2 is selected from the sequence of SEQ ID NO:719-818.

[0035] In some implementations, the target cell marker is a tumor antigen. In some implementations, the target cell markers are selected from 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A-kinase ankylosing 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipocyte differentiation-associated protein, adrenergic receptor β3 (ADRB3), AGS-22M6, alpha-folate receptor, alpha-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoietin-binding cell surface receptor 2 (Tie 2), anthrax toxin, AOC3 (VAP-1), B cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthrax, B cell activating factor (BAFF), B lymphoma cells, bone marrow stromal cell antigen 2 (BST2), and homologues of the regulator of imprinted sites. Sites (BORIS), C242 antigen, C5, CA-125, Cancer antigen 125 (CA-125 or MUC16), Cancer / Testis antigen 1 (NY-ESO-1), Cancer / Testis antigen 2 (LAGE-1a), Carbonic anhydrase 9 (CA-IX), Carcinoembryonic antigen (CEA), Cardiac myosin, CCCTC binding factor (CTCF), CCL11 (eosinophil chemokine-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, CD3ε, CD30, CD300 molecular-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44v7, CD44v8, CD44v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-associated antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), blocking protein 18.2 (CLDN18.2), blocking protein 6 (CLDN6), Clostridium difficile, aggregation 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, exo-ADP-ribosyltransferase 4 (ART4), mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2), EGF-like domain polyploid 7 (EGFL7), mutated elongation factor 2 (ELF2M), endotoxin, hepatocyte glycoprotein A2, hepatocyte glycoprotein B2, hepatocyte glycoprotein type A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial salivary proteins, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), *Escherichia coli*, ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML), respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (F CAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II β chain, fibroblast activation protein α (FAP), fibronectin outer 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-associated antigen 1, coiled receptor, fucose GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C5 member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), phosphatidylinositol proteoglycan- 3 (GPC3), GMCSF receptor α-chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, mutant heat shock protein 70-2 (muthsp70-2), hemagglutinin, hepatitis A virus cell receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, globoH glycoceramide hexose moiety (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 scattering 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 lamina-like peptide-1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin α4β7, integrin β2, integrin α2, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor Interferon-γ induced proteins, interleukin-11 receptor α (IL-11Rα), interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxylate esterase, kinase domain (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), asparagine endopeptidase, 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 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, breast 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 (ML-IAP), melanoma Related 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-acetylglucosamine-transferase V (NA17), NCA-90 (granulocyte antigen), connexin-4, nerve growth factor (NGF), protease 1 regulating neuronal apoptosis, neuronal adhesion molecule (NCAM), neurite growth inhibitors (e.g., NOGO-A, ...).NOGO-B, NOGO-C), neurocilia protein-1 (NRP1), N-hydroxyacetylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), oncogene (h5T4), oncogene fusion protein (bcr-abl) composed of the split cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl), rabbit, OX-40, oxLDL, p53 mutant, pairing box protein Pax-3 (PAX3), pairing box protein Pax-5 (PAX5), pan-connector protein 3 (PANX3), P-cadherin, sodium phosphate cotransporter White, phosphatidylserine, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor α (PDGF-Rα), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid, acrosome protein-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostate enzyme, prostate cancer tumor antigen-1 (PCTA-1 or galactolectin 8), melanoma antigen-1 recognized by T cells (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA) Prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate cancer cells, prostein, protease serine 21 (testisin or PRSS21), proteasome (Macropain) subunit β-type 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), receptor activator of nuclear factor κ-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, macaque Factors, sarcoma translocation breakpoint, sclerosin (SOST), select protein P, sialylated Lewis adhesion molecule (sLe), spermin 17 (SPA17), sphingosine-1-phosphate, squamous cell carcinoma antigens 1, 2, and 3 recognized by T cells (SART1, SART2, and SART3), stage-specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1, cohesinic glycan 1 (SDC1) + A314, SOX10, susceptin, susceptin-2B, synovial sarcoma, X-breakpoint 2 (SSX2), T cell receptor, TCRΓ alternating reading frame protein (TARP), telomerase, TEM1, tendinin C, TGF-β (e.g., TGF-β1,TGF-β2, TGF-β3), thyroid-stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen ((TnAg) 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 protein 2 (TROP-2), tumor-associated glycoprotein 72 (TAG72), tumor-associated glycoprotein 72 (TAG-72)+A327, TWE AK receptor, tyrosinase, tyrosinase-associated protein 1 (TYRP1 or glycoprotein 75), tyrosinase-associated protein 2 (TYRP2), uroplastic protein 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 myeloma virus oncogene neuroblastoma-derived homolog (MYCN), von Willebrand factor (VWF), nephroblastoma protein (WT1), X antigen family member 1A (XAGE1) ), β-amyloid protein, κ-light chain, fibroblast growth factor receptor 2 (FGFR2), estrogen-regulated LIV-1 protein (LIV1, also known as SLC39A6), neurotrophic receptor tyrosine kinase 1 (NTRK1, also known as TRK), Ret proto-oncogene (RET), B cell maturation antigen (BCMA, also known as TNFRSF17), transferrin receptor (TFRC, also known as CD71), activated leukocyte cell adhesion molecule (ALCAM, also known as CD166), somatostatin receptor 2 (SSTR2), KIT proto-oncogene receptor tyrosine kinase (cKIT), V-Set immunomodulatory receptor (VSIR, also known as The following proteins are listed: VISTA, glycoprotein Nmb (GPNMB), delta-like canonical Notch ligand 3 (DLL3), interleukin-3 receptor subunit α (IL3RA, also known as CD123), lysosome-associated membrane protein 1 (LAMP1), cadherin type 3 1, P-cadherin (CDH3), liver ligand A4 (EFNA4), protein tyrosine kinase 7 (PTK7), solute carrier family 34 member 2 (SLC34A2, also known as NaPi-2b), guanylate cyclase C (GCC), PLAUR domain-containing protein 3 (LYPD3, also known as LY6 or C4.4a), mucin 17, and cell surface-associated protein (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 α2 (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 α (IL3RA), CD5 molecule (CD5), UL16 binding protein 1 (ILBP1), T cell activation inhibitor 1 containing V-Set domain (VTCN1, also known as B7-H4), chondroitin sulfate proteoglycan 4 (CSPG4), cohesin glycan 1 (SDC1, also known as CD138), interleukin-1 receptor cofactor The protein contains IL1RAP, BIRC5 (also known as survivin), CD74 molecule, hepatitis A virus cell 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 αV (ITGAV), teratogenic growth factor 1 (TDGF1, also known as Crypto1), SLAM family member 6 (SLAMF6, also known as CD352), and Notch receptor 3 (NOTCH3).

[0036] In some embodiments, the CDR of AF2 is selected from the CDR sequence of SEQ ID NO:719-918. In some embodiments, AF2 comprises VL and VH of a monoclonal antibody having binding affinity for a target cell marker. In some embodiments, the VL sequence is selected from the sequence of SEQ ID NO:719-818 and the VH sequence is selected from the sequence of SEQ ID NO:819-918.

[0037] In some implementations, AF2 has a K0 value between about 0.1 nM and about 100 nM. dAF2 specifically binds to target cell markers, as determined in an in vitro antigen-binding assay containing the target cell marker. In some embodiments, AF2 has a binding affinity for the target cell marker that is at least 10-fold, at least 100-fold, or at least 1000-fold greater than that of AF1 for CD3, as measured in an in vitro antigen-binding assay. In some embodiments, AF2 comprises a CDR of a monoclonal antibody having binding affinity for the target cell marker.

[0038] In some embodiments, the polypeptide disclosed herein further comprises a second release segment (RS2), wherein said RS2 is a substrate for cleavage by a mammalian protease. In some embodiments, RS2 is a substrate for a protease selected from asparagine endopeptidase, 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 with a sequence selected from SEQ ID NO:42-660. In another embodiment, the sequences of RS1 and RS2 are identical. In yet another embodiment, the sequences of RS1 and RS2 are different. In some embodiments, RS1 and RS2 are each a substrate for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence.

[0039] In some embodiments, the polypeptide disclosed herein further comprises a second extended recombinant polypeptide (XTEN2), wherein XTEN2 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 of the XTEN2 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (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 wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence comprises non-overlapping sequences selected from at least three of SEQ ID NO: 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 with sequences selected from SEQ ID NO:665-718 and 922-926. In some embodiments, the XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the following sequences: 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 as shown in Table 7.

[0040] In other embodiments, the polypeptide has the following N-terminal to C-terminal 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-dimer-RS1-XTEN1, or XTEN1-RS1-dimer-RS2-XTEN2, wherein the dimer comprises VL and VH of AF1 and AF2, wherein AF1 specifically binds CD3 and AF2 specifically binds target cell markers, and wherein XTEN1 and XTEN2 have different amino acid lengths or sequences.

[0041] In some other embodiments, AF1 is fused to AF2 via a flexible peptide linker, wherein a. AF2 specifically binds to a second reference antigen other than CD3, such that the polypeptide is a bispecific antigen-binding fragment capable of binding to both CD3 and the second reference antigen; b. the bispecific antigen-binding fragment exhibits higher thermal stability compared to a control bispecific antigen-binding fragment, such as by measuring the melting temperature (T) in vitro. m The addition of ) is determined, wherein the control bispecific antigen-binding fragment comprises SEQ ID NO:41 and AF2.

[0042] In some embodiments, the second reference antigen is a target cell marker selected from the following: 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A-kinase ankylosing 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipocyte differentiation-associated protein, adrenergic receptor β3 (ADRB3), AGS-22M6, alpha-folate receptor, alpha-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, and 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), homologue of the regulator of blot sites (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a) Carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCCTC-binding factor (CTCF), CCL11 (eosinophil chemokine-1), CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD171, CD179a, CD18, CD19, CD2, CD 20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (α chain of IL-2 receptor), CD27, CD274, CD28, CD3, CD3ε, CD30, CD300 molecular-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44v7, CD44v8, CD44v6, CD5, CD51, CD5 2, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-associated antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), blocking protein 18.2 (CLDN18.2), blocking protein 6 (CLDN6), Clostridium difficile, aggregation 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, exo-ADP-ribosyltransferase 4 (ART4), mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2), EGF-like domain polyploid 7 (EGFL7), mutant elongation factor 2 (ELF2M), endotoxin, hepatocyte glycoside A2, hepatocyte glycoside B2, hepatocyte glycoside B2, etc. Protein A receptor 2, epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), epithelial salivary protein, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation-variant gene 6 located on chromosome 12p (ETV6-AML), respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FC) RL5), fetal acetylcholine receptor, fibrin II β chain, fibroblast activation protein α (FAP), fibronectin extradomain-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-associated antigen 1, coiled receptor, fucose GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C5 member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), phosphatidylinositol proteoglycan-3 (GPC3), G MCSF receptor α-chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, mutant heat shock protein 70-2 (muthsp70-2), hemagglutinin, hepatitis A virus cell receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, globoH glycoceramide hexose moiety (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 scattering 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, IGH, 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 λ-like polypeptide 1 (IGLL1), Influenza A hemagglutinin, Insulin-like growth factor 1 receptor (IGF-I receptor), Insulin-like growth factor 2 (ILGF2), Integrin α4β7, Integrin β2, Integrin α2, Integrin α4, Integrin α5β1, Integrin α7β7, Integrin αIIbβ3, Integrin αvβ3, Interferon α / β receptor Interferon-γ induced proteins, interleukin-11 receptor α (IL-11Rα), interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxylate esterase, kinase domain (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), asparagine endopeptidase, 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 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, breast 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 (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, cell Cell surface-associated mucin 1 (MUC1), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, mucin CanAg, myelin-associated glycoproteins, myostatin, N-acetylglucosamine transferase V (NA17), NCA-90 (granulocyte antigen), connexin-4, nerve growth factor (NGF), protease 1 regulating neuronal apoptosis, neuronal adhesion molecule (NCAM), neurite growth inhibitors (e.g., NOGO-A, NOGO-B, NOGO-C), neurociliacin-1 (NRP1), N-hydroxyacetylneuraminic acid,NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), oncogene (h5T4), oncogene fusion protein (bcr-abl) composed of the split cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl), rabbit, OX-40, oxLDL, p53 mutant, pairing box protein Pax-3 (PAX3), pairing box protein Pax-5 (PAX5), pan-connecting protein 3 (PANX3), P-cadherin, sodium phosphate cotransporter, phosphatidylserine, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor α (PGFα). PDGF-Rα), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid, acrosome protein-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostate enzymes, prostate cancer tumor antigen-1 (PCTA-1 or galactolectin 8), melanoma antigen-1 recognized by T cells (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate Cancer cells, prostein, serine 21 (testisin or PRSS21), proteasome (Macropain) subunit β-type 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), receptor activator of nuclear factor κ-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, macaque factor, sarcoma translocation breakpoint, sclerosin (SOST), select protein P, sialyl Lew The following are listed: adhesion molecule (SLE), spermin 17 (SPA17), sphingosine-1-phosphate, squamous cell carcinoma antigens 1, 2, and 3 recognized by T cells (SART1, SART2, and SART3), stage-specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1, cohesinic glycan 1 (SDC1)+A314, SOX10, susceptin, susceptin-2B, synovial sarcoma, X-breakpoint 2 (SSX2), T cell receptor, TCRΓ alternating reading frame protein (TARP), telomerase, TEM1, tendinin C, TGF-β (e.g., TGF-β1, TGF-β2, TGF-β3), and thyroid-stimulating hormone receptor (TSHR).Tissue factor pathway inhibitors (TFPIs), Tn antigen ((TnAg) 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 protein 2 (TROP-2), tumor-associated glycoprotein 72 (TAG72), tumor-associated glycoprotein 72 (TAG-72)+A327, TWEAK receptor, tyrosinase Tyrosinase-associated protein 1 (TYRP1 or glycoprotein 75), tyrosinase-associated protein 2 (TYRP2), uroplastic protein 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 myeloma virus oncogene neuroblastoma-derived homolog (MYCN), von Willebrand factor (VWF), nephroblastoma protein (WT1), X antigen family member 1A (XAGE1), β-amyloid protein, κ-light chain, fibroblasts Cell growth factor receptor 2 (FGFR2), estrogen-regulated LIV-1 protein (LIV1, also known as SLC39A6), neurotrophic receptor tyrosine kinase 1 (NTRK1, also known as TRK), Ret proto-oncogene (RET), B cell maturation antigen (BCMA, also known as TNFRSF17), transferrin receptor (TFRC, also known as CD71), activated leukocyte cell adhesion molecule (ALCAM, also known as CD166), somatostatin receptor 2 (SSTR2), KIT proto-oncogene receptor tyrosine kinase (cKIT), V-Set immunomodulatory receptor (VSIR, also known as VISTA), glycoprotein Nmb (GPNMB), delta-like typical N otch ligand 3 (DLL3), interleukin-3 receptor subunit α (IL3RA, also known as CD123), lysosome-associated membrane protein 1 (LAMP1), cadherin type 3 1, P-cadherin (CDH3), liver ligand A4 (EFNA4), protein tyrosine kinase 7 (PTK7), solute carrier family 34 member 2 (SLC34A2, also known as NaPi-2b), guanylate cyclase C (GCC), LYPD3 (LY6 or C4.4a) containing the PLAUR domain, mucin 17, cell surface-associated (MUC17), Fms-associated receptor tyrosine kinase 3 (FLT3), NKG2D ligand (e.g., ULBP1,ULBP2, ULBP3, H60, Rae-1α, Rae-1β, Rae-1δ, Rae-1γ, MICA, MICB, hHLA-A), SLAM family member 7 (SLAMF7), interleukin-13 receptor subunit α2 (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 α (IL3RA), CD5 molecule (CD5), UL16 binding protein 1 (ILBP1), T cell activation inhibitor 1 containing V-Set domain (VTCN1, also known as B7-H4), chondroitin sulfate proteoglycan 4 (CSPG4), cohesin glycan 1 (SDC1, also known as CD138), interleukin-1 receptor accessory protein (IL1RAP), containing baculoviruses The following receptors are involved in the hepatitis A virus (HAV) cell receptor 1 (BIRC5, also known as survivin), CD74 molecule (CD74), HAVCR1 (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 αV (ITGAV), teratogenic growth factor 1 (TDGF1, also known as Crypto1), SLAM family member 6 (SLAMF6, also known as CD352), and Notch receptor 3 (NOTCH3).

[0043] In some implementations, (1) the AF2 fragment disclosed herein is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, single-domain antibody and single-chain variable fragment (scFv), or (2) the AF1 and AF2 disclosed herein are constructed as (Fab′)2 or single-chain dimorphisms.

[0044] In some implementations, AF2 has a binding affinity for target cell markers that is at least 10 times, at least 100 times, or at least 1000 times greater than that of AF1 for CD3, as measured in an in vitro antigen binding assay.

[0045] In some other embodiments, AF1 and AF2 each exhibit an isoelectric point (pI) of less than or equal to 6.6. In another embodiment, AF1 and AF2 each exhibit a pI between 5.5 and 6.6, including the endpoints. In some 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.

[0046] In another aspect, a polypeptide is disclosed herein 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 ε subunit of CD3; and b. comprises the VH amino acid sequence of SEQ ID NO: 920. In some embodiments, the antigen-binding fragment comprises the VL amino acid sequence of SEQ ID NO: 919. In some embodiments, the antigen-binding fragment is composed of SEQ ID NO: 921.

[0047] In one aspect, pharmaceutical compositions are disclosed herein comprising the polypeptides disclosed herein and one or more pharmaceutically suitable excipients. In some embodiments, the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intrathecal, intrasheathal, or intramuscular administration. In another embodiment, the pharmaceutical composition is in liquid form. In yet another embodiment, the pharmaceutical composition is in a pre-filled syringe for a single injection. In still another embodiment, the pharmaceutical composition is formulated as a lyophilized powder to be reconstituted prior to administration.

[0048] In another aspect, this document discloses the use of the disclosed peptides 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: carcinomas, 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 sarcoma, metastatic peritoneal cancer, uterine cancer. Serous carcinoma, parathyroid carcinoma, endometrial carcinoma, cervical cancer, colorectal cancer, intraperitoneal malignant tumors with malignant ascites, uterine cancer, mesothelioma in peritoneal renal carcinoma, lung cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, gastric 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. Cancer), ovarian androma, adenocarcinoma, sarcoma of any origin, primary hematologic malignancies including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, B-cell-derived chronic lymphocytic leukemia, hairy cell leukemia, myeloproliferative disorders or myelodysplastic disorders, myasthenia gravis, Graves' disease (Morbus Basedow), Kaposi's sarcoma, neuroblastoma, Hashimoto's thyroiditis, nephroblastoma, or Goodpassuia syndrome.

[0049] In another aspect, methods for treating diseases in subjects are disclosed herein, comprising administering one or more therapeutically effective doses of the pharmaceutical composition disclosed herein to a subject in need. In some embodiments, the subjects are selected from a group consisting of mice, rats, monkeys, and humans. In some embodiments, the diseases are selected from the group consisting of: epithelial cancer, 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 cancer metastasis, serous uterine cancer, endometrial cancer, cervical cancer, colorectal cancer, intraperitoneal malignant tumors with malignant ascites, uterine cancer, peritoneal kidney. Mesothelioma, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, gastric cancer, small intestinal 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 malignancies including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, myeloproliferative disorders or myelodysplastic disorders, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpassuia syndrome.

[0050] In other embodiments, the drug composition is administered to the subject at one or more therapeutically effective doses, administered twice weekly, once weekly, once every two weeks, once every three weeks, once every four weeks, or once monthly. In some embodiments, the drug composition is administered to the subject at one or more therapeutically effective doses, administered over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. In some embodiments, the dose is administered intradermally, subcutaneously, intravenously, intra-arterially, intraperitoneally, intraperitoneally, intrathecally, or intramuscularly.

[0051] In one aspect, isolated nucleic acids are disclosed herein, said nucleic acids comprising (a) a polynucleotide encoding a polypeptide disclosed herein; or (b) a complement of the polynucleotide of (a).

[0052] In one relevant aspect, expression vectors comprising the polynucleotide sequences disclosed herein and recombinant regulatory sequences operatively linked to said polynucleotide sequences are disclosed herein.

[0053] In another aspect, isolated host cells containing the expression vectors disclosed herein are disclosed. In some embodiments, the host cell is a prokaryote. In one embodiment, the host cell is *Escherichia coli*.

[0054] By incorporating via reference

[0055] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is expressly and individually cited and incorporated herein by reference. Brief description of the attached diagram

[0057] Various features of this disclosure are specifically set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of exemplary embodiments (in which the principles of the invention are utilized) and the accompanying drawings, wherein the drawings are:

[0058] Figure 1 ( Figure 1 It describes the various components of a bispecific antigen-binding fragment composition. Figure 1 A describes an antigen-binding fragment with affinity for target cell markers. Figure 1 B describes an antigen-binding fragment with affinity for effector cells. Figure 1 C and 1D depict XTEN peptides of different lengths. Figure 1 E describes the cuttable release section.

[0059] Figure 2 ( Figure 2 This describes two different forms of the polypeptide composition described herein. Figure 2 On the left, A depicts the antigen-binding fragment that binds to effector cells and is fused with the release segment and XTEN, while the arrow depicts the effect of the protease cleavage of the release segment, resulting in the release of XTEN from the antigen-binding fragment of the peptide on the right. This allows the antigen-binding fragment to regain its full binding affinity potential as it is no longer shielded by XTEN. Figure 2 B depicts a bispecific composition on the left, having an antigen-binding fragment that binds to effector cells, fused to an antigen-binding fragment with binding affinity for target cell markers. A release segment and XTEN are also fused to the antigen-binding fragment with effector cell affinity, and the arrows depict the role of the protease cleavage release segment, resulting on the right, the release of XTEN and the fused antigen-binding fragment from the peptide. The fused antigen-binding fragment then regains its full binding affinity potential because it is no longer shielded by XTEN.

[0060] Figure 3 ( Figure 3This describes two different forms of bispecific antigen-binding peptides. On the left, the bispecific composition has an antigen-binding fragment that binds to effector cells, which is fused to an antigen-binding fragment with binding affinity for target cell markers, wherein a release segment (where scissors indicate sensitivity to protease cleavage) and XTEN are fused to the antigen-binding fragment with binding affinity for effector cells. On the right, the bispecific composition has an antigen-binding fragment that binds to effector cells, which is fused to an antigen-binding fragment with binding affinity for target cell markers, wherein a release segment and XTEN are fused to the antigen-binding fragment with binding affinity for target cell markers.

[0061] Figure 4 ( Figure 4 It describes three different forms of bispecific antigen-binding peptides. Figure 4 A describes a bispecific composition having an scFv antigen-binding fragment that binds to effector cells, the fragment being fused to an scFv antigen-binding fragment having binding affinity for target cell markers, wherein a release segment (in which scissors indicate sensitivity to protease cleavage) and XTEN are fused to each antigen-binding fragment. Figure 4 B and 4C are variations of 4A, in which the antigen-binding fragment is in a dimer configuration, wherein the release region (in which the scissors indicate sensitivity to protease cleavage) and XTEN are fused to the antigen-binding fragment, which binds to effector cell or target cell markers, respectively.

[0062] Figure 5 ( Figure 5 The diagram shows a schematic representation of a bispecific antigen-binding peptide near tumor tissue (at the top) and normal tissue (at the bottom). Compared to normal tissue, the bispecific antigen-binding peptide is preferentially cleaved at tumor tissue to release one or more XTEN moieties. The cleaved bispecific antigen-binding peptide is able to bind to T cells and tumor cells expressing tumor-specific markers.

[0063] Figure 6 ( Figure 6 The amino acid sequence of the control release segment RSR-1517 (SEQ ID NO:42) is depicted, showing the peptide cleavage site of the listed protease.

[0064] Figure 7 ( Figure 7The results show the in vitro cytotoxic activity of the N- and C-terminal XTEN-treated antiHER2-antiCD3 XPAT construct (“HER2-XPAT”) compared to the un-XTEN-treated identical construct (“HER2-PAT”) in PBMC / SK-OV-3 cells (A) or PBMC / BT-474 cells (B) in cytotoxicity assays. Cytotoxicity was assessed by caspase 3 / 7 assay or luminescent ATP assay, respectively.

[0065] Figure 8 ( Figure 8 The results indicate that the in vitro toxicity of the un-XTENized HER2-CD3 construct is correlated with HER2 expression. (A) shows the dose response of the un-XTENized HER2-CD3 construct (“HER2-PAT”) in cell lines with different HER2 expression in the presence of PBMCs. (B) shows the dose response of the un-XTENized HER2-CD3 construct (“HER2-PAT”) versus the XTENized HER2-CD3 construct (“HER2-XPAT”) in selected cell lines with different HER2 expression in the presence of PBMCs.

[0066] Figure 9 A( Figure 9 A) This illustration shows a model of how the proposed HER2-PAT molecule forms an immune synapse between T cells and HER2-positive cells.

[0067] Figure 9 B( Figure 9 Figure B shows that the HER2-PAT and HER2-XPAT constructs are able to induce conventional markers of T cell activation in “native” T cells (provided as PBMCs). Figure B shows the dose-response of the un-XTENized HER2-CD3 construct (“HER2-PAT”) versus the XTENized HER2-CD3 construct (“HER2-XPAT”) in the presence of HER2+ cells (SK-OV-3 cells) to upregulate CD69+ T cells (an early marker of T cell activation) in PBMCs, as assessed by flow cytometry.

[0068] Figure 10 A( Figure 10 A) indicates that, compared with unXTENized HER2-PAT, HER2-XPAT showed significantly reduced cytotoxicity in an in vitro PBMC / cardiomyocyte model.

[0069] Figure 11 ( Figure 11(A) shows that T cell activation of HER2-PAT and HER2-XPAT constructs depends on the involvement of HER2-positive cells. (B) shows the activation of T cells by HER2-PAT or HER2-XPAT in the in vitro Jurkat T cell / BT-474 model with or without BT-474 HER2+ cells, as measured by luciferase activity driven by NFAT response elements in Jurkat T cells.

[0070] Figure 12 ( Figure 12 The results show that, in the presence of HER2-positive cells, single (N- or C-)terminal XTENation results in a moderate reduction in the activation of XTENated HER2-CD3 molecules in Jurkat cells. (A) shows the dose-response of Jurkat cell activation in the presence of BT-474 cells for unXTENated HER2-CD3 constructs, single-terminal XTENated HER2-CD3 constructs, or N-terminal and C-terminal XTENated HER2-CD3 molecules. (B) shows the dose-response of Jurkat cell activation in the presence of SK-OV-3 cells for unXTENated HER2-CD3 constructs, single-terminal XTENated HER2-CD3 constructs, or N-terminal and C-terminal XTENated HER2-CD3 molecules.

[0071] Figure 13 ( Figure 13 The results showed that N-terminal and C-terminal XTENated anti-HER2-CD3 molecules (“XPAT”) and unXTENated HER2-CD3 molecules (“PAT”) were effective in reducing tumor burden in the BT-474 / human PBMC xenograft model, and the antitumor activity of XTENated HER2-CD3 molecules (“XPAT”) depended on the cleavage of the XTEN molecule. (A) shows the tumor volume after 25 days of treatment with mediator + / - PBMCs, XTENated HER2-CD3 molecules (“XPAT”), and unXTENated HER2-CD3 molecules (“PAT”). (B) shows the tumor volume after 25 days of treatment with mediator + PBMCs, XTENated HER2-CD3 molecules (“XPAT”), and XTENated HER2-CD3 molecules with an uncleavable XTEN.

[0072] Figure 14 ( Figure 14The results show that N-terminal and C-terminal XTENated HER2-CD3 molecules (“HER2-XPAT”) and un-XTENated HER2-CD3 molecules (“HER2-PAT”) effectively increase the population of activated CD4+ and CD8+ tumor-infiltrating lymphocytes in tumor tissue derived from a post-treatment BT-474 / human PBMC xenograft model. (A) shows the percentage of “activated” (e.g., CD25+) CD4+ cells in tumors after treatment with the vector, HER2-XPAT, or HER2-PAT, as assessed by flow cytometry. (B) shows the percentage of “activated” (e.g., CD25+) CD8+ cells in tumors after treatment with the vector, HER2-XPAT, or HER2-PAT, as assessed by flow cytometry.

[0073] Figure 14 C( Figure 14 C) indicates that the alternative dosing regimen of HER2-XPAT effectively reduced the tumor burden in humanized BT-474 xenograft mice.

[0074] Figure 15 ( Figure 15 The figure illustrates how XTENation of the HER2-CD3 molecule reduces the toxicity of the HER2-CD3 construct in cynomolgus monkeys. (A) shows the maximum tolerated dose (MTD) protocol in monkeys for XTENated (“HER2-XPAT”) or non-XTENated (“HER2-PAT”) constructs. (B) shows the plasma levels of the XTENated or non-XTENated molecule in monkeys after administration, indicating that the maximum tolerated dose of the XTENated construct is >1000x that of the non-XTENated molecule.

[0075] Figure 16 ( Figure 16 (A) shows that in cynomolgus monkeys, the HER2-XTEN construct induced T cell margination at doses greater than 2.5 mpk, but did not activate peripheral T cells at doses up to 50 mpk. (B) shows the effect on total lymphocytes, indicating that the XTEN-encoded construct induced a reduction in total blood lymphocytes at doses greater than 2.5 mpk. (C) shows the effect of HER2-XPAT2275 on activated CD4+ and CD8+ T cell populations, indicating that the effect on systemic T cell activation after administration was within the pre-administration range.

[0076] Figure 17 ( Figure 17This study demonstrates that XTENation of the HER2-CD3 molecule alleviates cytokine release syndrome when the agent is administered to cynomolgus monkeys. (A), (B), and (C) show the maximum concentrations of serum IL-6, TNFα, or IFNγ induced by dose series of HER2-PAT or HER2-XPAT in cynomolgus monkeys, indicating that HER2-XPAT does not induce detectable cytokine release at the test dose. Invention Details

[0078] Although preferred embodiments of the invention have been shown and described herein, those skilled in the art will understand that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions will be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The appended claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials to those described herein may be used to practice or test the invention, suitable methods and materials are described below. In case of conflict, the patent specification (including definitions) shall prevail. Furthermore, the materials, methods, and embodiments described are merely exemplary and not intended to be limiting. Numerous variations, modifications, and substitutions will now be made by those skilled in the art without departing from the invention.

[0080] definition

[0081] In the context of this application, unless otherwise stated, the following terms shall have the meanings assigned to them:

[0082] As used throughout the specification and claims, the terms “a,” “an,” and “the” are used to mean “at least one,” “at least the first,” “one or more,” or “more than one,” except where a higher limit is specifically stated thereafter. Thus, as used herein, a “release segment” means “at least the first release segment,” but includes more than one release segment. Those skilled in the art will recognize the operational limitations and parameters of the combinations, as well as the amount of any single agent, in light of this disclosure.

[0083] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of any length of amino acids. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also covers modified amino acid polymers, for example, modified by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation, such as conjugation with a labeled component.

[0084] The term “monomer” applied to polypeptides refers to the state of a polypeptide as a single continuous amino acid sequence that is substantially not associated with one or more additional polypeptides having the same or different sequences.

[0085] As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including, but not limited to, both D or L optical isomers, as well as amino acid analogs and peptide mimics. Standard single-letter or three-letter codes may be used to designate amino acids.

[0086] The term “natural L-amino acid” or “L-amino acid” refers to the following L optical isomers: 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).

[0087] The term "antibody" is used in the broadest sense herein and encompasses a wide 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, provided they exhibit the desired antigen-binding or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. Full-length antibodies can be, for example, monoclonal, recombinant, chimeric, deimmunized, humanized, and human antibodies. Antibodies represent a large class of molecules, including several types such as IgD, IgG, IgA, IgM, and IgE. The term "immunoglobulin molecule" includes, for example, hybrid antibodies or modified antibodies and their fragments. It has been demonstrated that the antigen-binding function of antibodies can be performed by naturally occurring antibodies or fragments of monoclonal antibodies.

[0088] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from nonhuman complementarity-determining regions (CDRs) and amino acid residues from human framework regions (FRs). In some embodiments, a humanized antibody will comprise at least one and typically substantially all of two variable domains, wherein all or substantially all of the CDRs correspond to those of nonhuman antibodies (which may include amino acid substitutions), and all or substantially all of the FRs correspond to those of human antibodies (which may include amino acid substitutions).

[0089] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles), which are typically present in trace amounts. Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the characteristic of an antibody derived from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to the invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are known in the art or described herein.

[0090] As used herein, “antigen-binding fragment” refers to immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that specifically binds to an antigen (“generates an immune response”). Examples include, but are not limited to: Fv, Fab, Fab′, Fab′-SH, F(ab′)2, dimers, linear antibodies (see U.S. Patent No. 5,641,870), single-domain antibodies, single-domain camelid antibodies, variable single-chain fragment (scFv) antibody molecules, and multispecific antibodies formed from antibody fragments that retain the ability to specifically bind antigens. The term “antigen-binding fragment” also encompasses any polypeptide chain-containing molecular structure having a specific shape suitable for recognizing and binding epitopes, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. An antigen-binding fragment binds an antigen with greater affinity or affinity than other reference antigens (including polypeptides or other substances) to which it binds, then the antigen-binding fragment “specifically binds” or “generates an immune response” with the antigen.

[0091] "scFv" or "variable single-chain fragment" is used interchangeably herein to refer to an antibody fragment containing a variable region of a heavy ("VH") and light ("VL") chain linked together by a short, flexible peptide linker, or two copies of the VH or VL chain, which enables the scFv to form the structure required for antigen binding. scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin and can be readily expressed in a functional form in *E. coli* or other host cells.

[0092] "Dimeric" refers to a small antibody fragment prepared by constructing a short linker (approximately 5-10 residues) between the VH and VL domains of an scFv fragment, thereby achieving interchain rather than intrachain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. A bispecific dimeric antibody is a heterodimer of two "crossed" scFv fragments, in which the VH and VL domains of the two antibodies reside on different polypeptide chains. A more complete description of dimeric antibodies can be found, for example, in US7635475.

[0093] The term "bispecific antigen-binding fragment" should be understood as an antigen-binding fragment that has binding specificity to at least two different antigens.

[0094] The terms “antigen,” “target antigen,” and “immunogen” are used interchangeably herein to refer to the structure or binding determinant to which an antibody, antibody fragment, or antibody fragment-based molecule binds or has specific binding characteristics. 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) and other homologs from humans, non-human primates, and mice.

[0095] The term "CD3 antigen-binding fragment" refers to an antigen-binding fragment that is capable of binding with sufficient affinity to a member of differentiation cluster 3 (CD3) or the CD3 complex, such that the antigen-binding fragment can be used as a diagnostic and / or therapeutic agent targeting CD3.

[0096] “Target cell markers” refer to molecules expressed by target cells, including, but not limited to, cell surface receptors, antigens, glycoproteins, oligonucleotides, enzyme substrates, antigenic determinants or binding sites that may be present inside or on the surface of target tissues or cells and can act as ligands for antibodies.

[0097] "Target tissue" or "target cell" refers to tissue or cell that is the cause or part of a disease condition (such as, but not limited to, cancer or inflammatory conditions). Sources of diseased target tissue or cells include body organs, tumors, cancer cells or clusters of cancer cells, or cells that form the matrix or are found to be associated with cancer cell clusters, bone, skin, cells that produce cytokines or factors that contribute to the disease condition.

[0098] The term "epitope" refers to a specific site on an antigen molecule that an antibody, antibody fragment, or binding domain binds to. An epitope is a ligand for an antibody or antibody fragment.

[0099] “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 noted, “binding affinity” as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the dissociation constant (K0). d () indicates. "Greater binding affinity" as used in this paper refers to a lower K... d Value; for example, 1x10 -9 M is more than 1x10 -8 M has a greater binding affinity. An antibody that binds to an antigen of interest (e.g., a tumor-associated target cell antigen) is an antibody that binds to the antigen with sufficient affinity so that it can be used as a diagnostic and / or therapeutic agent targeting cells or tissues expressing that antigen, and does not significantly cross-react with other proteins.

[0100] "Dissociation constant" or "K" d "Used interchangeably, it represents the affinity between the ligand "L" and the protein "P"; that is, the tightness with which the ligand binds to a specific protein. It can be expressed using the formula K. d = [L][P] / [LP], where [P], [L] and [LP] represent the molar concentrations of the protein, ligand and complex, respectively.

[0101] The terms “hypervariant region,” “HVR,” or “CDR,” as used herein, interchangeably refer to regions in the sequence that are hypervariable and / or form structurally defined loops and / or participate in antigen recognition of antibody variable domains. Typically, an antibody contains six hypervariable regions; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Many CDR descriptions are in use and are included herein; for example, CDR-L1 represents the first hypervariable CDR region of the light chain, CDR-H2 represents the second hypervariable CDR region of the heavy chain, and so on. The Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0102] "Frame" or "FR" residues are those variable domain residues in an antigen-binding fragment, other than the hypervariable region residues defined herein, and are typically located between or flanking CDRs. Many FR descriptions are in use and are included herein; for example, FR-L1 represents the first FR region of the light chain, FR-H2 represents the second FR region of the heavy chain, and so on.

[0103] The term "isoelectric point" or "pI" is used interchangeably throughout this document to indicate the pH at which a particular molecule carries no net charge or is electrically neutral in statistical mean. The standard nomenclature for isoelectric point is pH, with units in pH units; for example, an antigen-binding fragment with a pI of 6.3 would be neutrally charged in a solution at pH 6.3. Isoelectric points can be determined mathematically, including numerous algorithms for estimating the isoelectric points of peptides and proteins; for example, the Henderson-Hasselbalch equation with different pK values. Isoelectric points can also be determined experimentally in vitro, such as by capillary electrophoresis focusing.

[0104] The term "release fragment" or "RS" refers to a peptide in the subject composition having one or more sites in its sequence that can be recognized and cleaved by one or more proteases, thereby enabling the release of the antigen-binding fragment and XTEN from the composition. As used herein, "mammalian protease" refers to a protease commonly found in the body fluids, cells, and tissues of mammals and may be present at higher levels in certain target tissues or cells (e.g., in diseased tissues, such as tumors). The RS sequence can be engineered to be cleaved by various mammalian proteases or multiple mammalian proteases present in or near the subject's target tissue or introduced in an in vitro assay. Other equivalent proteases (endogenous or exogenous) capable of recognizing defined cleavage sites may be used. In particular, the RS sequence can be tailored and customized according to the protease used and can incorporate linker amino acids to connect with adjacent peptides.

[0105] The term "cleavage site" refers to the location in a peptide or polypeptide between adjacent amino acids that can be broken or cleaved by an enzyme (such as a protease); the breakage of the peptide bond between adjacent amino acids.

[0106] The term "within" when referring to a first polypeptide linked to a second polypeptide encompasses the linking or fusion of additional components that link the N-terminus of the first or second polypeptide to the C-terminus of the second or first polypeptide, as well as the insertion of the first polypeptide into the sequence of the second polypeptide. For example, when the RS component is linked "within" a chimeric polypeptide assembly, the RS can be linked to the N-terminus, the C-terminus, or can be inserted between any two amino acids of the XTEN polypeptide.

[0107] When used in the form of the compositions provided herein, “activity” means action or effect, including, but not limited to, antigen binding, antagonist activity, agonist activity, cellular or physiological response, cell lysis, cell death, or any effect known in the art for the effector component of the composition, whether measured in vitro, ex vivo, or in vivo or by clinical efficacy.

[0108] As used in this article, "effect cells" include any eukaryotic cell capable of conferring effects on target cells. For example, effector cells can induce loss of membrane integrity, condensation, nuclear fragmentation, apoptosis, lysis, and / or death of target cells. In another instance, effector cells can induce the division, growth, differentiation, or otherwise alter 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), mast cells, dendritic cells, regulatory T cells (RegT cells), helper T cells, myeloid cells, macrophages, and NK cells.

[0109] "Effective cell antigen" refers to molecules expressed by effector cells, including, but not limited to, cell surface molecules such as proteins, glycoproteins, or lipoproteins. Exemplary effector cell antigens include proteins of the CD3 complex or T cell receptors (TCRs), CD4, CD8, CD25, CD38, CD69, CD45RO, CD57, CD95, CD107, and CD154, as well as effector molecules such as cytokines associated with and bound to effector cells, cytokines expressed within effector cells, or cytokines expressed and released by effector cells. Effector cell antigens can act as binding counterparts to the binding domains of a subject-specific chimeric polypeptide assembly.

[0110] As used in this article, "CD3" or "differentiation cluster 3" refers to the T-cell surface antigen CD3 complex, which includes all known CD3 subunits, such as CD3ε, CD3δ, CD3γ, CD3δ, CD3α, and CD3β, either individually or in independent combinations. The extracellular domains of CD3ε, γ, and δ contain immunoglobulin-like domains and are therefore considered part of the immunoglobulin superfamily. CD3 includes, for example, human CD3ε protein (NCBI RefSeq No. NP_000724), which is 207 amino acids in length, and human CD3γ protein (NCBI RefSeq No. NP_000064), which is 182 amino acids in length.

[0111] As used herein, the term "ELISA" refers to an enzyme-linked immunosorbent assay as described herein or otherwise known in the art.

[0112] "Host cell" includes a single cell or cell culture that may be, or has been, a recipient of a subject vector (such as those described herein) incorporating exogenous nucleic acids. Host cells include the progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original maternal cell (morphologically or genomically in terms of total DNA complement). Host cells include cells transfected in vivo with the vectors of the present invention.

[0113] When used to describe the various polypeptides disclosed herein, "isolated" means a polypeptide that has been identified, isolated, and / or recovered from components of its native environment or from more complex mixtures, such as during protein purification. Contaminating components of its native environment are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other protein- or non-protein-based solutes. It will be apparent to those skilled in the art that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not require "isolation" to distinguish them from their naturally occurring counterparts. Furthermore, "concentrated," "isolated," or "diluted" polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof can be distinguished from their naturally occurring counterparts because the concentration or number of molecules per volume is generally greater than that of their naturally occurring counterparts. Generally, polypeptides prepared recombinantly and expressed in host cells are considered "isolated."

[0114] "Isolated nucleic acid" refers to a nucleic acid molecule identified and isolated from at least one contaminating nucleic acid molecule that is typically present in the natural source of a nucleic acid encoding a polypeptide. For example, the isolated nucleic acid molecule encoding a polypeptide differs from its naturally occurring form or environment. Therefore, the isolated nucleic acid molecule encoding a polypeptide is distinct from the nucleic acid molecule encoding a specific polypeptide present in natural cells. However, the isolated nucleic acid molecule encoding a polypeptide includes nucleic acid molecules encoding a polypeptide that are present in cells that typically express said polypeptide, wherein, for example, said nucleic acid molecule is located at a chromosomal or extrachromosomal location different from that of nucleic acid molecules in natural cells.

[0115] A "chimeric" protein or polypeptide contains at least one fusion polypeptide that includes at least one region in its sequence at a location different from its naturally occurring position. The regions may typically be present in individual proteins and aggregated together in the fusion polypeptide; or they may typically be present in the same protein but in a new arrangement within the fusion polypeptide. Chimeric proteins can be produced, for example, through chemical synthesis or by generating and translating polynucleotides in which the peptide regions encode in a desired relationship.

[0116] The terms “fusion” and “fusion body” are used interchangeably herein and refer to the linking of two or more peptide or polypeptide sequences together by recombination. A “fusion protein” or “chimeric protein” contains a first amino acid sequence linked to a second amino acid sequence that is not naturally linked to it in nature.

[0117] "XTENized" is used to refer to a peptide or polypeptide that has been modified by linking or fusing one or more XTEN polypeptides (described below) to the peptide or polypeptide, whether by recombination or chemical cross-linking.

[0118] "Operationally ligated" means that the ligated DNA sequences are within the reading phase or frame. "In-frame fusion" indicates that two or more open reading frames (ORFs) are ligated in a manner that maintains the correct reading frame of the original ORF to form a continuous, longer ORF. For example, if a promoter or enhancer affects the transcription of a polypeptide sequence, then the promoter or enhancer is operatively ligated to the coding sequence of the polypeptide. Therefore, the resulting recombinant fusion protein is a single protein containing two or more segments corresponding to the polypeptide encoded by the original ORF (segments that are not typically ligated in this way in nature).

[0119] In the context of polypeptides, a "linear sequence" or "sequence" is the amino acid sequence of a polypeptide in the direction from the amino terminus to the carboxyl terminus (N-terminus to C-terminus), where adjacent residues in the sequence are continuous in the primary structure of the polypeptide. A "partial sequence" is a straight-chain sequence of a polypeptide portion known to contain additional residues in one or two directions.

[0120] "Heterologous" refers to an entity that is genotyped differently from the entity it is comparing. For example, a glycine-rich sequence that is taken from its natural coding sequence and operatively linked to a non-natural coding sequence is a heterologous glycine-rich sequence. When applied to polynucleotides and polypeptides, the term "heterologous" refers to a polynucleotide or polypeptide that is genotyped differently from the entity it is comparing.

[0121] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “oligonucleotide” are used interchangeably. They refer to nucleotides of any length, including single nucleic acids and multiple nucleic acids, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene segments, multiple loci (single loci) defined by 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. Modifications to the nucleotide structure can be conferred before or after polymer assembly, if present. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.

[0122] The term "polynucleotide complement" refers to a polynucleotide molecule that has a complementary base sequence and reverse orientation compared to a reference sequence, allowing it to hybridize with the reference sequence with perfect fidelity.

[0123] When applied to polynucleotides, “recombination” means that the polynucleotide is the product of various combinations of recombination steps, which may include cloning, restriction and / or ligation steps, as well as other procedures that result in the expression of the recombinant protein in the host cell.

[0124] The terms "gene" and "gene segment" are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame (ORF) that, upon transcription and translation, encodes a specific protein. A gene or gene segment can be genomic or cDNA, provided that the polynucleotide contains at least one ORF and can cover an entire coding region or a segment thereof. A "fusion gene" is a gene consisting of at least two heterologous polynucleotides linked together.

[0125] As used herein, a “coding region” or “coding sequence” is a portion of a polynucleotide consisting of codons that can be translated into amino acids. While a “stop codon” (TAG, TGA, or TAA) is not typically translated into an amino acid, it can be considered part of a coding region, but any flanking sequences (e.g., promoters, ribosome binding sites, transcription terminators, introns, etc.) are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' end (encoding the amino terminus of the resulting polypeptide) and a translation stop codon at the 3' end (encoding the carboxyl terminus of the resulting polypeptide). Two or more coding regions of the present invention can exist in a single polynucleotide construct, for example, on a single vector, or in separate polynucleotide constructs, for example, on separate (different) vectors. Thus, a single vector may contain only a single coding region, or may contain two or more coding regions; for example, a single vector may encode binding domain-A and binding domain-B, as described below, respectively. Furthermore, the vectors, polynucleotides, or nucleic acids of the present invention can encode heterologous coding regions that are fused to or not fused with a nucleic acid encoding the binding domains of the present invention. Heterogeneous coding regions include, but are not limited to, specialized elements or motifs, such as secretory signal peptides or heterogeneous functional domains.

[0126] The term "downstream" refers to the nucleotide sequence located at the 3' end of a reference nucleotide sequence. In some embodiments, the downstream nucleotide sequence refers to the sequence following the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0127] The term "upstream" refers to the nucleotide sequence located at the 5' end of a reference nucleotide sequence. In some embodiments, the upstream nucleotide sequence refers to the sequence located 5' to the side of the coding region or transcription start site. For example, most promoters are located upstream of the transcription start site.

[0128] "Homology" or "homology" refers to the 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 can be used, or an appropriate scoring matrix, such as blosum45 or blosum80, can be selected to optimize the identity, similarity, or homology score. Preferably, homologous polynucleotides are those that 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 those sequences. When optimally aligned over sequences of a considerable length, homologous polypeptides preferably have at least 70%, preferably at least 80%, even more preferably at least 90%, and even more preferably at least 95%-99% identical sequence identity.

[0129] In the context of polynucleotides, "ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments or genes, thereby linking them together. For DNA fragments or genes to be ligated, the ends of the DNA must be compatible. In some cases, the ends will be directly compatible after digestion with an endonuclease. However, it may be necessary to first convert the interlaced ends, which typically result from endonuclease digestion, into blunt ends to make them compatible for ligation.

[0130] The term "stringent conditions" or "stringent hybridization conditions" refers to conditions that hybridize a polynucleotide to its target sequence to a greater extent than other sequences can detect (e.g., at least twice the background). Typically, the stringency of hybridization is expressed in part with reference to the temperature and salt concentration used in the washing step. Typically, stringent conditions are conditions where the salt concentration is less than about 1.5 M Na ions, typically about 0.01 M to 1.0 M Na 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., greater than 50 nucleotides)—for example, "stringent conditions" may include hybridization at 37 °C in 50% formamide, 1 M NaCl, and 1% SDS, and washing three times at 60 °C to 65 °C in 0.1 × SSC / 1% SDS, each wash lasting 15 minutes. Alternatively, temperatures of about 65 °C, 60 °C, 55 °C, or 42 °C may be used. SSC concentrations can vary from about 0.1 × SSC to 2 × SSC, with SDS present at about 0.1%. Such washing temperatures are typically chosen to be about 5°C to 20°C lower than the pyrolysis point of the specific sequence at a given ionic strength and pH. Tm is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at a given ionic strength and pH). Equations for calculating Tm and nucleic acid hybridization conditions are well-known and can be found in: Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3rd ed., Cold Spring Harbor Laboratory Press, 2001. Blocking reagents are typically used to block nonspecific hybridization. Such blocking reagents include, for example, approximately 100–200 μg / ml of cleaved and denatured salmon sperm DNA. Organic solvents, such as formamide at concentrations of about 35%–50% v / v, may also be used in specific cases, such as for RNA:DNA hybridization. Useful variations of these washing conditions will be apparent to those skilled in the art.

[0131] When applied to polynucleotide sequences, the terms "percentage of identity," "percentage of sequence identity," and "% identity" refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm can insert gaps in the compared sequences in a standardized and reproducible manner to optimize the alignment between the two sequences, thus enabling a more meaningful comparison. The percentage of identity can be measured over the entire length of the defined polynucleotide sequence, or over a shorter length, such as over a fragment taken from a larger, defined polynucleotide sequence, for example, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210, or at least 450 consecutive residues. Such lengths are merely exemplary, and it should be understood that any fragment length supported by the sequences shown herein, in tables, figures, or sequence listings can be used to describe lengths on which the percentage of identity can be measured. The percentage of sequence identity is calculated as follows: Two best-aligned sequences are compared within a comparison window. The number of matching positions (where the same residue appears in both polypeptide sequences) is determined. This number of matching positions is divided by the total number of positions within the comparison window (i.e., the window size), and the result is multiplied by 100 to produce the percentage of sequence identity. When comparing sequences of different lengths, the shortest sequence defines the length of the comparison window. Conservative substitutions are not considered when calculating sequence identity.

[0132] The terms “percentage of identity,” “sequence identity percentage,” and “% identity” used in relation to the polypeptide sequences identified herein are defined as, after aligning sequences and introducing gaps where necessary to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity, resulting in an optimal alignment, the percentage of amino acid residues in the query sequence that are identical to those in a second reference polypeptide sequence or a portion thereof of comparable length. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways within 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 suitable parameters for measuring the alignment, including any algorithm required to achieve optimal alignment over the full length of the compared sequence. The percentage of identity can be measured over the entire length of the defined polypeptide sequence, or over a shorter length, for example, over a fragment taken from a larger, defined polypeptide sequence, such as a fragment of at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues. Such lengths are merely exemplary and should be understood that any segment length supported by the sequences shown in this document, in tables, figures, or sequence lists may be used to describe lengths on which a percentage of identity can be measured.

[0133] In the context of polynucleotide sequences, "reproducibility" refers to the degree of homology within a sequence, such as, for example, the frequency of identical nucleotide sequences of a given length. For instance, reproducibility can be measured by analyzing the frequency of identical sequences.

[0134] As used herein, the term "expression" refers to the process of producing a gene product (e.g., RNA or polypeptide) from a polynucleotide. It includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of mRNA into a polypeptide. Expression produces a "gene product." The gene product used herein can be a nucleic acid, such as messenger RNA produced by gene transcription, or a polypeptide translated from a transcript. The gene products described herein further include nucleic acids with post-transcriptional modifications (e.g., polyadenylation or splicing), or polypeptides with post-translational modifications (e.g., methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage).

[0135] The terms "vector" and "expression vector" are used interchangeably and refer to nucleic acid molecules that preferably self-replicate in a suitable host, transferring inserted nucleic acid molecules into host cells and / or between host cells. The term includes vectors whose primary function is to insert DNA or RNA into cells, replication vectors whose primary function is to replicate DNA or RNA, and expression vectors whose function is to transcribe and / or translate DNA or RNA. It also includes vectors that provide more than one function. An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into one or more polypeptides. An "expression system" generally refers to a suitable host cell containing an expression vector that can function to produce a desired expression product.

[0136] When applied to peptides, "serum degradation resistance" refers to the peptide's ability to resist degradation in blood or its components, typically involving proteases in serum or plasma. Serum degradation resistance can be measured as follows: the protein is combined with human (or mouse, rat, dog, monkey, where appropriate) serum or plasma, typically for several days (e.g., 0.25, 0.5, 1, 2, 4, 8, 16 days), typically at about 37°C. Samples at these time points can be run on a Western blot assay, and the protein can be detected with an antibody. The antibody can target a tag in the protein. If the protein shows a single band on the Western blot, where the protein size is the same as the injected protein size, degradation has not occurred. In this exemplary method, the time point at which 50% of the protein is degraded, determined by Western blotting or equivalent techniques, is the serum degradation half-life or "serum half-life" of the protein.

[0137] The term "t" 1 / 2 The terms “half-life”, “terminal half-life”, “elimination half-life”, and “cyclic half-life” are used interchangeably in this paper, and as used herein, they refer to the calculation as ln(2) / K. el The terminal half-life of K. el The terminal clearance rate constant is calculated through linear regression of the terminal linear portion of the logarithmic concentration versus time curve. Half-life typically represents the time required for half the amount of administered substance deposited in a living organism to be metabolized or eliminated through normal biological processes. When the clearance curve of a given peptide is constructed as a function of time, it is typically biphasic, with a rapid α phase and a longer β phase. The typical β-phase half-life of human antibodies in humans is 21 days. Half-life can be measured using timed samples from any bodily fluid, but is most commonly measured in plasma samples.

[0138] The term "molecular weight" generally refers to the sum of the atomic masses of the component atoms in a molecule. Theoretically, the molecular weight can be determined by adding the atomic masses of the component atoms in the molecule. When applied in the context of peptides, the molecular weight is calculated as follows: based on the amino acid composition, the molecular weight of each type of amino acid in the composition is summed, or estimated by comparison with a molecular weight standard in an SDS electrophoresis gel. The calculated molecular weight of a molecule may differ from the molecule's "apparent molecular weight," which typically represents the molecular weight of a molecule 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 peptides, these terms represent a measure of the relative increase or decrease in the apparent molecular weight exhibited by a particular amino acid or peptide sequence. Apparent molecular weight can be determined, for example, using size exclusion chromatography (SEC) or similar methods, by comparison with globular protein standards, such as measurements in "apparent kD" units. The apparent molecular weight factor is the ratio of the apparent molecular weight to the "molecular weight"; the latter is calculated by summing the amino acid composition as described above, or estimated by comparison with a molecular weight standard in an SDS electrophoresis gel. The determination of apparent molecular weight and apparent molecular weight factor is described in U.S. Patent No. 8,673,860.

[0139] "Definitive composition medium" refers to a medium that contains the nutrients and hormones necessary for the survival and / or growth of cells in the culture, such that the components of the medium are known. Definitive composition media have traditionally been formulated by adding the nutrients and growth factors required for growth and / or survival. Typically, a specific composition medium provides at least one component from one or more of the following categories: a) all essential amino acids, and typically the basic set of twenty amino acids plus cysteine; b) an energy source, typically in the form of carbohydrates such as glucose; c) vitamins and / or other organic compounds required in low concentrations; d) free fatty acids; e) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range. Definitive composition media may also optionally be supplemented with one or more components from any of the following categories: a) one or more mitogenic agents; b) salts and buffers, such as calcium, magnesium, and phosphates; c) nucleosides and bases, such as adenosine and thymidine, hypoxanthine; d) proteins and tissue hydrolysates.

[0140] The term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of the natural polypeptides disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments, or amino acid sequence variants of natural polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of natural polypeptides may include contacting the natural polypeptide with a candidate agonist molecule and measuring detectable changes in one or more biological activities typically associated with the natural polypeptide.

[0141] The terms “treatment” or “treating,” or “relief” or “improvement” as used herein are used interchangeably. These terms refer to a protocol that achieves a beneficial or desired outcome, including, but not limited to, therapeutic and / or preventative benefits. A therapeutic benefit is the eradication or improvement of the underlying disorder being treated. Furthermore, a therapeutic benefit is achieved by eradicating or improving one or more physical symptoms or improving one or more clinical parameters associated with the underlying disorder, thereby observing improvement in the subject, even though the subject may still be suffering from the underlying disorder. For preventative benefits, the composition may be administered to subjects at risk of developing a specific disease, or to subjects who report one or more physical symptoms of a disease, even if a diagnosis of the disease may not yet have been made.

[0142] As used herein, “therapeutic effect” or “therapeutic benefit” refers to physiological effects, including, but not limited to, the reduction, improvement, or prevention of disease or improvement of one or more clinical parameters associated with an underlying disorder in a subject, or otherwise enhancing the subject’s physical or mental health due to the administration of the polypeptide of the present invention, rather than the ability to induce the production of antibodies against an antigenic epitope of a bioactive protein. For preventative benefits, the composition may be administered to subjects at risk of developing a specific disease, a prior disease, a condition of the disease, or a recurrence of symptoms, or to subjects who report one or more physiological symptoms of the disease, even if the disease may not yet have been diagnosed.

[0143] As used herein, the terms "therapeutic effective amount" and "therapeutic effective dose" refer to an amount of a drug or bioactive protein, alone or as part of a composition, that, when administered to a subject in one or repeated doses, is capable of having any detectable beneficial effect on any symptom, aspect, measurement parameter, or characteristic of a disease state or condition. Such an effect is not necessarily absolutely beneficial. The determination of a therapeutically effective amount is well within the competence of those skilled in the art, particularly based on the detailed disclosure provided herein.

[0144] As used herein, the term "therapeuticly effective and non-toxic dose" means a tolerable dose of the composition as defined herein that is sufficiently high to cause exhaustion, elimination, shrinkage, or stabilization of the tumor or cancer cells without significant or substantially no major toxic effects in subjects. Such a therapeutically effective and non-toxic dose can be determined by dose escalation studies as described in the art and should be below doses that induce serious adverse side effects.

[0145] The term "therapeutic index" as used in this article refers to the ratio of the blood concentration at which a drug becomes toxic to the concentration at which the drug is effective. An exemplary ratio of the therapeutic index is the LD50. 50 :ED 50 , of which LD 50 This is the dose that causes a 50% mortality rate in the subject population, and ED 50 It is the dose that produces effectiveness in the subject population.

[0146] As used herein, the term "dosing regimen" refers to a schedule of multiple doses (i.e., at least two or more) of a composition administered consecutively, wherein the dose is administered at a therapeutically effective amount to produce a sustained beneficial effect on any symptom, aspect, measurement parameter, endpoint, or characteristic of a disease state or condition in a subject.

[0147] As used herein, “administration” means the method of administering a dose of a compound (e.g., the anti-CD3 antibody of the present invention) or a composition (e.g., a pharmaceutical composition comprising the anti-CD3 antibody of the present invention) to a subject.

[0148] "Subject" is a mammal. Mammals include, but are not limited to, domesticated 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 some embodiments, the subject or individual is a human.

[0149] The terms "cancer" and "cancerous" indicate or describe a physiological condition in mammals typically characterized by disordered cell growth / proliferation. Examples of cancer include, but are not limited to: epithelial 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, metastatic peritoneal cancer, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, intraperitoneal malignant tumors with malignant ascites, uterine cancer, and interstitial renal cancer. Skin tumors, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, gastric cancer, small intestinal cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, salivary gland cancer, thyroid cancer, epithelial cancer, adenocarcinoma, sarcoma of any origin, primary hematologic malignancies including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, myeloproliferative disorders or myelodysplastic disorders, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, or Goodpassuia syndrome.

[0150] As used in this article, “tumor” refers to all proliferative cell growth and proliferation (whether malignant or benign), as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferation disorder,” “proliferative disorder,” and “tumor” used in this article are not mutually exclusive.

[0151] As used in this article, “tumor-specific markers” refers to antigens found on or in cancer cells that may, but not necessarily, be found in or on cancer cells in higher numbers than in normal cells or tissues.

[0152] I) General Technology

[0153] Unless otherwise stated, the practice of this invention employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the scope of the art. See Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3rd ed., Cold Spring Harbor Laboratory Press, 2001; “Current protocols in molecular biology,” edited by F.M. Usubel, et al., 1987; series “Methods in Enzymology,” Academic Press, San Diego, CA; “PCR 2: a practical approach,” edited by M.J. MacPherson, B.D. Hames, and G.G. Taylor, Oxford University Press, 1995; “Antibodies, a laboratory manual,” edited by Harlow, E., and Lane, D., Cold Spring Harbor Laboratory, 1988; “Goodman & Gilman’s The Pharmacological Basis of Therapeutics,” 11th ed., McGraw-Hill, 2005; and Freshney, R.I., “Culture of Animal Cells: A Manual of Basic Technique,” ​​4th ed., John Wiley & Sons, Somerset, NJ, 2000, the contents of which are incorporated herein by reference in their entirety.

[0154] Host cells can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are suitable for culturing eukaryotic cells. Furthermore, animal cells can be grown in serum-deficient media supplemented with hormones, growth factors, or any other factors required for the survival and / or growth of specific cell types. Definitive media supporting cell survival maintain cell viability, morphology, metabolic capacity, and potential differentiation capacity, while definitive media promoting cell growth provide all the chemical substances required for cell proliferation or reproduction. General parameters for controlling the in vitro survival and growth of mammalian cells are well established in the art. Physicochemical parameters that can be controlled in different cell culture systems include, for example, pH, pO2, temperature, and osmotic pressure. The nutritional requirements of cells are typically provided in standard culture medium formulations developed to provide optimal conditions. Nutrients can be classified into several categories: amino acids and their derivatives, carbohydrates, sugars, fatty acids, complex lipids, nucleic acid derivatives, and vitamins. In addition to nutrients necessary for maintaining cellular metabolism, most cells require one or more hormones from at least one of the following groups: steroids, prostaglandins, growth factors, pituitary hormones, and peptide hormones, to proliferate in serum-free media (Sato, GH, et al., "Growth of Cells in Hormonally Defined Media", Cold Spring Harbor Press, NY, 1982). Besides hormones, cells may also require transport proteins such as transferrin (plasma iron transporter), ceruloplasmin (a copper transporter), and high-density lipoprotein (a lipid carrier) to survive and grow in vitro. The optimal set of hormones or transport proteins varies for each cell type. Most of these hormones or transport proteins are added exogenously, or, in rare cases, mutant cell lines that do not require specific factors have been discovered. Those skilled in the art can understand the other factors required to maintain cell culture without extensive experimentation.

[0155] Growth media for prokaryotic host cell growth include nutrient broth (liquid nutrient medium) or LB medium (Luria Bertani). Suitable media include defined-component media and undefined-component media. Generally, media contain carbon sources such as glucose, water, and salts required for bacterial growth. Media may also include sources of amino acids and nitrogen, such as beef or yeast extract (in undefined-component media) or known amounts of amino acids (in defined-component 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 digest of casein), yeast extract, and sodium chloride. In some embodiments, selective media containing antibiotics are used. In such media, only desired cells resistant to the antibiotic will grow.

[0156] II) CD3 antigen-binding fragment composition

[0157] In a first aspect, this disclosure provides a polypeptide comprising an antigen-binding fragment (AF1) having a 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 a binding affinity for an effector cell antigen expressed on the surface of a T cell. In another embodiment, this disclosure provides a polypeptide comprising an antigen-binding fragment having a binding affinity for CD3. In yet another embodiment, the antigen-binding fragment has a binding affinity for a member of the CD3 complex, including all known CD3 subunits of the CD3 complex in individual or independently combined forms; for example, CD3ε, CD3δ, CD3γ, CD3δ, CD3α, and CD3β.

[0158] Antigen-binding fragments that bind to CD3 antigens are particularly well-suited for pairing with a second antigen-binding fragment (AF2) in combination. This second antigen-binding fragment has a binding affinity for target cell markers or antigens in diseased cells or tissues, thereby achieving cytotoxicity of diseased cells or tissues. Binding specificity can be determined by complementarity-determining regions or CDRs (such as light chain CDRs or heavy chain CDRs). In many cases, binding specificity is determined by both light chain CDRs and heavy chain CDRs. A given combination of heavy chain CDRs and light chain CDRs will provide a given binding groove with greater affinity and / or specificity for CD3 compared to other reference antigens.

[0159] The antigen-binding fragments covered by this 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 prepared using the hybridoma method described in Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (US Patent No. 4,816,567). The structure of antibodies and their fragments, the variable regions (VH and VL) of the heavy and light chains of antibodies, the single-chain variable region (scFv), the complementarity-determining region (CDR), and the domain antibodies (dAb) are well understood. Methods for generating peptides with desired antigen-binding fragments of target cell markers are known in the art.

[0160] Certain CD3-binding antigen-binding fragments of this disclosure have been specifically modified to enhance their stability in the peptide embodiments described herein, compared to those known in the art. Protein aggregation of monoclonal antibodies and other antibodies remains a significant issue in their developability and a major area of ​​concern in antibody production. Antibody aggregation can be triggered by partial unfolding of their domains, leading to monomer-monomer binding, followed by nucleation and aggregate growth. While the aggregation tendency of antibodies and antibody-based proteins can be influenced by external experimental conditions, they are strongly dependent on inherent antibody properties determined by their sequence and structure. Although it is known that proteins are only slightly stable in their folded state, it is generally not understood that most proteins are inherently prone to aggregation in their unfolded or partially unfolded state, and the resulting aggregates can be extremely stable and long-lived. Reduced aggregation tendency has also been shown to be accompanied by increased expression titers, suggesting that reducing protein aggregation is beneficial throughout the development process and could provide a more efficient pathway for clinical research. For therapeutic proteins, aggregates are a significant risk factor for generating harmful immune responses in patients and can form through a variety of mechanisms. Controlling aggregation can improve protein stability, manufacturability, loss rate, safety, formulation, titer, immunogenicity, and solubility. Intrinsic properties of proteins, such as size, hydrophobicity, electrostatic properties, and charge distribution, play a significant role in protein solubility. Low solubility of therapeutic proteins due to surface hydrophobicity has been shown to complicate formulation development and can lead to undesirable biodistribution, undesirable pharmacokinetic behavior, and immunogenicity in vivo. Reducing the overall surface hydrophobicity of candidate monoclonal antibodies can also provide benefits and cost savings associated with purification and dosing regimens. Individual amino acids can be identified through structural analysis as having aggregation potential in antibodies and can be located in the CDR as well as the frame region. Specifically, residues at high risk of causing hydrophobicity problems in a given antibody can be predicted. In one embodiment, this disclosure provides AF1 with the ability to specifically bind CD3, wherein AF1 has at least one amino acid substitution in the frame region relative to the parent antibody or antibody fragment, wherein the hydrophobic amino acid is selected from isoleucine, leucine, or methionine. In another embodiment, CD3 AF1 has at least two amino acid substitutions of hydrophobic amino acids in one or more framework regions, wherein the hydrophobic amino acids are selected from isoleucine, leucine, or methionine.

[0161] In the context of antigen-binding fragments, the isoelectric point (pI) is the pH at which an antibody fragment has no net charge. If the pH is below the pI of the antibody fragment, it will have a net positive charge. A larger positive charge tends to be associated with increased blood clearance and tissue retention, and typically has a shorter half-life. If the pH is above the pI of the antibody fragment, it will have a negative charge. A negative charge typically results in reduced tissue uptake and a longer half-life. This charge can be manipulated by mutations in framework residues. These considerations inform the design of the sequence of the antigen-binding fragment of the embodiments described herein, where individual amino acid substitutions are performed relative to the parent antibody used as the starting point. The isoelectric point of a peptide can be determined mathematically or experimentally in an in vitro assay. The isoelectric point (pI) is the pH at which a protein has zero net charge and can be calculated using the charge of a specific amino acid in the protein sequence. An estimate of the charge is called the acid dissociation constant, or pKa value, and is used to calculate the pI. pI can be determined in vitro by methods such as capillary isoelectric focusing (see Datta-Mannan, A., et al. The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies. mAbs 7:1084 (2015); Li, B., et al. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge. mAbs 6,1255-1264 (2014)) or other methods known in the art.

[0162] In some aspects of any of the embodiments disclosed herein, the subject polypeptide containing AF1 includes a light chain complementarity-determining region (CDR-L) and a heavy chain complementarity-determining region (CDR-H), wherein the AF1(a) specifically binds to the differentiation cluster 3 T cell receptor (CD3), which may include all known CD3 subunits, such as CD3ε, CD3δ, CD3γ, CD3δ, CD3α, and CD3β, either individually or in independent combinations. In one embodiment, the antigen-binding fragment of any subject composition embodiment described herein is a chimeric or humanized antigen-binding fragment. In another embodiment, the antigen-binding fragment of any subject composition embodiment described herein is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibodies, and single-chain variable fragments (scFv). The antigen-binding fragment having CDR-H and CDR-L can be constructed at the N-terminus to the C-terminus in a (CDR-H)-(CDR-L) or (CDR-H)-(CDR-L) orientation.

[0163] In one embodiment, this disclosure provides a polypeptide comprising AF1 containing CDR-L and CDR-H, wherein the AF1 (a) specifically binds to the differentiation cluster 3 T cell receptor (CD3); and (b) comprises CDR-H3 having the amino acid sequence of SEQ ID NO: 10. In some embodiments of this disclosure, AF1 comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NO: 8, 9, and 10, respectively. In another embodiment, the polypeptide of any of the subject composition embodiments described herein may comprise AF1, wherein the AF1 comprises CDR-L and CDR-H, wherein the 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) comprises heavy chain framework regions (FR-H) FR-H1, FR-H2, FR-H3, and FR-H4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with or identical to the amino acid sequences of SEQ ID NO:22, 23, 25, and 26.In another embodiment, this disclosure provides a polypeptide comprising AF1, wherein the AF1 comprises CDR-L and CDR-H, wherein the 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, and FR-H4, and further comprises light chain framework regions (FR-L) FR-L1, FR-L2, FR-L3, and FR-L4, wherein each of the heavy chain framework regions (FR-H) FR-H1, FR-H2, FR-H3, and FR-H4 exhibits an amino acid sequence consistent with SEQ ID NO:10. At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequences of SEQ ID NO: 22, 23, 25, and 26 are identical to or the same as the amino acid sequences of SEQ ID NO: 22, 23, 25, and 26, and the light chain framework regions (FR-L) FR-L1, FR-L2, FR-L3, and FR-L4 each exhibit at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequences of SEQ ID NO: 12, 13, 18, and 19 are identical to or the same as the amino acid sequences of SEQ ID NO: 12, 13, 18, and 19.

[0164] In another embodiment, the polypeptide of the subject composition embodiment described herein comprises AF1, wherein AF1 comprises CDR-L and 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 NO:8, 9, and 10, respectively. In another embodiment described above, the polypeptide comprises AF1, which further comprises (a) CDR-L1 having the amino acid sequence of SEQ ID NO:1 or 2, (b) CDR-L2 having the amino acid sequence of SEQ ID NO:4 or 5, and (c) CDR-L3 having the amino acid sequence of SEQ ID NO:6. In yet another embodiment, the polypeptide of any of the subject composition embodiments described herein may comprise AF1 containing CDR-L and CDR-H, wherein the AF1 (a) specifically binds CD3; (b) comprises CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of SEQ ID NO:8, 9, and 10, respectively, and further comprises (c) CDR-L1 having the amino acid sequence of SEQ ID NO:1; (d) CDR-L2 having the amino acid sequence of any one of SEQ ID NO:4 or 5; and (e) CDR-L3 having the amino acid sequence of SEQ ID NO:6 or 7. In yet another embodiment, this disclosure provides a polypeptide comprising AF1 containing CDR-L and CDR-H, wherein the AF1 (a) specifically binds to CD3; (b) comprises CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1, CDR-H2, and CDR-H3 each comprise the amino acid sequences of SEQ ID NO:8, 9, and 10, 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 NO:4 or 5; and (e) CDR-L3 having the amino acid sequence of SEQ ID NO:6. In another embodiment, the peptide of any of the subject composition embodiments described herein may comprise AF1 containing CDR-L and CDR-H, wherein the AF1 (a) specifically binds CD3; (b) comprises CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1, CDR-H2, and CDR-H3 comprise the amino acid sequences of SEQ ID NO:8, 9, and 10, respectively, and further comprises (c) CDR-L1 having the amino acid sequence of SEQ ID NO:1; and (d) CDR-L2 having the amino acid sequence of SEQ ID NO:4.(e) CDR-L3 having the amino acid sequence of SEQ ID NO:6. In another embodiment, this disclosure provides a polypeptide comprising AF1 containing CDR-L and CDR-H, wherein the AF1 (a) specifically binds CD3; (b) comprises CDR-H1, CDR-H2, and CDR-H3, wherein the CDR-H1, CDR-H2, and CDR-H3 each contain the amino acid sequences of SEQ ID NO:8, 9, and 10, 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 SEQ ID NO:5; and (e) CDR-L3 having the amino acid sequence of SEQ ID NO:6. In the foregoing embodiments of the paragraph, AF1 may further comprise a light chain framework region (FR-L) and a heavy chain framework region (FR-H) which are connected to their respective CDR regions. In certain embodiments described above, AF1 further comprises: FR-L1, which 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:12; FR-L2, which 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:12; FR-L3, which 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:13; and ... At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of any one of SEQ ID NO:14-17 is identical to the amino acid sequence of any one of SEQ ID NO:14-17; FR-L4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO:19, or is identical to the amino acid sequence of SEQ ID NO:19; FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO:20, SEQ ID NO:19, or is identical to the amino acid sequence of SEQ ID NO:19; The amino acid sequence of any one of SEQ ID NO:21 is identical to or is identical to the amino acid sequence of any one of SEQ ID NO:20 or SEQ ID NO:21 by at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence.FR-H2, 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; FR-H3, 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:24; and FR-H4, exhibiting... At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of NO:26 are identical or the same as the amino acid sequence of SEQ ID NO:26. In other instances of the foregoing embodiments of the paragraph, AF1 further comprises: FR-L1, which 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:12; FR-L2, which 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:13; FR-L3, which 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:13; and .... At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of SEQ ID NO:14 are identical or are the same as the amino acid sequence of SEQ ID NO:14; FR-L4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of SEQ ID NO:19 are identical or are the same as the amino acid sequence of SEQ ID NO:19; FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of SEQ ID NO:19 are identical or are the same as the amino acid sequence of SEQ ID NO:19; At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of NO:20 are identical or the same as the amino acid sequence of SEQ ID NO:20;FR-H2, 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; FR-H3, 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:24; and FR-H4, 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:24; and FR-H4, exhibiting... At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of NO:26 are identical or the same as the amino acid sequence of SEQ ID NO:26. In other instances of the foregoing embodiments described in the paragraph, AF1 further comprises: FR-L1, which 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:12; FR-L2, which 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:12; FR-L3, which 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:13; and ...2. At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of SEQ ID NO:15 are identical or are the same as the amino acid sequence of SEQ ID NO:15; FR-L4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of SEQ ID NO:19 are identical or are the same as the amino acid sequence of SEQ ID NO:19; FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of SEQ ID NO:19 are identical or are the same as the amino acid sequence of SEQ ID NO:15. At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of NO:21 are identical or the same as the amino acid sequence of SEQ ID NO:21;FR-H2, 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; FR-H3, 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:24; and FR-H4, exhibiting... At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of NO:26 are identical or the same as the amino acid sequence of SEQ ID NO:26. In other instances of the foregoing embodiments of the paragraph, AF1 comprises: FR-L1, which 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:12; FR-L2, which 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:12; FR-L3, which 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:13; and ...2%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of SEQ ID NO:16 are identical or are the same as the amino acid sequence of SEQ ID NO:16; FR-L4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of SEQ ID NO:19 are identical or are the same as the amino acid sequence of SEQ ID NO:19; FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of SEQ ID NO:19 are identical or are the same as the amino acid sequence of SEQ ID NO:19; At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of NO:21 are identical or the same as the amino acid sequence of SEQ ID NO:21;FR-H2, 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; FR-H3, 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:24; and FR-H4, exhibiting... At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of NO:26 are identical or the same as the amino acid sequence of SEQ ID NO:26. In other embodiments described above, the polypeptide comprising AF1 comprises: FR-L1, which 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:12; FR-L2, which 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:12; FR-L3, which 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:13; and ... The amino acid sequence of NO:17 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:17; FR-L4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:19; FR-H1 exhibits the same amino acid sequence as SEQ ID NO:17. At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of NO:21 are identical or the same as the amino acid sequence of SEQ ID NO:21;FR-H2, 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; FR-H3, 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:24; and FR-H4, 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:24; and FR-H4, exhibiting... At least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of the amino acid sequence of SEQ ID NO:26 are identical or identical to the amino acid sequence of SEQ ID NO:26.

[0165] In some aspects of any of the embodiments disclosed herein, the subject polypeptide may comprise an AF1 that binds to CD3, wherein the AF1 comprises a VL region and a VH region, the regions conferring the ability to specifically bind to CD3. The AF1 may be constructed in a VL-VH or VH-VL orientation and fused via a linker peptide.

[0166] In one embodiment, this disclosure provides a polypeptide comprising AF1 containing a VH amino acid sequence, the VH amino acid sequence having at least 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:28 or SEQ ID NO:31. In another embodiment, this disclosure provides a polypeptide comprising AF1 containing a VL amino acid sequence, the VL amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequence of ... In another instance, the polypeptide of any embodiment of the subject composition described herein comprises an AF1 that binds CD3, wherein the AF1 comprises a VL region and a VH region, the regions conferring specific binding ability to CD3 and each having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequences of SEQ ID NO:27 and 28, respectively. In other instances, this disclosure provides polypeptides comprising an AF1 that binds CD3, wherein the AF1 comprises a VL region and a VH region, the regions conferring specific binding ability to CD3 and each having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequences of SEQ ID NO:29 and 28, respectively. In another instance, this disclosure provides a polypeptide comprising CD3-binding AF1, wherein the AF1 comprises a VL region and a VH region, the regions conferring the ability to specifically bind CD3 and each having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences of SEQ ID NO:30 and 31, respectively.In another instance, the polypeptide of any embodiment of the subject composition described herein comprises an AF1 that binds CD3, wherein the AF1 comprises a VL region and a VH region, the regions conferring specific binding ability to CD3 and each having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequences of SEQ ID NO: 32 and 31, respectively. In other instances, this disclosure provides polypeptides comprising an AF1 that binds CD3, wherein the AF1 comprises a VL region and a VH region, the regions conferring specific binding ability to CD3 and each having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with or identical to the amino acid sequences of SEQ ID NO: 33 and 31, respectively.

[0167] In some aspects of any of the embodiments disclosed herein, the subject polypeptide comprises an AF1 that binds to CD3, wherein the AF1 is configured to have an scFv that specifically binds to CD3. In one embodiment, the AF1 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of any one of SEQ ID NO: 36-40, or the same amino acid sequence as the amino acid sequence of any one of SEQ ID NO: 36-40.

[0168] In some cases, the CD3 AF1 of the peptide embodiments described herein specifically binds to human or cynomolgus monkey CD3. In other cases, the CD3 AF1 of the peptide embodiments described herein specifically binds to human and cynomolgus monkey CD3. In one embodiment, the CD3 AF1 of the peptide embodiments described herein binds to a CD3 complex subunit selected from the CD3ε, CD3δ, CD3γ, CD3δ, CD3α, and CD3βε units of CD3. In one embodiment, the AF1 of the peptide embodiments described herein binds to the CD3ε fragment of CD3.

[0169] In another aspect, this disclosure provides peptides comprising AF1, which binds to CD3 protein complexes and exhibits enhanced stability compared to CD3-binding antibodies or AF1s known in the art. Furthermore, certain CD3 AF1s of this disclosure are designed to confer a higher degree of stability to chimeric bispecific antigen-binding compositions in which they are integrated, leading to improved expression and recovery of the fusion protein, increased shelf life, and enhanced stability when administered to a subject. In one method, certain CD3 AF1s of this disclosure are designed to have a higher degree of thermal stability compared to certain CD3-binding antibodies and antigen-binding fragments known in the art. Therefore, CD3 AF1s used as components in chimeric bispecific antigen-binding fragment compositions in which they are integrated exhibit advantageous 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 the variable domains of antibodies, and their inherent properties vary considerably. High thermal stability is often associated with high expression levels and other desired properties, including resistance 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). This is achieved by measuring the "melting temperature" (T0). m To determine thermal stability, Tm is defined as the temperature at which half of the molecules denature. The melting temperature of each heterodimer indicates its thermal stability. m The in vitro determination of the heterodimer is known in the art and includes the methods described in the following examples. The melting point of the heterodimer can be measured using techniques such as differential scanning calorimetry (Chen et al. (2003) Pharm Res 20:1952-60; Ghirlando et al. (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of the heterodimer can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9).

[0170] The thermal denaturation curves of the CD3-binding fragment of this disclosure and the anti-CD3 bispecific antibody comprising the anti-CD3-binding fragment, as well as the reference binding, demonstrate that the various constructs of this disclosure are more resistant to thermal denaturation than the antigen-binding fragment or control bispecific antibody comprising the sequence shown in SEQ ID NO:41, wherein the control bispecific antigen-binding fragment comprises SEQ ID NO:41 and a reference antigen-binding fragment binding antigens other than CD3. In one embodiment, the peptide of the embodiments described herein comprises anti-CD3 AF1, wherein the AF1 comprises CDR-L and CDR-H, and wherein the AF1: specifically binds CD3; comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO:10, and exhibits high thermal stability, as confirmed in in vitro assays, wherein (i) the peptide exhibits a higher melting temperature (T0) relative to the antigen-binding fragment comprising the sequence shown in SEQ ID NO:41. m (ii) after incorporating the anti-CD3 AF1 into the anti-CD3 bispecific antibody, the bispecific antibody exhibits a higher T value compared to the control bispecific antibody. m The anti-CD3 bispecific antibody comprises the anti-CD3 binding fragment and a reference antigen binding fragment that binds to antigens other than CD3, and the control bispecific antigen binding fragment consists of SEQ ID NO:41 and the reference antigen binding fragment. For example, in some cases, the control bispecific antibody is the same as the subject peptide, but AF1 is replaced by the antigen binding fragment of SEQ ID NO:41. The reference antigen binding fragment of the embodiments is intended to include an antigen binding fragment that binds to any target cell markers described herein (including, but not limited to, EGFR, HER2, EpCAM, and CD19, and other disclosed target cell markers). In one embodiment, this disclosure provides a peptide comprising anti-CD3 AF1, wherein the T of AF1 is... m T compared to the antigen-binding fragment composed of the sequence of SEQ ID NO:41 m The temperature is at least 2°C higher, or at least 3°C ​​higher, or at least 4°C higher, or at least 5°C higher, or at least 6°C higher, or at least 7°C higher, or at least 8°C higher, or at least 9°C higher, or at least 10°C higher. In another embodiment, this disclosure provides a polypeptide comprising anti-CD3 AF1, wherein the T of AF1 is... m T compared to the antigen-binding fragment composed of the sequence of SEQ ID NO:41 mThe temperature is at least 2°C-10°C higher, or at least 3°C-9°C higher, or at least 4°C-8°C higher, or at least 5°C-7°C higher. In another embodiment, this disclosure provides a bispecific antigen-binding polypeptide comprising anti-CD3 AF1, wherein the AF1 comprises CDR-L and CDR-H, and wherein the AF1 specifically binds CD3; comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H3 comprises the amino acid sequence of SEQ ID NO:10 and a second antigen-binding fragment binding antigens other than CD3, and exhibits high thermal stability, as demonstrated in in vitro assays, wherein the bispecific antigen-binding polypeptide exhibits a higher melting temperature (T0) relative to a control bispecific antibody. m The control bispecific antibody comprises the sequence shown in SEQ ID NO:41 and a reference antigen-binding fragment that binds antigens other than CD3.

[0171] In one relevant aspect, this disclosure provides various polypeptides comprising CD3-binding AF1, said polypeptides being incorporated into chimeric, bispecific antigen-binding fragment compositions designed to have an isoelectric point (pI) that imparts enhanced stability to the compositions of this disclosure compared to corresponding compositions comprising CD3-binding antibodies or antigen-binding fragments known in the art. In one embodiment, the polypeptide embodiments described herein may comprise a CD3-binding antigen-binding fragment wherein AF1 exhibits a pI between 5.8 and 6.6, including the endpoints. In another embodiment, this disclosure provides polypeptides comprising CD3-binding AF1 wherein AF1 exhibits a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units lower than the pI of a reference antigen-binding fragment comprising the sequence shown in SEQ ID NO:41. In another embodiment, the polypeptide of any of the subject composition embodiments described herein may comprise an AF1 that binds CD3, said AF1 being fused to a second antigen-binding fragment that binds an antigen other than CD3, wherein the pI exhibited by CD3 AF1 is within at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units of the pI of the antigen-binding fragment that does not bind CD3. In another embodiment, this disclosure provides a polypeptide comprising an AF1 that binds CD3, said AF1 being fused to a second antigen-binding fragment that binds an antigen other than CD3, wherein the pI exhibited by CD3 AF1 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 confirmed by calculation (see Examples) or in vitro assays. In one embodiment, the second antigen-binding fragment has a specific binding affinity for a non-CD3 antigen selected from the group consisting of EpCAM, EGFR, HER2, CD19, or any target cell marker embodiment disclosed herein (including, but not limited to, the target cell markers in Table 8). It is particularly intended that by designing the two antigen-binding fragments such that their pIs are within a certain range, the resulting fused antigen-binding fragments will impart a higher degree of stability to the chimeric bispecific antigen-binding fragment composition in which they are integrated, 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 peptide composition, and enhanced stability when the composition is administered to a subject.

[0172] In some aspects of any of the embodiments disclosed herein, the subject polypeptide comprises a dissociation constant (Ki) between about 10 nM and about 400 nM, or between about 50 nM and about 350 nM, or between about 100 nM and 300 nM. d AF1 specifically binds to human or cynomolgus monkey CD3, as determined in an in vitro antigen binding assay containing human or cynomolgus monkey CD3 antigen. In another embodiment, the peptide of any of the subject composition embodiments described herein may contain a dissociation constant (K0) of less 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 less than about 400 nM. d It specifically binds to AF1 on human or cynomolgus monkey CD3, as determined in an in vitro antigen binding assay. For clarity, K with 400 nM... d The antigen-binding fragment is more than 10 nM K d The antigen-binding fragment binds to its ligand more weakly.

[0173] In another embodiment, this disclosure provides a polypeptide comprising AF1, said AF1 exhibiting a binding affinity for CD3 that is at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times weaker than the binding affinity of the antigen-binding fragment comprising the amino acid sequence of SEQ ID NO:41, as measured by their respective dissociation constants (K0) in an in vitro antigen binding assay. d As determined by [the relevant authority]. In another embodiment, this disclosure provides a polypeptide comprising AF1, said AF1 exhibiting a binding affinity for CD3 that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or at least 1000 times weaker than the binding affinity of a second antigen-binding fragment, the second antigen-binding fragment being incorporated into a polypeptide that specifically binds antigens other than CD3, as determined by their respective dissociation constants (K0) in an in vitro antigen binding assay. dThe antigen, other than CD3, is selected from, but not limited to, HER2, EGFR, EpCAM, or CD19, or any target cell marker embodiments disclosed herein, including, but not limited to, the target cell markers listed in Table 8. The binding affinity of the subject composition to the target ligand can be determined using binding or competitive binding assays, such as the Biacore assay or ELISA assay (as described in U.S. Patent 5,534,617), the assays described in the embodiments herein, the radioreceptor assay, or other assays known in the art. The binding affinity constant can then be determined using standard methods, such as the Scatchard assay, as described in van Zoelen, et al., Trends Pharmacol Sciences (1998) 19) 12:487, or other methods known in the art. The same method can be used to prepare bispecific antigen-binding fragment constructs having antigen-binding fragments against CD3 and the target cell markers described herein in any combination or orientation (i.e., AF1-AF2 or AF2-AF1 orientation from the N-terminus to the C-terminus).

[0174] Table 1: CD3 CDR Sequences

[0175] Construct CDR area amino acid sequence SEQ ID NO: 3.23,3.30,3.31,3.32 CDR-L1 RSSNGAVTSSNYAN 1 3.24 CDR-L1 RSSNGEVTTSNYAN 2 3.9 CDR-L1 RSSTGAVTTSNYAN 3 3.23,3.30,3.31,3.32,3.9 CDR-L2 GTNKRAP 4 3.24 CDR-L2 GTIKRAP 5 3.23,3.24,3.30,3.31,3.32 CDR-L3 ALWYPNLWVF 6 3.9 CDR-L3 ALWYSNLWVF 7 3.23,3.24,3.30,3.31,3.32,3.9 CDR-H1 GFTFNTYAMN 8 3.23,3.24,3.30,3.31,3.32,3.9 CDR-H2 RIRSKYNNYATYYADSVKD 9 3.23.3.24,3.30,3.31,3.32 CDR-H3 HENFGNSYVSWFAH 10 3.9 CDR-H3 HGNFGNSYVSWFAY 11

[0176] Table 2: CD3 FR sequences

[0177]

[0178]

[0179] Table 3: VL & VH sequences

[0180]

[0181]

[0182] Table 4: scFv sequences

[0183]

[0184]

[0185]

[0186] III) Release Section

[0187] In another aspect, this disclosure relates to release segment (RS) peptides suitable for inclusion in the subject compositions described herein, which are substrates of one or more mammalian proteases associated with or produced by diseased tissues or cells found near or near diseased tissues. Such proteases may include, but are not limited to, protease classes such as metalloproteinases, cysteine ​​proteases, aspartic proteases, and serine proteases. RS is particularly used to confer a prodrug form upon which the subject compositions can be activated by cleavage of the RS by a mammalian protease. As described herein, incorporating RS into embodiments of the subject compositions involves linking the incorporated antigen-binding fragment to an XTEN (the configuration of which is described more fully below), such that upon cleavage of the RS by one or more RS-based proteases, the antigen-binding fragment and the XTEN are released from the composition, and the antigen-binding fragment, no longer shielded by the XTEN, regains its full potential to bind its respective ligand. In one particular feature, RSs act as substrates for discovering proteases closely associated with or co-located in diseased tissues or cells (such as, but not limited to, tumors, cancer cells, and inflammatory tissues), and upon cleavage of RSs, antigen-binding fragments that would otherwise be shielded by the XTEN of the subject composition (and thus have lower binding affinity for their respective ligands) are released from the composition and regain their full potential to bind to their targets and / or effector cell ligands. In another embodiment, the RSs of the subject polypeptide composition comprise an amino acid sequence that serves as a substrate for cellular proteases located within the target cell. In another particular feature of the subject compositions described herein, RSs serving as substrates for two or three classes of proteases are engineered to have sequences capable of being cleaved by different proteases at different positions in the RS sequence; a representative example is depicted in Figure 6 Therefore, RS, which serves as a substrate for two, three, or more classes of proteases, has two, three, or more distinct cleavage sites in its RS sequence, but cleavage by a single protease still results in the release of the antigen-binding fragment and XTEN from the composition containing RS.

[0188] In one embodiment, this disclosure provides an activatable polypeptide comprising one or more release segments, wherein the release segments are substrates cleaved by one or more mammalian proteases. In another embodiment, this disclosure provides a polypeptide comprising a first release segment (RS1) sequence, wherein RS1 is a substrate cleaved by a mammalian protease, wherein RS1 is a substrate of a protease selected from the group consisting of asparagine endopeptidase, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase.In other cases, the polypeptide of any embodiment of the subject composition described herein comprises a first release segment (RS1) sequence, wherein RS1 is a substrate cleaved by one or more mammalian proteases selected from the group consisting of: transmembrane peptidase, neprilysin (CD10), PSMA, BMP-1, A integrin and metalloproteinase (ADAM), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM with a thrombin-sensitive protein motif (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS 5, MMP-1 (collagenase 1), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3 (MMP-3, matrix lysin 1), matrix metalloproteinase-7 (MMP-7, matrix lysin 1), matrix metalloproteinase-8 (MMP-8, collagenase 2), matrix metalloproteinase-9 (MMP-9, gelatinase B), matrix metalloproteinase-10 (MMP-10, matrix lysin 2), matrix metalloproteinase-11 (MMP-11, matrix lysin 3), matrix metalloproteinase-12 (MMP-12, macrophage elastase), matrix metalloproteinase-13 (MMP-13, collagenase 3), matrix 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, matrix lysis factor 2), matrix metalloproteinase-27 (MMP-27, CMMP), asparagine endopeptidase, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X, cathepsin D, cathepsin E, secretase, urokinase (u Peptide (PA), tissue plasminogen activator (tPA), plasmin, thrombin, prostate-specific antigen (PSA, KLK3), human neutrophil elastase (HNE), elastase, trypsin, transmembrane serine protease type II (TTSP), DESC1, hepsin (HPN), matriptase, matriptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast activating protein (FAP), kallikrein-associated peptidases (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14.

[0189] In another embodiment, this 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 wherein RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from SEQ ID NO:42-660. In another embodiment, RS1 comprises an amino acid sequence selected from the following sequences: 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 shown in Table 5. As described more fully in the following description of the configuration and properties of the subject peptide composition, the release segment is fused between the antigen-binding fragment and the XTEN peptide, such that upon cleavage of the release segment, XTEN is released from the composition.

[0190] In other embodiments, this 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, this 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 of the foregoing embodiments, RS1 and RS2 are each substrates cleaved by mammalian proteases selected from the group consisting of asparagine endopeptidase, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In another embodiment, this disclosure provides a polypeptide comprising RS1 and RS2 sequences for incorporation into the subject polypeptide compositions described herein, wherein RS1 and RS2 are each substrates cleaved 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 with a sequence selected from SEQ ID NO:42-660. In another embodiment, RS1 and RS2 each comprise an amino acid sequence selected from the following sequences: 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 as shown in Table 5. As described more fully in the following paragraphs relating to the configuration and properties of the subject peptide composition, the release segment is fused between the antigen-binding fragment and the XTEN peptide such that upon cleavage of each release segment, the adjacent XTEN is released from the composition.

[0191] Table 5: Release segments and amino acid sequences

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] In another aspect, the release segments (RS1 and / or RS2) in peptides incorporated into any of the subject composition embodiments described herein can be designed to be selectively sensitive to different cleavage rates and efficiencies against various proteases for which they serve as substrates. Since different concentrations of a given protease can be found in diseased tissues (including, but not limited to, tumors, leukemia, or inflammatory tissues or sites of inflammation) compared to healthy tissue or in circulation, this disclosure provides RSs having a single amino acid sequence engineered to have a higher or lower cleavage efficiency against a given protease, to ensure that, upon approaching target cells or tissues and their co-localized proteases, the peptide preferentially converts from a prodrug form to its active form (i.e., separation and release of the peptide after cleavage of the release segment via an antigen-binding fragment and XTEN) compared to the cleavage rate of the release segment in healthy tissue or in circulation, such that the released antigen-binding fragment has a greater ability to bind ligands in diseased tissue compared to the prodrug form retained in circulation. Through such selective design, the therapeutic index of the resulting composition can be improved, resulting in reduced side effects relative to conventional therapeutic agents that do not contain such site-specific activation.

[0213] The cleavage efficiency used in this article is defined as the log2 value of the ratio of the percentage of the test substrate containing the cleaved release fragment to the percentage of the cleaved control substrate AC1611 when subjected to the protease in a biochemical assay of the reaction (further detailed in the examples), wherein the initial substrate concentration was 6 μM, the reaction was incubated at 37°C for 2 hours, and then terminated by the addition of EDTA. The percentage ratio of the cleaved release fragment was established using the amount of digested product and uncleaved substrate analyzed by non-reducing SDS-PAGE. The cleavage efficiency was calculated as follows:

[0214]

[0215] Therefore, a cleavage efficiency of -1 means that the amount of test substrate cleaved is 50% compared to the control substrate, while a cleavage efficiency of +1 means that the amount of test substrate cleaved is 200% compared to the control substrate. A higher cleavage rate of the test protease relative to the control will result in a higher cleavage efficiency, and a slower cleavage rate of the test protease relative to the control will result in a lower cleavage efficiency. As detailed in the examples, the control RS sequence AC1611 (RSR-1517) having the amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO:42) was identified as having an appropriate baseline cleavage efficiency for proteases asparagine endopeptidase, MMP-2, MMP-7, MMP-9, MMP-14, uPA, and matriptase when testing the cleavage rates of various proteases in in vitro biochemical assays. A library of RS peptides was created by selectively substituting amino acids at various positions and evaluated against seven proteases (described in more detail in the examples), resulting in a map for establishing appropriate amino acid substitutions to achieve RS peptides with desired cleavage efficiency. When preparing RS peptides with desired cleavage efficiency, substitutions of hydrophilic amino acids A, E, G, P, S, and T are preferred; however, other L-amino acids can be substituted at a given position to adjust the cleavage efficiency so that the released fragment retains at least some sensitivity to protease cleavage.

[0216] IV) XTEN peptides

[0217] In another aspect, this disclosure relates to polypeptides comprising at least a first extended recombinant polypeptide (XTEN), the first extended recombinant polypeptide (XTEN) being 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 an antigen-binding fragment to bind its ligand when the molecule is in its intact, uncleaved state, as described more fully below. In some embodiments, this disclosure provides a polypeptide comprising a single XTEN fused to the terminal of an RS located between the antigen-binding fragment and the XTEN. In other embodiments, this disclosure provides a polypeptide comprising first and second XTENs (XTEN1 and XTEN2) fused to the N-terminus and C-terminus of RS1 and RS2 located between each antigen-binding fragment and the XTEN, respectively.

[0218] Unconstrained by theory, the incorporation of XTENs can be integrated into the design of subject compositions to impart certain properties: ii) providing peptide compositions containing XTENs that, when the composition is in its intact prodrug form, shield antigen-binding fragments and reduce their binding affinity for target cell markers and effector cell antigens; ii) providing peptide compositions containing XTENs that provide an extended 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 composition; and iv) providing peptide compositions containing XTENs that reduce extravasation in normal tissues and organs, still allowing some extravasation in diseased tissues (e.g., tumors) with larger pore sizes in the vascular system, but still allowing release from the composition by the action of certain mammalian proteases, thereby allowing antigen-binding fragments of the composition to more easily penetrate diseased tissues such as tumors and bind and link together with target cell markers on effector cells and tumor cells. To meet these needs, this disclosure provides compositions comprising one or more XTENs, wherein the XTENs provide increased mass and hydrodynamic radius to the resulting composition. The XTEN peptides of the embodiments offer several advantages in the design of the subject composition, namely, not only providing increased mass and hydrodynamic radius to the composition, but also that their flexible, unstructured characteristics can provide a shielding effect on the antigen-binding fragments of the composition, thereby reducing binding to antigens in normal tissue or the vascular system of normal tissue that do not express or express low levels of target cell markers and / or effector cell antigens. Furthermore, the incorporation of XTEN into the subject composition can improve the solubility and proper folding of single-chain antibody-binding fragments during their expression and recovery.

[0219] XTENs are polypeptides having a non-naturally occurring, substantially non-repetitive sequence, exhibiting low or no secondary or tertiary structure under physiological conditions, and one or more additional properties described in the following paragraphs. In some embodiments, this 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, this disclosure provides a polypeptide comprising XTEN1, wherein XTEN1 is characterized in that it has at least about 36 or 100 amino acid residues, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), and it has at least 4-6 different amino acids selected from G, A, S, T, E, and P. In some embodiments, this disclosure provides a polypeptide comprising XTEN1, wherein XTEN1 has 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 288-868 amino acid residues. In other instances, this disclosure provides a polypeptide comprising XTEN1 having at least about 36 to about 1000, at least about 100 to about 1000, or at least 100 to about 900, or at least about 144 to about 868, or at least about 288-868 amino acid residues, wherein 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues are selected from 4-6 classes 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, this disclosure provides a polypeptide comprising XTEN1, wherein XTEN1 is characterized in that it has at least about 36 to about 1000 amino acid residues or at least about 100 to about 1000 amino acid residues, and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from six classes of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P).

[0220] In another embodiment, this disclosure provides a polypeptide of any embodiment described herein, the polypeptide comprising XTEN1, wherein XTEN1 is characterized in that it has at least about 36 to about 1000, at least about 100 to about 1000, at least about 100 to about 900, or at least 144 to about 868 amino acid residues, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from at least three of the sequences of SEQ ID NO: 661-664. In some cases, the XTEN1 sequence can be assembled by any combination of 12 amino acid units of SEQ ID NO: 661-664, such that any length of at least 36 amino acids or longer can be achieved in increments of 12 amino acids; for example, 36, 48, 60, 72, 84, 96 amino acids, etc. In other cases, the polypeptide of any embodiment of the subject composition described herein may comprise XTEN1, wherein XTEN1 is characterized in that it 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, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 sequence are selected from the sequences of SEQ ID NO: 665-718 and 922-926. In another embodiment, the XTEN of any of the subject composition embodiments described herein may have an affinity tag of HHHHHH (SEQ ID NO: 1150), HHHHHHHH (SEQ ID NO: 1151), or the sequence EPEA (SEQ ID NO: 1149), which is attached to the N- or C-terminus of the XTEN of the composition to facilitate purification of the composition to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% purity by chromatographic methods known in the art; for example, IMAC chromatography or C-tagXL chromatography, or the methods described in the following examples.

[0221] In another embodiment, this disclosure provides a polypeptide comprising XTEN1, wherein XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with AE36 (including amino acids selected from SEQ ID NO: 100%). The sequence can be any three of the sequences NO:661-664, or a sequence selected from the following sequences: AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which is shown in Table 7.

[0222] In some aspects of any of the embodiments disclosed herein, the subject polypeptide comprises XTEN1 and XTEN2. The conformations of polypeptides comprising XTEN1 and XTEN2, as well as other components, are described below. In one embodiment, this disclosure provides a polypeptide comprising XTEN1 and XTEN2, wherein each of XTEN1 and XTEN2 is characterized in that it has at least about 36 to about 1000 amino acid residues or at least about 100 to about 1000 amino acid residues, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 and XTEN2 sequences are selected from at least three sequences of SEQ ID NO: 661-664. In another embodiment, this disclosure provides a polypeptide comprising XTEN1 and XTEN2, wherein each of XTEN1 and XTEN2 is characterized in that it has at least about 36 to about 1000 amino acid residues or at least about 100 to about 1000 amino acid residues, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the XTEN1 and XTEN2 sequences are selected from the sequences of SEQ ID NO: 665-718 and 922-926. In another embodiment, the polypeptide of any of the subject composition embodiments described herein may comprise XTEN1 and XTEN2, wherein XTEN1 and XTEN2 each comprise an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the following sequences: AE144_1A, AE144_2A, AE144_2B, AE1 44_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each as shown in Table 7. In some embodiments of the foregoing paragraph, XTEN1 and XTEN2 are identical. In other embodiments of the foregoing paragraph, XTEN1 and XTEN2 of the foregoing embodiments of the foregoing paragraph have different amino acid sequences. In some cases, XTEN1 of any polypeptide composition embodiment having 2 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, in any peptide composition embodiment having two XTENs, XTEN2 is fused to the N-terminus of the peptide and is selected from the group consisting of AE144_7A, AE292, AE576, and AE864. In other cases, in any peptide composition embodiment having two XTENs, XTEN1 is fused to the C-terminus of the peptide 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.

[0223] Table 6: XTEN sequence motifs

[0224] motif name amino acid sequence SEQ ID NO: AE1 GSPAGSPTSTEE 661 AE2 GSEPATSGSETP 662 AE3 GTSESATPESGP 663 AE4 GTSTEPSEGSAP 664

[0225] Table 7: XTEN sequences

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245] This disclosure covers compositions of any embodiment described herein that comprise intermediate lengths of XTENs of the lengths in Table 7, as well as XTENs of longer lengths than those in Table 7, such as those in which a motif of 12 amino acids from Table 6 is added to the N- or C-terminus of the XTEN in Table 7.

[0246] In another embodiment, this disclosure covers peptide compositions of any embodiment described herein, comprising XTEN1 and XTEN2, which may further comprise a His tag of HHHHHH (SEQ ID NO: 1150) or HHHHHHHH (SEQ ID NO: 1151) at the N-terminus of the peptide composition and / or the sequence EPEA (SEQ ID NO: 1149) at the C-terminus 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, including, but not limited to, IMAC chromatography, C-tagXL affinity matrix, and other such methods, including, but not limited to those described in the following examples.

[0247] Other examples of XTEN sequences that can be used according to this disclosure are disclosed in U.S. Patent Publications 2010 / 0239554 A1, 2010 / 0323956 A1, 2011 / 0046060 A1, 2011 / 0046061 A1, 2011 / 0077199 A1 or 2011 / 0172146 A1 or International Patent Publications WO 2010091122 A1, WO 2010144502 A2, WO2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, WO 2011028344 A2, WO 2014 / 011819 A2 or WO 2015 / 023891.

[0248] V) Target cell marker antigen-binding fragments

[0249] In another aspect, this disclosure relates to antigen-binding fragments having specific binding affinity for target cell marker antigens other than CD3, which can be incorporated into any of the subject composition embodiments described herein. The resulting bispecific composition (a first antigen-binding fragment (AF1) having binding affinity for CD3 linked via a short, flexible peptide linker to a second antigen-binding fragment (AF2) having binding affinity for a second non-CD3 antigen) is bispecific, wherein each antigen-binding fragment has specific binding affinity for its respective ligand. It should be understood that in such compositions, the antigen-binding fragment targeting a target cell marker of diseased tissue is used in combination with the second antigen-binding fragment targeting an effector cell marker to bring effector cells close to cells of the diseased tissue, thereby achieving cell lysis of the diseased tissue cells. Furthermore, AF1 and AF2 can be incorporated into a specially designed polypeptide comprising a cleavable release segment and XTEN to impart a prodrug character to the composition, which becomes activated upon proximity to diseased tissue following cleavage of the release segment by the release of fused AF1 and AF2, the diseased tissue having a protease capable of cleaving the release segment at one or more sites in the release segment sequence.

[0250] In one embodiment, the peptide of any of the subject composition embodiments described herein may comprise AF2 having a specific binding affinity for a target cell marker expressed on the cell surface, in the cell membrane, or within target cells associated with cancer, autoimmune diseases, inflammatory diseases, and other conditions requiring locally activated peptides. In one embodiment, the antigen to which AF2 has a specific binding affinity is selected from antigens including, but not limited to, 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A-kinase ankylosing protein 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipocyte differentiation-associated protein, adrenergic receptor β3 (ADRB3), AGS-22M6, α-folate receptor, alpha-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, and 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), homologue of the regulator of blot sites (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCCTC binding factor (CTCF), CCL11 (eosinophil chemokine-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, CD3ε, CD30, CD300 molecular-like family members f (CD300LF), CD319(SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44v7, CD44v8, CD44v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-associated antigen, CFD, ch4D5Chromosome X open reading frame 61 (CXORF61), blocking protein 18.2 (CLDN18.2), blocking protein 6 (CLDN6), Clostridium difficile, aggregation 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, Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 2 ... Hepatitis B virus type 2, exo-ADP-ribosyltransferase 4 (ART4), mucin-like hormone receptor-like 2 (EMR2) containing an EGF-like module, EGF-like domain polyploid 7 (EGFL7), mutant elongation factor 2 (ELF2M), endotoxin, hepatocyte glycoside A2, hepatocyte glycoside B2, hepatocyte glycoside A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial salivary proteins, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protease), serine 2 (TMPRSS2) ET S fusion gene), E. coli, ETS translocation-variant gene 6 (ETV6-AML) located on chromosome 12p, F protein of respiratory syncytial virus, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II β chain, fibroblast activation protein α (FAP), fibronectin outer 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-associated antigen 1, coiled receptor, fucose GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor Group C5 members D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), phosphatidylinositol proteoglycan-3 (GPC3), GMCSF receptor α-chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, mutant heat shock protein 70-2 (muthsp70-2), hemagglutinin, hepatitis A virus cell receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, globoH (the hexose moiety of glycoceramide), 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 scattering 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 lamina-like peptide-1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin α4β7, integrin β2, integrin α2, integrin α4, integrin α5β1, integrin α7β7, integrin αIIbβ3, integrin αvβ3, interferon α / β receptor Interferon-γ induced proteins, interleukin-11 receptor α (IL-11Rα), interleukin-13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxylate esterase, kinase domain (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), asparagine endopeptidase, 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 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, breast 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 (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-acetylglucosamine-transferase V (NA17), NCA-90 (granulocyte antigen), connexin 4, nerve growth factor (NGF), protease 1 regulating neuronal apoptosis, neuronal cell adhesion molecule (NCAM), neurite growth inhibitors (e.g., NOGO-A, NOGO-B, NOGO-C), neurociliacin-1 (NRP1), N-hydroxyacetylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), oncogene (h5T4), and a tumor cell line composed of the split cluster region (BCR) and Abelson murine leukemia virus oncogene homolog 1 (Abl). Gene fusion protein (bcr-abl), rabbit, OX-40, oxLDL, p53 mutant, Pax-3 (PAX3), Pax-5 (PAX5), pan-connector 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, acrosome protease-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), programmed cell death-ligand 1 (PD-L1), proprotein convertase subtilisin / Kexin type 9 (PCSK9), prostate enzymes, prostate cancer tumor antigen-1 (PCTA-1 or galactolectin 8), melanoma antigen 1 recognized by T cells (MelanA or MART1), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate cancer cells, prostein, protease serine 21 (testisin or PRSS21), proteasome (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 receptor 1 (RU1), renal ubiquitous receptor 2 (RU2), respiratory syncytial virus, Rh blood group D antigen, macaque factor, sarcoma translocation breakpoint, sclerosingin (SOST), select protein P, sialylated Lewis adhesion molecule (sLe), spermin 17 (SPA17), sphingosine-1-phosphate, squamous cell carcinoma antigens 1, 2, and 3 (SART1, SART2, and SART3) recognized by T cells, stage-specific embryonic antigen-4 (SSEA-4), Staphylococcus aureus, STEAP1.Cohesinic glycan 1 (SDC1) + A314, SOX10, susceptin, susceptin-2B, synovial sarcoma, X-breakpoint 2 (SSX2), T cell receptor, TCRΓ alternating reading frame protein (TARP), telomerase, TEM1, tendinin 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 protein 2 (TROP-2), tumor-associated glycoprotein 72 (TAG72), tumor Related glycoprotein 72 (TAG-72)+A327, TWEAK receptor, tyrosinase, tyrosinase-associated protein 1 (TYRP1 or glycoprotein 75), tyrosinase-associated protein 2 (TYRP2), uroplastic protein 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 myeloma virus oncogene, neuroblastoma-derived homolog ( MYCN), von Willebrand factor (VWF), nephroblastoma protein (WT1), X antigen family member 1A (XAGE1), β-amyloid protein, κ-light chain, fibroblast growth factor receptor 2 (FGFR2), estrogen-regulated LIV-1 protein (LIV1, also known as SLC39A6), neurotrophic receptor tyrosine kinase 1 (NTRK1, also known as TRK), Ret proto-oncogene (RET), B cell maturation antigen (BCMA, also known as TNFRSF17), transferrin receptor (TFRC, also known as CD71), activated leukocyte cell adhesion. Molecules (ALCAM, also known as CD166), somatostatin receptor 2 (SSTR2), KIT proto-oncogene receptor tyrosine kinase (cKIT), V-Set immunomodulatory receptor (VSIR, also known as VISTA), glycoprotein Nmb (GPNMB), delta-like canonical Notch ligand 3 (DLL3), interleukin-3 receptor subunit α (IL3RA, also known as CD123), lysosome-associated membrane protein 1 (LAMP1), cadherin type 3 1, P-cadherin (CDH3), liver ligand A4 (EFNA4), protein tyrosine kinase 7 (PTK7).Solute carrier family 34 members 2 (SLC34A2, also known as NaPi-2b), guanylate cyclase C (GCC), LYPD3 (LY6 or C4.4a) containing a PLAUR domain, mucin 17, cell surface-associated (MUC17), Fms-associated 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 α2 (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 α (IL3RA), CD5 molecule (CD5), UL16 binding protein 1 (ILBP1), T cell activation inhibitor 1 containing V-Set domain (VTCN1, also known as B7-H4) Chondroitin sulfate proteoglycan 4 (CSPG4), cohesin glycan 1 (SDC1, also known as CD138), interleukin-1 receptor cofactor (IL1RAP), BIRC5 (also known as survivin) containing baculovirus IAP repeats, CD74 molecule (CD74), hepatitis A virus cell 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 αV (ITGAV), teratogenic growth factor 1 (TDGF1, also known as Crypto1), SLAM family member 6 (SLAMF6, also known as CD352), and Notch receptor 3 (NOTCH3).

[0251] Therapeutic monoclonal antibodies that can be derived from this for incorporation into any polypeptide embodiment of the subject composition described herein are known in the art. Such therapeutic antibodies may include, but are not limited to: rituximab, IDEC / Genentech / Roche (see, for example, U.S. Patent No. 5,736,137), a chimeric anti-CD20 antibody for the treatment of many lymphomas, leukemias, and some autoimmune disorders; ofamumumab, an anti-CD20 antibody approved for chronic lymphocytic leukemia and developed for follicular non-Hodgkin lymphoma, diffuse large B-cell lymphoma, rheumatoid arthritis, and relapsing-remitting multiple sclerosis; rukatumumab (HCD122), an anti-CD40 antibody for non-Hodgkin or Hodgkin lymphoma (see, for example, U.S. Patent No. 6,899,879); AME-133, an antibody that binds to CD20-expressing cells to treat non-Hodgkin lymphoma; and vetozumab (hA20), which binds to CD20-expressing cells to treat... Antibodies for the treatment of immune thrombocytopenic purpura; HumaLYM, developed for the treatment of low-grade B-cell lymphoma; and oligrin, an anti-CD20 monoclonal antibody for the treatment of rheumatoid arthritis (see, for example, U.S. Patent Application 20090155257); trastuzumab (see, for example, 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, for example, U.S. Patent No. 4,753,894); cetuximab, an anti-EGFR antibody for the treatment of KRAS wild-type metastatic colorectal and head and neck cancers expressing epidermal growth factor receptor (EGFR) (see, for example, U.S. Patent No. 4,943,533; PCT WO96 / 40210); panitumumab, a fully human monoclonal antibody specific to epidermal growth factor receptor (also known as EGF receptor, EGFR, ErbB-1, and HER1, currently marketed for the treatment of metastatic colorectal cancer (see US Patent No. 6,235,883)); zalumumab, a fully human IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR) for the treatment of squamous cell carcinoma of the head and neck (see, for example, US Patent No. 40210). 7,247,301); Nimotuzumab, a chimeric antibody against EGFR developed for the treatment of squamous cell carcinoma, nasopharyngeal carcinoma, and glioma of the head and neck (see, for example, U.S. Patent No. 5,891,996; U.S. Patent No. 6,506,883); Matozumab, a humanized monoclonal antibody against epidermal growth factor receptor (EGFR) developed for the treatment of colorectal cancer, lung cancer, esophageal cancer, and gastric cancer (see, for example, U.S. Patent Application 20090175858A1);Cetuximab, a chimeric (mouse / human) monoclonal antibody against epidermal growth factor receptor (EGFR) for the treatment of metastatic colorectal cancer, metastatic non-small cell lung cancer, and head and neck cancer (see, for example, U.S. Patent No. 6,217,866); alenzumab, a humanized monoclonal antibody against CD52 for the treatment of chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL), and T-cell lymphoma; and tiimozumab, developed for the treatment of certain forms of anti-CD20 monoclonal antibodies against B-cell non-Hodgkin lymphoma. Long antibody; Gestuzumab oxamicin, an anti-CD33 (p67 protein) antibody linked to the cytotoxic chelator tiuxetan, which is attached with a radioactive isotope for the treatment of acute myeloid leukemia; ABX-CBL, anti-CD147 antibody; ABX-IL8, anti-IL8 antibody; ABX-MA1, anti-MUC18 antibody; Pantomumab (R1549, 90Y-muHMFG1), an anti-MUC1 antibody under development; Therex (R1550), an anti-MUC1 antibody; AngioMa b(AS1405), developed by Antisoma; HuBC-1, developed by Antisoma; Thioplatin (AS1407), developed by Antisoma; ANTEGREN (natalizumab), anti-α-4-β-1 (VLA4) and α-4-β-7 antibody; VLA-1mAb, anti-VLA-1 integrin antibody; LTBRmAb, anti-lymphotoxin β receptor (LTBR) antibody; CAT-152, anti-TGF-β2 antibody; J695, anti- -IL-12 antibody; CAT-192, developed anti-TGFβ1 antibody; CAT-213, developed anti-eosinophil chemokine 1 antibody; LYMPHOSTAT-B, anti-Blys antibody; TRAIL-R1mAb, 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; Anti-CD4 antibody; tocilizuma, anti-IL6R antibody; HuMax-IL15, anti-IL15 antibody, HuMax-Inflam; HuMax-cancer, anti-heparinase I antibody; HuMax-lymphoma, HuMax-TAC; IDEC-131, anti-CD40; IDEC-151 (crixacillin), 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 (lapezizumab), anti-carcinoembryonic antigen (CEA) antibody developed by Immunomedics; Yervoy (ipilimumab), anti-CTLA4 antibody for the treatment of melanoma; (Epazizumab), anti-CD22 antibody; AFP-Cide, developed by Immunomedics; MyelomaCide, developed by Immunomedics; LkoCide, developed by Immunomedics; ProstaCide, developed by Immunomedics; MDX-010, anti-CTLA4 antibody; MDX-060, anti-CD30 antibody; MDX-070; MDX-018 developed by Medarex; OSIDEM (IDM-1), anti-HER2 antibody; -CD4, anti-CD4 antibody; HuMax-IL15, anti-IL15 antibody; anti-intercellular adhesion molecule-1 (ICAM-1)(CD54) antibody, MOR201; trimelimumab, anti-CTLA-4 antibody; anti-α5β1 integrin, developed by Protein Design Labs; anti-IL-12, developed by Protein Design Labs; ING-1, anti-Ep-CAM antibody developed by Xoma; and MLN01, anti-Beta2 integrin antibody; all antibody references cited above in this paragraph are expressly incorporated herein by reference. The sequences of the above antibodies are available from publicly available databases, patents, or references. Furthermore, non-limiting examples of monoclonal antibodies and VH and VL sequences (and, in some cases, CDR sequences indicating incorporability into AF2) suitable for use in the subject compositions of this disclosure are presented in Table 8.

[0252] According to the antigen-binding fragment implementation scheme mentioned above, it may be advantageous if the binding site recognizing the target cell marker antigen has a high binding affinity to efficiently capture the target cells to be destroyed. The subject peptides of any embodiment of this disclosure have the advantage that they can be used multiple times to kill tumor cells because, in a preferred embodiment, the AF2 target cell antigen-binding fragment has a binding affinity of 10... -7 Up to 10 -10 K within the range of M d The affinity of the bispecific antigen-binding fragment to the target cell marker is determined as in an in vitro binding assay. If the affinity of the bispecific antigen-binding fragment to the target cell marker is too high, the composition binds to the expressed target cell and remains on its surface, preventing it from being released and binding to another cell. In one embodiment, the polypeptide of any of the subject compositions described herein comprises AF2, wherein AF2 is in the form of K+ at a concentration between about 0.1 nM and about 100 nM, or about 0.5 to about 50 nM, or about 1.0 to about 10 nM. d AF2 specifically binds to target cell markers, as determined in an in vitro antigen-binding assay containing the target cell marker. In another embodiment, AF2 binds with binding affinity 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 (as determined by K in an in vitro binding assay). d The identified specific binding affinity to target cell markers. In another embodiment, this disclosure provides a polypeptide comprising AF2, wherein the binding affinity of AF2 to the target cell marker is at least 10-fold, at least 100-fold, or at least 1000-fold greater than the binding affinity of AF1 to CD3, as measured in an in vitro antigen binding assay. In another embodiment, the AF1 antigen-binding fragment of any subject embodiment of this disclosure has a lower binding affinity to the CD3 antigen by at least one order of magnitude, at least two orders of magnitude, or at least three orders of magnitude lower than the greater binding affinity of AF2 to the target cell marker antigen, as measured in an in vitro assay as K. d This is determined by a constant. It should be understood that a higher binding affinity refers to a lower K. d Value; for example, 1×10 -9 M is more than 1×10 -8 M has a greater binding affinity.

[0253] In another embodiment, this disclosure provides a polypeptide comprising AF2, wherein the AF2 comprises a CDR of a monoclonal antibody having binding affinity for a target cell marker antigen. In another embodiment, a polypeptide comprising any embodiment of the subject matter compositions described herein comprises AF2, wherein the AF2 comprises a CDR derived from a monoclonal antibody having binding affinity for a target cell marker antigen, wherein the CDR of AF2 is selected from the CDRs within the VL and VH sequences of SEQ ID NO:719-918.

[0254] In some aspects of any of the embodiments disclosed herein, the subject polypeptide comprises AF2, wherein the AF2 comprises VL and VH of monoclonal antibodies having binding affinity for target cell marker antigens. In some cases, the peptide of any embodiment of the subject composition described herein may comprise AF2, wherein the AF2 comprises VL and 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 with or identical to the amino acid sequence of SEQ ID NO: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 with or identical to the amino acid sequence of SEQ ID NO:719-818.

[0255] It should be understood that the term "antigen-binding fragment" used in the embodiments of the compositions disclosed herein is intended to include antibody portions or fragments that retain the ability to bind antigens as ligands of the corresponding intact antibodies. In such embodiments, the antigen-binding fragment can be, but is not limited to, CDRs and intercalation frames, variable or hypervariable regions (VL, VH) of the light and / or heavy chains of an antibody, variable fragments (Fv), Fab' fragments, F(ab')2 fragments, Fab fragments, single-chain antibodies (scAb), VHH camelid antibodies, single-chain variable fragments (scFv), linear antibodies, single-domain antibodies, complementarity-determining regions (CDRs), domain antibodies (dAb), single-domain heavy chain immunoglobulins of the BHH or BNAR type, single-domain light chain immunoglobulins, or other polypeptides known in the art containing antibody fragments capable of binding antigens. The VL and VH of two antigen-binding fragments can also be configured in a single-chain dimeric configuration; that is, the VL and VH of AF1 and AF2 are configured with linkers of appropriate length to allow for a dimeric arrangement.

[0256] In some embodiments, the VL and VH of the antigen-binding fragment are fused together by a relatively long linker consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids, which, when joined together, possess flexible properties. In one embodiment, the VL and VH of any scFv embodiment described herein are joined by a linker of hydrophilic amino acids selected from the sequences GSGEGGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 1142), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 1143), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 1144), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 1145). In other cases, the AF1 and AF2 of the subject composition are joined together by a short linker of hydrophilic amino acids having 3, 4, 5, 6, or 7 amino acids. In one embodiment, the short connector sequence is selected from the group consisting of sequences SGGGGS (SEQ ID NO: 1146), GGGGS (SEQ ID NO: 1147), GGSGGS (SEQ ID NO: 1148), GGS, or GSP. In another embodiment, this disclosure provides a composition comprising a single-stranded dimeric compound, wherein, upon folding, a first domain (VL or VH) pairs with a last domain (VH or VL) to form an scFv, and two intermediate domains pair to form another scFv, wherein the first and second domains, as well as the third and last domains, are fused together via one of the aforementioned short connectors, and the second and third variable domains are fused together via one of the aforementioned long connectors. The selection of short and long connectors prevents mispairing of adjacent variable domains, thereby facilitating the formation of single-stranded dimeric configurations of VL and VH containing a first antigen-binding fragment and a second antigen-binding fragment.

[0257] Table 8: Antibodies and Sequences of Target Cell Markers

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297] VI) Bispecific antigen-binding compositions - configuration and functional properties

[0298] In another aspect, this 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. Therefore, they may be referred to as T-cell adaptors. As described more fully below, the bispecific antigen-binding compositions are formulated in an activated prodrug form, which imparts advantages over bispecific T-cell adaptors and related compounds known in the art. The various compositions of this disclosure possess properties including enhanced stability during their production and purification, enhanced stability in circulation and increased half-life when administered to a subject, the ability to be activated at the intended treatment site rather than in normal healthy tissue, and binding affinity to target and effector cells at least comparable to that of corresponding conventional bispecific IgG antibodies when activated by proteolytic cleavage of the release segment and release of fused AF1 and AF2. Upon binding of AF1 and AF2 to effector and target cells, an immune synapse is formed, which enables the activation of effector cells and promotes subsequent destruction of target cells via apoptosis or cell lysis.

[0299] The various subject matter bispecific antigen-binding compositions described herein are specifically designed in prodrug form because one or more XTEN components shield the antigen-binding fragments, thereby reducing their ability to bind their ligands until they are released from the composition by protease cleavage at any protease cleavage site located within the release segment. Known proteases associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine ​​proteases, aspartic proteases, and metalloproteinases, including, but not limited to, the specific proteases described herein. This prodrug property of the bispecific antigen-binding compositions enhances the specificity of the composition to diseased tissues or cells compared to non-prodrug forms of bispecific T-cell adaptor therapeutics. Conversely, by specifically activating the bispecific antigen-binding composition in the microenvironment of target cells or diseased tissues, where target cell markers and proteases capable of cleaving the release segment are highly expressed, the bispecific antigen-binding fragments and XTENs of the construct are released upon cleavage of the release segment, and the fused antigen-binding fragments can crosslink cytotoxic effector cells with cells expressing target cell markers in a highly specific manner, thereby directing the cytotoxic potential of T cells to target cells. Following protease cleavage, the antigen-binding fragment is no longer shielded and effectively regains its full potential to bind to target cells and effector cells carrying target cell markers (such as cytotoxic T cells, which bind to the CD3 antigen, forming part of the T cell receptor complex), thereby activating T cells that mediate subsequent cleavage of target cells expressing specific target cell markers. Therefore, the bispecific antigen-binding compositions of this disclosure are expected to exhibit strong, specific, and effective target cell killing. In such cases, cells are selectively eliminated, thereby reducing the likelihood of toxic side effects.

[0300] In one aspect, this disclosure provides an activatable bispecific antigen-binding fragment composition comprising two antigen-binding fragments, wherein the first antigen-binding fragment targets effector cells and the second antigen-binding fragment targets cellular markers associated with diseased tissues or cells; both of them have specific binding affinity for their respective ligands. In designing subject compositions having first and second antigen-binding fragments (AF1 and AF2, respectively), considerations for at least three properties are noteworthy: 1) compositions having bispecific antigen-binding fragments capable of binding and linking effector cells and target cells together to form an immune synapse; 2) compositions having XTEN i) shielding both antigen-binding fragments and reducing their ability to bind target and effector cell ligands when the composition is in its intact prodrug form, ii) providing an extended half-life when administered to a subject, iii) reducing extravasation of the intact composition from circulation in normal tissues and organs compared to diseased tissues (e.g., tumors), and iv) conferring an increased safety profile compared to conventional bispecific cytotoxic antibody therapeutics; and 3) activation when RS is cleaved by one or more mammalian proteases in the vicinity of diseased tissue, thereby releasing the fused bispecific antigen-binding fragments and allowing them to regain their full binding affinity potential for target ligands. The subject composition is designed to take advantage of the properties of the XTEN and release segment (RS) components, and their positioning relative to the bispecific antigen-binding fragment achieves the aforementioned properties, as demonstrated by the results in the following exemplary embodiments.

[0301] In one embodiment, the polypeptide of any bispecific antigen-binding fragment composition embodiment described herein has two antigen-binding fragments (AF1 and AF2), a single RS and a single XTEN, said polypeptide in the uncleaved state may have an N-terminal to C-terminal arrangement of AF2-AF1-RS1-XTEN1, AF1-AF2-RS1-XTEN1, XTEN1-RS1-AF2-AF1, XTEN1-RS1-AF1-AF2, or a dimer-RS1-XTEN1 or XTEN1-RS1-dimer, said dimer comprising VL and VH of AF1 and AF2.

[0302] In other respects, this disclosure provides bispecific antigen-binding compositions having two antigen-binding fragments (AF1 and AF2), two RSs, and two XTENs. In the design of these compositions, considerations are noteworthy regarding the following: by adding a second XTEN, the binding affinity of the uncleaved composition to the corresponding ligands of the AF1 and AF2 antibody fragments is further reduced to further decrease the unintended binding of the composition to healthy tissue or cells when administered to a subject, thereby further improving the therapeutic index of the subject composition compared to compositions having only one RS and one XTEN. When measured in vitro, the addition of a second RS and a second XTEN results in a striking reduction in the binding affinity of the intact uncleaved peptide to the corresponding ligands of the AF1 and AF2 antibody fragments relative to those compositions having a single RS and XTEN, and also results in reduced toxicity in animal models of disease when administered at a therapeutically effective dose, as described in the examples below. In embodiments of the subject composition having two antigen-binding fragments, two RS and two XTEN, in the uncut state, the composition may have 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-Dual-RS1-XTEN1 (wherein the dual comprises VL and VH of AF1 and AF2), or XTEN1-RS1-Dual-RS2-XTEN2 (wherein the dual comprises VL and VH of AF1 and AF2).

[0303] A characteristic of the various designed compositions is that, when the RS of the bispecific antigen-binding composition is cleaved by a mammalian protease in the target cell environment and converted from a prodrug form to an activated or apoprotein form, after the bispecific antigen-binding fragment and XTEN are cleaved and released from the composition, fused AF1 and AF2 bind effector cells (e.g., CD3-carrying T cells) and diseased cells (e.g., tumors or cancer cells) carrying target cell marker antigens (which are capable of being bound by AF2) and link them together, thereby activating the effector cells. In one embodiment, wherein the RS of the bispecific antigen-binding composition is cleaved and the antigen-binding fragment is released, and the subsequent simultaneous binding of effector cells and target cells can lead to at least 3-fold, or 10-fold, or 30-fold, or 1000-fold activation of the effector cells, wherein activation is assessed by the production of cytokines, cell lysing proteins, or lysis of target cells, which is evaluated in an in vitro cell-based assay. In another embodiment, the released, fused AF1 and AF2 simultaneously bind to T cells carrying CD3 antigens and diseased cells carrying target cell marker antigens to form an immune synapse, wherein the binding leads to the release of T cell-derived effector molecules capable of lysing diseased cells. Non-limiting examples of in vitro assays for measuring effector cell activation and / or cell lysis include cell membrane integrity assays, mixed cell culture assays, FACS-based propidium iodide assays, trypan blue influx assays, photometric enzyme release assays, ELISA, radiation-based 51Cr release assays, luminescent europium release assays, calcein AM release assays, photometric MTT assays, XTT assays, WST-1 assays, Alamar blue assays, radiation-based 3H-Thd incorporation assays, clonogen assays for measuring cell division activity, luminescent rhodamine 123 assays for measuring mitochondrial transmembrane gradients, apoptosis assays monitored by FACS-based phosphatidylserine exposure, ELISA-based TUNEL assays, caspase activity assays, and cell morphology assays, or other assays known in the art for measuring cytokines, cell lysis proteins, or cell lysis, or methods described in the examples below.

[0304] Not limited to any particular theory, it is believed that, using the bispecific antigen-binding composition form described above, the released fused AF1 and AF2, upon cleavage of the RS, are capable of killing target cells by recruiting cytotoxic effector cells without any pre-stimulation and / or co-stimulation. Furthermore, the independence of pre-stimulation and / or co-stimulation of effector cells may significantly contribute to the exceptionally high cytotoxicity mediated by the released, fused AF1 and AF2 antigen-binding fragments. In some embodiments, the released AF1 and AF2 (wherein AF1 remains fused to AF2 via a linker peptide) are engineered to have binding specificity, enabling them to bind and link closely adjacent cytotoxic effector cells (e.g., T cells, NK cells, cytokine-induced killer cells (CIK cells)) together, via AF2 to a preselected target cell marker, which has binding specificity to target cell markers associated with tumor cells, cancer cells, or cells associated with diseased tissue, thereby influencing the immune synapse and the selective, targeted, and localized action of released cytokines and effector molecules on the target tumor or cancer cells, resulting in damage or destruction of the tumor or cancer cells, thus producing a therapeutic benefit for the subject. Released AF1 bound to effector cell antigens can modulate one or more functions of effector cells, thereby causing or facilitating cell lysis of target cells (e.g., tumor cells) to which AF2 is bound. Effector cell antigens can be expressed by effector cells or other cells. In one embodiment, the effector cell antigen is expressed on the cell surface of the effector cell. Non-limiting examples of effector cell antigens are CD3, CD4, CD8, CD16, CD25, CD38, CD45RO, CD56, CD57, CD69, CD95, CD107, and CD154. Therefore, those skilled in the art will understand that the configuration of the subject composition is designed to selectively or disproportionately deliver the active form of the composition to target tumor tissue or cancer cells, compared to healthy tissue or healthy cells of a subject having the desired therapeutic benefit in which the composition is administered. As will be apparent from the foregoing, this disclosure provides a large class of peptides in engineered configurations to achieve desired performance.

[0305] One object of this disclosure is to design a subject bispecific antigen-binding composition having the shielding effect conferred by the XTEN of the complete circulating composition and the associated reduced binding effector cells and target tissue potential, resulting in a reduction in the production of Th1T cell-associated cytokines or other pro-inflammatory mediators during systemic exposure when administered to a subject, compared to bispecific antigen-binding compositions not connected to a shielding portion (such as XTEN), thereby improving the overall side effects and safety profile (e.g., therapeutic index). As important components of cellular immunity, the production of IL-2, TNF-α, and IFN-γ is a marker of Th1 response (Romagnani ST-cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol. 2000. 85(1): 9-18), especially 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), and IL-4, IL-6, and IL-10 are also important pro-inflammatory cytokines in the cytotoxic response of bispecific antibody combinations (Zimmerman, Z., et al. Unleashing the clinical power of T cells: CD19 / CD3 bi-specific T cell engager). (Antibody composition blinatumomab as a potential therapy. Int. Immunol. (2015) 27(1):31-37). In one embodiment, compared with the corresponding released AF1 and AF2 (which remain fused together after proteolytic release via RS) of the corresponding protease-treated composition in an in vitro cell-based cytokine stimulation assay performed under comparable conditions (e.g., equivalent molar concentrations) to stimulate Th1 and / or cytokine levels, when the intact, uncut peptide is contacted with effector and target cells in an in vitro cell-based cytokine stimulation assay, the intact, uncut bispecific antigen-binding composition of the embodiments described herein may exhibit a reduction of at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold, or at least 7-fold, or at least 8-fold, or at least 9-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 100-fold, or at least 1000-fold in the potential to induce Th1 and / or pro-inflammatory cytokine production. Non-limiting examples of Th1 and / or pro-inflammatory cytokines are IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ. In one of the foregoing embodiments, the production of Th1 cytokines is measured in an in vitro assay comprising effector cells such as PBMCs or CD3+ T cells and target cells having target cell marker antigens disclosed herein. In another embodiment, cytokines can be assessed from blood, fluid, or tissue samples taken from a subject in which the peptide composition has been administered. In the foregoing embodiments, the subject can be a mouse, rat, monkey, or human. However, among the advantages of the subject bispecific antigen-binding composition of the embodiments described herein, it has been found that the cell-lytic performance of the composition does not require pre-stimulation with cytokines; cell lysis or apoptosis in the target cells is achieved by the formation of an immune synapse between the antigen-binding fragment and the effector cell bound to the target cells. Nevertheless, the production of pro-inflammatory cytokines is a useful marker for assessing the efficacy or effect of the subject peptide composition, whether by in vitro assay or by monitoring treatment of a subject with diseased tissue (e.g., a tumor) after administration of the subject bispecific antigen-binding composition.

[0306] In the context of the use of bispecific antigen-binding compositions in subjects, one objective of this disclosure is to design subject bispecific antigen-binding compositions to utilize the pore size difference of vascular systems in tumor or inflamed tissues compared to healthy vascular systems via the addition of XTEN, thereby reducing the extravasation of intact bispecific antigen-binding compositions in normal tissues. However, in the leaky environment of tumor vascular systems or other inflammatory areas in diseased tissues, intact assemblies can extravasate and be activated by proteases in the diseased tissue environment, thereby releasing antigen-binding fragments to effectors and target cells (see, e.g., Figure 5In the case of RS in a bispecific antigen-binding composition, this design takes advantage of the fact that when the bispecific antigen-binding composition is near a diseased tissue (e.g., a tumor) that produces one or more proteases, the RS sequence sensitive to one or more proteases expressed by the tumor can be cleaved by the proteases (described more fully above). The action of the proteases cleaves the release segment (RS) of the composition, separating the antigen-binding fragment from the XTEN, resulting in a reduction in the molecular weight and hydrodynamic radius of the components, particularly for the released fused AF1 and AF2. As will be understood, the reduction in the molecular weight and hydrodynamic radius of the composition also confers the property that the released, fused AF1 and AF2 can move more freely in solution, traverse smaller pore spaces in tissues and tumors, and more easily extravasate from the larger pores of the tumor vascular system and penetrate into the tumor more readily, thereby increasing the ability to bind and attach to effector cells and tumor cells. Such properties can be measured by various assays. Therefore, those skilled in the art will understand that, in the context of treating a subject with the subject composition, the bispecific antigen-binding composition exists in prodrug form and transforms into a more active form upon entering a cellular environment via the action of a protease co-localized with diseased tissue or cells. After being released from the composition by the action of one or more proteases in the target tissue, the fused AF1, which binds specifically to effector cell antigens, and the AF2, which binds specifically to target cell marker antigens of diseased cells, regain their full ability to bind to effector cells and target cells and connect together to form an immune synapse. The formation of the immune synapse leads to the activation of effector cells, the initiation of new gene transcription through various signaling pathways, and the release of effector molecular contents from their vesicles via exocytosis. Depending on the type of effector cell, different cytokines and lymphokines are released; for example, type 1 helper T cells (Th1) release cytokines such as IFN-γ, IL-2, and TNF-α, while type 2 helper T cells (Th2) release cytokines such as IL-4, IL-5, IL-10, and IL-13 (which stimulate B cells), and cytotoxic T lymphocytes (CTLs) release cytotoxic molecules such as perforin and granzymes (which kill their targets) (collectively referred to as "effect molecules"). Specifically, it is anticipated that after the bispecific antigen-binding fragments released via the bispecific antigen-binding composition simultaneously bind to and link effector cells and target tumor cells together, effector molecules released by effector cells into the intercellular immune synapses act on tumor cells at a very low effector-to-target (E:T) ratio, leading to damage, perforin-mediated lysis, granzyme B-induced cell death, and / or tumor cell apoptosis.Therefore, in another aspect, the various designed compositions are characterized in that, when the 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 of normal tissue but is able to extravasate into the more permeable vascular system of the tumor. This allows the prodrug form of the assembly to be activated by a protease co-localized with the tumor, and the released antigen-binding fragment binds and connects effector cells (e.g., T cells) and tumor cells expressing a target cell marker targeted by the composition's AF2, thereby activating the effector cells and achieving tumor cell lysis. In other words, in some cases, the more permeable vascular system in tumor tissue may allow the bispecific antigen-binding peptide to extravasate into the tissue, where tumor-associated proteases can act on the release segment (RS), cleave it, and release the binding moiety, which in turn can bind and link effector cells and tumor-associated cells together. In the case of normal tissue, extravasation may be blocked by a tighter vascular barrier, or, if the bispecific antigen-binding peptide does extravasate to a certain extent, the bispecific antigen-binding peptide may remain primarily in its "original" form because there may not be sufficient proteases in healthy tissue to release the binding moiety, resulting in a net effect without the formation of an immune synapse. In some cases, fused AF1 and AF2 released from a subject's tumor bound to both tumor cells and effector cells exhibit at least 10-fold, at least 30-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, or at least 1000-fold enhanced ability to activate effector cells compared to the corresponding intact, uncut bispecific antigen-binding composition in the tumor. In other cases, linked AF1 and AF2 released from a subject's tumor bound to both tumor cells and effector cells exhibit at least 10-fold, at least 30-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, or at least 1000-fold enhanced ability to lyse tumor cells compared to the corresponding intact, uncut bispecific antigen-binding composition in the tumor. In the foregoing embodiments, effector cell activation and / or cytotoxicity can be determined by conventional methods known in the art, such as cytometry of activated effector cells, cytokine assays, tumor size measurements, or by histopathology. In the aforementioned implementation scheme, the subjects can be mice, rats, dogs, monkeys, and humans.Specifically, the subject composition is particularly considered to be designed such that, upon administration to a subject with a disease having a target cell marker that can be bound by AF2, the bispecific antigen-binding composition exhibits an enhanced therapeutic index and a reduced incidence of side effects compared to conventional bispecific antibodies known in the art. This is achieved through a combination of shielding effect of XTEN on the binding affinity of the antigen-binding fragment in prodrug form and steric hindrance, but is able to release bispecific AF1 and AF2 near or within a target tissue (e.g., a tumor) (achieved by including a cleavage sequence in RS), which produces a protease for which RS is a substrate.

[0307] VII) Methods and uses of bispecific antigen-binding compositions

[0308] In another aspect, this disclosure provides activatable bispecific antigen-binding compositions and pharmaceutical compositions comprising bispecific antigen-binding compositions that are particularly useful in medical settings; for example, for the prevention, treatment, and / or improvement of certain diseases, such as, but not limited to, cancer, tumors, or inflammatory diseases. For use in treating diseases, the bispecific antigen-binding compositions of the present invention will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific disorder being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disorder, the delivery site of the medicament, the method of administration, the administration schedule, and other factors known to a medical practitioner.

[0309] Many therapeutic strategies have been used to design peptide compositions for treating subjects with cancerous diseases, including modulating T-cell responses by targeting TCR signaling, particularly 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 antibody approved for human use (Sgro, Toxicology 105 (1995), 23-29) and is widely used clinically as an immunosuppressant in transplantation (Chatenoud L: Immunologic monitoring during OKT3 therapy. ClinTransplant 7:422-430, 1993). Furthermore, anti-CD3 monoclonal drugs can induce partial T-cell signaling and clonal unresponsiveness (Smith, J. Exp. Med. 185 (1997), 1413-1422). OKT3 reacts with and blocks the function of the CD3 complex in the T-cell membrane; the CD3 complex is associated with the T-cell antigen recognition structure (TCR), which is crucial for signal transduction. These and other 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)), proliferative and suppressor T cell induction. In cancer, attempts have been made to utilize cytotoxic T cells to lyse cancer cells. Unbound by theory, it is believed that cytotoxic T cells require direct cell-to-cell contact to achieve target cell lysis; the TCR on the cytotoxic T cell must recognize and bind to the appropriate antigen on the target cell. This creates an immune synapse, which in turn initiates a signaling cascade within the cytotoxic T cell, resulting in T cell activation and the production of various cytotoxic cytokines and effector molecules. Perforin and granzymes are highly toxic molecules stored in pre-formed granules present in activated cytotoxic T cells. Following recognition of the target cell, the cytoplasmic granules of the attached cytotoxic T cell migrate towards the cytotoxic T cell membrane, eventually fusing with it and releasing its contents into the immune synapse in a directed manner. This creates a pore within the target cell membrane, thereby disrupting the tumor cell membrane. The resulting pore serves as an entry point for granzymes, which are serine proteases that induce apoptosis in tumor cells.

[0310] The subject matter described herein, a bispecific antigen-binding composition (in which AF1, having a specific binding affinity for CD3 on T cells, is tightly fused with AF2, having a specific binding affinity for target cell markers), is a T cell adaptor that, once released from the composition in its intact prodrug form by cleavage of the release segment, is able to regain their full potential to bind to both T cells and target cells, thereby forming an immune synapse that promotes T cell activation and subsequently facilitates the destruction of tumor cells through apoptosis or cell lysis.

[0311] This disclosure covers methods of using bispecific antigen-binding compositions engineered to target a range of malignant cells, such as tumors, in addition to effector cells, to initiate target cell lysis and achieve beneficial therapeutic outcomes. This is because the bispecific antigen-binding compositions are designed such that AF1 binds to and links CD3 to activate cytotoxic T cells, while AF2 can be engineered to target a variety of different target cell markers with specific malignant tumor characteristics; these are linked together to create an immune synapse. A particular advantage of this design is that the physical binding of cytotoxic effector cells to cells carrying target cell markers eliminates the need for antigen processing, MHC1 / β2-microglobulin, and co-stimulatory molecules. Examples of important target cell markers include, but are not limited to, those disclosed herein. Given the range of such target cell markers that can be engineered to the various embodiments of the subject bispecific antigen-binding compositions (described more broadly above), it should be understood that the resulting compositions will have utility against a variety of diseases, including hematologic cancers and solid tumors. In one embodiment, this disclosure provides a method of treating a subject with a tumor. The treated tumor can comprise tumor cells derived from a group selected from the following: 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, gastric cells, genitourinary tract cells, cervical cells, uterine cells, small intestinal cells, hepatocytes, pancreatic cells, gallbladder cells, bile duct cells, esophageal cells, salivary gland cells, lung cells, and thyroid cells. Another advantage of the composition is that, since cytotoxic effector cells are not consumed during the damage / destruction of the bridging target cancer cells, after causing lysis of one target cell, activated effector cells can be released and migrate through local tissues to other target cancer cells, rebinding AF1-AF2 and the target antigen, and initiating additional cell lysis. Furthermore, it is anticipated that in local environments such as solid tumors, the release of effector cell molecules such as perforin and granzyme will lead to damage to tumor cells that are adjacent to but not bound by a given molecule with a bispecific binding domain, thereby causing tumor growth arrest or regression.

[0312] Therefore, the utility of this disclosure will be understood as follows: upon administration of a therapeutically effective dose of a pharmaceutical composition comprising the bispecific antigen-binding composition described herein to a subject with cancer or tumors exhibiting target cell markers, a protease associated with or co-localized to cancer or tumor cells can act on the composition to release fused AF1 and AF2, enabling the formation of an immune synapse through the connection between target cells and effector cells. As a result, effector cell-derived effector molecules capable of cleaving target cells are released into the synapse, leading to apoptosis, lysis, or death of the target cancer or tumor cells. Furthermore, those skilled in the art will understand that once an immune synapse is formed through the binding of the released binding domains to effector cells and target cancer cells, the use of the bispecific antigen-binding composition can result in a more durable and generalized beneficial therapeutic effect than "single-cell killing."

[0313] In one aspect, this disclosure relates to methods for treating a disease (such as cancer or an inflammatory disorder) in a subject. In some embodiments, this disclosure provides a method for treating a disease in a subject, comprising administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising a bispecific antigen-binding composition of any embodiment described herein. The therapeutically effective amount of the pharmaceutical composition may vary depending on factors such as an individual's disease state, age, sex, and weight, and the ability of the antibody or antibody moiety to elicit a desired response in the individual. A therapeutically effective amount is also the amount in which any toxic or adverse effects of the subject composition are exceeded by the therapeutically beneficial effects. A preventatively effective amount represents the amount of pharmaceutical composition required for the period necessary to achieve the desired preventative outcome.

[0314] The therapeutically effective doses of the bispecific antigen-binding compositions described herein will generally provide therapeutic benefit without significant toxicity. The toxicity and therapeutic efficacy of the bispecific antigen-binding compositions can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals. Cell culture assays and animal studies can be used to determine the LD50. 50 (50% lethal dose for the population) and ED 50 (The dose effective for 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50. 50 / ED 50 Bispecific antigen-binding compositions exhibiting a high therapeutic index are preferred. In one aspect, the bispecific antigen-binding molecules according to this disclosure exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to determine a dosage range suitable for human use. The dosage is preferably within the range of ED. 50Within the circulating concentration range, there is little or no toxicity. The dosage can vary within this range depending on various factors, such as the dosage form used, the route of administration employed, and the condition of the subject. Precise formulation, route of administration, and dosage can be chosen by an individual physician taking into account the patient's condition (see, for example, Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Chapter 1, page 1). Skilled technicians readily recognize that in many cases, a bispecific antigen-binding composition may not provide a cure but may only provide partial benefit. In some respects, physiological changes that have certain benefits are also considered therapeutically beneficial. Therefore, in some respects, the amount of a bispecific antigen-binding composition that provides physiological changes is considered an "effective amount" or "therapeuticly effective amount." Subjects, patients, or individuals requiring treatment are typically mice, rats, dogs, monkeys, or humans.

[0315] The bispecific antigen-binding compositions of the present invention can be administered in combination with one or more other agents in treatment. For example, the bispecific antigen-binding molecule of any embodiment described herein can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" covers any agent administered to treat symptoms or diseases in an individual in need of such treatment. Such additional therapeutic agents may contain any active ingredient suitable for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. In some aspects, the additional therapeutic agent is an immunomodulator, an immuno-oncology antibody, a cell inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis inducers. In one particular aspect, the additional therapeutic agent is an anticancer agent, such as a microtubule disruptor, antimetabolite, topoisomerase inhibitor, DNA intercalating agent, alkylating agent, hormone therapy, kinase inhibitor, receptor antagonist, tumor cell apoptosis activator, or antiangiogenic agent.

[0316] In one implementation of a method for treating a disease in a subject, the disease being treated may be epithelial cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, genitourinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, vaginal cancer, vulvar cancer, Ewing sarcoma, peritoneal cancer metastasis, serous uterine carcinoma, parathyroid cancer, endometrial cancer, cervical cancer, colorectal cancer, intraperitoneal malignant tumors with malignant ascites, uterine cancer, mesothelioma in peritoneal renal cancer, lung cancer, laryngeal cancer, small cell lung cancer, non- Small cell lung cancer, gastric cancer, esophageal cancer, gastric cancer, small intestinal 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, ovarian androma, adenocarcinoma, sarcoma of any origin, primary hematologic malignancies including acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, B-cell-derived chronic lymphocytic leukemia, hairy cell leukemia, myeloproliferative disorders or myelodysplastic disorders, myasthenia gravis, Graves' disease, Kaposi's sarcoma, neuroblastoma, Hashimoto's thyroiditis, nephroblastoma, or Goodpassuia syndrome. Effective doses of treatment can have beneficial effects in helping to treat (e.g., cure or reduce severity) or prevent (e.g., reduce the likelihood of recurrence) cancer or tumors. In another embodiment of the method of treating a disease in a subject, the pharmaceutical composition is administered to the subject at one or more therapeutically effective doses administered twice weekly, once weekly, once every two weeks, once every three weeks, once every four weeks, or once monthly. In another embodiment of the method, the pharmaceutical composition is administered to the subject at 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 lead 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 lead 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 one or more subsequent maintenance doses 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, and at least about 0.20 mg / kg. At least about 0.22 mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg, or at least 5.0 mg / kg. In another embodiment of the method, the drug composition is administered to the subject via dermal, subcutaneous, intravenous, arterial, peritoneal, peritoneal, sheath, or intramuscular administration. In another embodiment of the method, the drug composition is administered to the subject in one or more therapeutically effective bolus doses or via infusion over 5 minutes to 96 hours (provided maximum tolerable safety and efficacy). In another embodiment of the method, the pharmaceutical composition is administered to the subject in one or more therapeutically effective bolus doses or via infusion over 5 minutes to 96 hours, wherein said dose is selected from the group consisting of: at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.14 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.22 mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg, or at least about 5.0 mg / kg.In another embodiment of the method, the pharmaceutical composition is administered to the subject in one or more therapeutically effective bolus doses or by infusion over a period of 5 minutes to 96 hours, wherein administration to the subject results in a Cmax plasma concentration of at least about 0.1 ng / mL to at least about 2 μg / mL or more of the intact, uncut bispecific antigen-binding composition in the subject, which is maintained for at least about 3 days, at least about 7 days, at least about 10 days, at least about 14 days, or at least about 21 days. Therapeuticly effective doses are at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, at least about 0.1 mg / kg, at least about 0.12 mg / kg, at least about 0.14 mg / kg, at least about 0.16 mg / kg, at least about 0.18 mg / kg, at least about 0.20 mg / kg, at least about 0.22 mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg. In one embodiment, the initial dose is selected from the group consisting of at least about 0.005 mg / kg, at least about 0.01 mg / kg, at least about 0.02 mg / kg, at least about 0.04 mg / kg, at least about 0.08 mg / kg, 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, and at least about 0.20 mg / kg. The dosage is specified as g / kg, at least about 0.22 mg / kg, at least about 0.24 mg / kg, at least about 0.26 mg / kg, at least about 0.27 mg / kg, at least about 0.28 mg / kg, at least 0.3 mg / kg, at least 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1.0 mg / kg, at least about 1.5 mg / kg, or at least about 2.0 mg / kg. In the aforementioned embodiments, administration to the subject results in a plasma concentration of the peptide of at least about 0.1 ng / mL to at least about 2 ng / mL or more, sustained 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 aforementioned embodiments of the method, the subject may be a mouse, rat, dog, monkey, or human.

[0317] VIII) Nucleic acid sequence

[0318] In another aspect, the present invention relates to isolated polynucleotide sequences encoding polypeptides or bispecific antigen-binding compositions of any embodiment described herein, and sequences complementary to polynucleotide molecules encoding polypeptide composition embodiments.

[0319] In some embodiments, the present invention provides isolated polynucleotide sequences or complements thereof, said isolated polynucleotide sequences encoding an AF1 sequence, or an AF2 sequence, or a release segment sequence (RS1 and RS2), or an XTEN sequence of any embodiment described herein. In one embodiment, the present invention provides isolated polynucleotide sequences or complements thereof, said isolated polynucleotide sequences encoding a polypeptide or a bispecific antigen-binding composition of any embodiment described herein. In one embodiment, the present invention provides isolated polynucleotide sequences encoding a polypeptide or a bispecific antigen-binding composition, said polynucleotide sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polynucleotide sequences shown in Table 9.

[0320] In another aspect, this disclosure relates to methods for producing polynucleotide sequences encoding polypeptides or bispecific antigen-binding compositions of any embodiment described herein, or sequences complementary to said polynucleotide sequences (including homologs thereof), and methods for expressing proteins expressed by the polynucleotide sequences. Generally, the methods include generating polynucleotide sequences encoding proteinic polypeptides or bispecific antigen-binding compositions of any embodiment described herein, and incorporating the encoding gene into an expression vector suitable for host cells. To generate a polypeptide or bispecific antigen-binding composition encoding any embodiment described herein, the methods include transforming suitable host cells with the expression vector, and culturing the host cells under conditions that cause or allow the expression of the resulting polypeptide or bispecific antigen-binding composition of any embodiment described herein in the transformed host cells, thereby generating 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 manufacture the polynucleotides and expression vectors of this disclosure.

[0321] According to this disclosure, nucleic acid sequences (or their complements) encoding peptides or bispecific antigen-binding compositions of any embodiment described herein are used to generate recombinant DNA molecules that direct expression in appropriate host cells. A variety of cloning strategies are suitable for implementing this disclosure, many of which are used to generate constructs comprising genes encoding compositions of this disclosure or their complements. In one embodiment, a cloning strategy is used to generate a gene encoding a construct containing nucleotides encoding a peptide or bispecific antigen-binding composition for transforming host cells to express said composition. In the foregoing embodiments described in this paragraph, the gene may contain nucleotides encoding antigen-binding fragments, release segments, and XTENs in the configurations disclosed herein.

[0322] In one method, a construct is first prepared containing a DNA sequence encoding a polypeptide or a bispecific antigen-binding composition construct. Exemplary methods for preparing such constructs are described in the examples. This construct is then used to generate an expression vector suitable for transforming host cells, such as prokaryotic or eukaryotic host cells, to express and recover the polypeptide construct. If desired, the host cell is *E. coli*. In another embodiment, the host cell is selected from BHK cells, NSO 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 generating the expression vector, transforming host cells, and expressing and recovering XTEN are described in the examples.

[0323] Genes encoding peptides or bispecific antigen-binding compositions can be prepared in one or more steps, either entirely synthetically or through synthesis in combination with enzymatic processes such as restriction enzyme-mediated cloning, PCR, and overlap extension, including the methods described more fully in the examples. For example, the methods disclosed herein can be used to link polynucleotide sequences encoding various components (e.g., binding domains, linkers, release segments, and XTENs) of desired length and sequence. Genes encoding peptide 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 suitable for use in the production of peptides or bispecific antigen-binding compositions in *E. coli* or mammalian host cells. In one method of this disclosure, as described above, a polynucleotide library encoding components of the construct is created and then assembled. The resulting gene is then assembled and used to transform host cells to produce and recover the peptide composition for evaluating its performance, as described herein.

[0324] The resulting polynucleotide encoding a polypeptide or bispecific antigen-binding composition sequence can then be cloned individually into an expression vector. Nucleic acid sequences are inserted into the vector using various procedures. Generally, DNA is inserted into one or more suitable restriction endonuclease sites using techniques known in the art. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. The construction of suitable vectors containing one or more of these components employs standard ligation techniques known to a skilled craftsman. Such techniques are well known in the art and are well described in scientific and patent literature. Various vectors are publicly available. For example, vectors can be in the form of plasmids, granules, viral particles, or bacteriophages, which facilitate recombinant DNA procedures, and the choice of vector generally depends on the host cell into which it is to be introduced. Thus, a vector can be a self-replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, a vector can be one that, when introduced into a host cell, integrates into the host cell genome and replicates along with one or more chromosomes into which it has already been integrated. Once introduced into a suitable host cell, the expression of antigen-binding fragments or bispecific antigen-binding compositions can be determined using any nucleic acid or protein assay known in the art. For example, the presence of transcribed mRNA of light chain CDRs or heavy chain CDRs, antigen-binding fragments, or bispecific antigen-binding compositions can be detected and / or quantified using probes complementary to any region of the antigen-binding unit polynucleotide using conventional hybridization assays (e.g., RNA blot analysis), amplification procedures (e.g., RT-PCR), SAGE (US Patent No. 5,695,937), and array-based techniques (see, for example, US Patent Nos. 5,405,783, 5,412,087, and 5,445,934).

[0325] This disclosure provides the use of plasmid expression vectors containing replication and control sequences that are compatible with and recognized by host cells and operatively 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 a wide variety of bacteria, yeasts, and viruses. Useful expression vectors that can be used include, for example, segments of chromosomal, non-chromosomal, and synthetic DNA sequences. "Expression vector" refers to a DNA construct containing a DNA sequence operatively linked to a suitable control sequence capable of influencing the expression of DNA encoding a polypeptide in a suitable host. The requirement is that the vector is replicable and viable in the selected host cell. Low-copy-number or high-copy-number vectors can be used as needed.

[0326] Suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA, and known bacterial plasmids such as colEI, pCR1, pBR322, pMal-C2, pET, pGEX (described in Smith et al., Gene 57:31-40 (1988)), pMB9 and its derivatives, plasmids such as RP4, phage DNA such as numerous derivatives of phage I such as NM989, and other phage DNA such as M13 and filamentous single-stranded phage DNA; yeast plasmids such as derivatives of 2-micron or 2-m plasmids, as well as centromere and integrative yeast shuttle vectors; vectors suitable for eukaryotic cells such as vectors suitable for insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, such as plasmids modified to use phage DNA or express control sequences; etc. Yeast expression systems that can also be used in this disclosure include, but are not limited to, non-fusion pYES2 vectors (Invitrogen), fusion pYESHisA, B, C (Invitrogen), pRS vectors, etc. The control sequences of the vector include a promoter that enables transcription, an optional operon sequence that controls such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that controls the termination of transcription and translation. The promoter can be any DNA sequence that exhibits transcriptional activity in the selected host cell and can be derived from a gene encoding a protein homologous or heterologous to that of the host cell. Promoters suitable for prokaryotic hosts in expression vectors include β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase and tryptophan (trp) promoter systems [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36, 776], and heterozygous promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)], all of which are operatively linked to DNA encoding the XTEN polypeptide. Promoters for bacterial systems may also contain a Shine-Dalgarno (SD) sequence, which is operatively linked to DNA encoding the polypeptide.

[0327] The expression of a vector can also be determined by examining the antigen-binding fragments or components of the expressed bispecific antigen-binding composition. A variety of techniques are available for protein analysis in the art. These include, but are not limited to, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, immunoradioassay, in situ immunoassay (using, for example, colloidal gold, enzyme, or radioisotope labeling), Western blot analysis, immunoprecipitation assay, immunofluorescence assay, and SDS-PAGE.

[0328] IX). Method for preparing peptide and bispecific antigen-binding compositions

[0329] In another aspect, this disclosure provides methods for preparing the subject compositions. In one embodiment, the method includes: culturing host cells containing a nucleic acid construct encoding a peptide or bispecific antigen-binding composition of any embodiment described herein, under conditions promoting expression of a peptide or bispecific antigen-binding composition; and then recovering the peptide or bispecific antigen-binding composition using standard purification methods (e.g., column chromatography, HPLC, etc.), wherein the composition is recovered 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 binding fragments of the expressed peptide or bispecific antigen-binding composition are correctly folded. In another embodiment of the preparation method, the expressed peptide or bispecific antigen-binding composition is recovered, wherein at least 90%, or at least 95%, or at least 97%, or at least 99% of the peptide or bispecific antigen-binding composition is recovered in a monomeric, soluble form.

[0330] In another aspect, this disclosure relates to the preparation of peptides and bispecific antigen-binding compositions using *E. coli* or mammalian host cells at high fermentation expression levels of functional proteins, and to methods for providing expression vectors encoding constructs that can be used in the methods to produce cytotoxically active peptide construct compositions at high expression levels. In one embodiment, the method includes the steps of: 1) preparing a polynucleotide encoding a peptide of any embodiment disclosed herein; 2) cloning the polynucleotide into an expression vector, which may be a plasmid or other vector under the control of appropriate transcription and translation sequences for high-level expression of the protein in a biological system; 3) transforming suitable host cells with the expression vector; and 4) culturing the host cells in a conventional nutrient medium under conditions suitable for expressing the peptide composition. If desired, the host cells are *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 the host cell expression product, and 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 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 its target ligand. In another embodiment, this disclosure provides a method for producing a polypeptide or a bispecific antigen-binding composition, the method comprising: culturing host cells in a fermentation reaction under conditions of efficient expression of a polypeptide product, the host cells containing a vector encoding a polypeptide comprising a polypeptide or a bispecific antigen-binding composition, wherein the concentration of the polypeptide product exceeds about 10 mg / g dry weight of the host cells (mg / g), or at least about 250 mg / g, or about 300 mg / g, or about 350 mg / g, or about 400 mg / g, or about 450 mg / g, or about 500 mg / g of the polypeptide, and wherein the antigen-binding fragment of the expressed protein is correctly folded. In another embodiment, this disclosure provides a method for generating a polypeptide or a bispecific antigen-binding composition, the method comprising: culturing host cells in a fermentation reaction under conditions of efficient expression of a polypeptide product, the host cells containing a vector encoding the composition, wherein the concentration of the polypeptide product exceeds about 10 mg / g dry weight of the host cells (mg / g), or at least about 250 mg / g, or about 300 mg / g, or about 350 mg / g, or about 400 mg / g, or about 450 mg / g, or about 500 mg / g of the polypeptide, and wherein the expressed polypeptide product is soluble.

[0331] The following are examples of the composition and evaluation of the compositions disclosed herein. It should be understood that various other embodiments can be implemented in light of the general description provided above. Example

[0332] Example 1: Construction of a bispecific antigen-binding polypeptide with two release regions.

[0333] To generate a plasmid from which individual scFvs could be removed by restriction digestion, pCW1700 (encoding an anti-EpCAM-anti-CD3(UCHT1) bispecific tandem scFv with an RSR2486 release region, AE866XTEN, and a 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 the Bsu36I site. The synthesized DNA fragment was cloned into the digested backbone using an In-Fusion kit (New England Biolabs) to assemble pJB0035. pJB0035 was then digested with NheI and BsaI to remove the BSRS1 release region sequence. An overlapping single-stranded oligonucleotide encoding RSR2486 was synthesized, having a single-stranded tail annealed to the NheI and BsaI overhangs. The oligonucleotides were annealed together and ligated into digested pJB0035 to produce pCW1880, which encodes an anti-EpCAM-anti-CD3 (UCHT1) bispecific tandem scFv, RSR2486, XTEN866, and a 6X His-tagged affinity tag (SEQ ID NO: 1150).

[0334] To generate plasmids with various CD3-binding domain variants, pCW1880 was digested with Bsu36I and NheI to remove UCHT1 anti-CD3 scFv. DNA fragments encoding the designed CD3 variants were synthesized. Each gene fragment included 30 nucleotides at the 5' and 3' of the restriction sites to serve as DNA overlaps for Gibson DNA assembly. The synthesized DNA fragments were cloned into the digested backbone using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to assemble pJB0205, pJB0206, pJB0207, and pJB0208.

[0335] To generate a bispecific antigen-binding polypeptide with both N-terminal and C-terminal XTEN, AE292 XTEN was amplified from the plasmid by PCR using primers comprising a 17-21 bp 5' homologous region to the backbone DNA at the N-terminus and to the non-cleavable release region (RSR3058, amino acid sequence TTGEAGEAAGATSAGATGP (SEQ ID NO:100)) at the C-terminus. A second PCR product encoding the light chain and a portion of the heavy chain of the anti-EpCAM antibody 4D5MOCB was amplified using primers comprising a 16-21 bp 5' homologous region to both the RSR3058 at the N-terminus and the heavy chain of 4D5MOCB at the C-terminus. These PCR fragments were cloned into a BsiWI-SacII-digested backbone vector using the In-Fusion plasmid assembly kit (Takara Bio). This backbone vector encoded the remainder of the 4D5MOCB heavy chain / anti-CD3 tandem scFv, a second copy of the uncleavable release region of RSR3058, and AE837 XTEN with a 6xHIS (SEQ ID NO: 1150) affinity tag. The final vector encoded a bispecific antigen-binding polypeptide under the control of the PhoA promoter and STII secretion leader sequence, comprising (N-terminally to C-terminally) the following components: AE292 XTEN, the uncleavable RSR3058 release region, the anti-EpCAM-anti-CD3 bispecific tandem scFv, and RSR3058 fused to the AE867 XTEN with a 6xHIS (SEQ ID NO: 1150) affinity tag. The resulting construct was pJB0084 (Table 9).

[0336] pJB0084 was used as a template to generate a bispecific antigen-binding polypeptide construct encoding AE292XTEN, a cleavable release fragment RSR2295, an anti-EpCAM-anti-CD3 bispecific tandem scFv, and an RSR2295 fused to AE868XTEN. The plasmid utilized two PCR products templated with pJB0084; the first encoding a 6xHIS (SEQ ID NO: 1150) affinity tag and AE292XTEN, possessing a 5' homologous region with the vector backbone and a 3' homologous region encoding the first RSR2295; the second encoding an anti-EpCAM-anti-CD3 bispecific tandem scFv, possessing 5' and 3' homologous regions encoding the RSR2295 release fragment at the 5' and 3' of the tandem scFv. The third fragment, encoding AE868 XTEN, has a C-tag affinity tag (amino acid sequence EPEA (SEQ ID NO: 1149)), a 5' homologous region encoding a second RSR2295, and a 3' homologous region with the backbone vector. The three PCR fragments were cloned into pJB0084, which had been digested with BsiWI-NotI, using the In-Fusion plasmid assembly kit. The final vector, pJB0169, encodes a bispecific antigen-binding polypeptide molecule under the control of the PhoA promoter and the STII secretion leader sequence of the DNA sequence, having the following components (from N-terminus to C-terminus): a 6xHIS affinity tag (SEQ ID NO: 1150), AE292 XTEN, an RSR2295 release region, an anti-EGFR-anti-CD3 bispecific tandem scFv, RSR2295, and AE868 XTEN with the C-tag affinity tag.

[0337] To construct pJB0163 and pJB0179, pJB0169 was digested with DraIII and BtsI to remove 5' RSR2295, the anti-EGFR-anti-CD3 bispecific tandem scFv, RSR2295, and the first 72 amino acids of AE868XTEN. For pJB0163, a DNA fragment encoding RSR3058, the anti-CD3 light chain, the anti-EGFR light and heavy chains, the anti-CD3 heavy chain, RSR3058, and the first 72 amino acids of AE868XTEN was synthesized. For pJB0179, a DNA fragment encoding RSR2295, the anti-CD3 light chain, the anti-EGFR light and heavy chains, the anti-CD3 heavy chain, RSR2295, and the first 72 amino acids of AE868XTEN was synthesized. The gene fragments also included 30 nucleotides at the 5' and 3' restriction sites to serve as DNA overlaps for Gibson DNA assembly. The synthesized DNA fragment was cloned into the digested pJB0169 backbone using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to assemble pJB0163 and pJB0179.

[0338] pJB0179 was digested with BsaI and BbvCI to remove the coding sequences for the anti-CD3 and anti-EGFR binding domains. The PCR products encoding the anti-HER2 light and heavy chains were amplified using primers containing an 18 bp 5' homologous region to the N-terminal backbone DNA and a 21 bp 3' homologous region to the second PCR product. Using pJB0205 as a template, a second PCR product encoding the anti-CD3 scFv sequence variant (CD3.23) was amplified using primers containing an 18 bp 5' homologous region to the N-terminal first PCR product and a 23 bp 3' homologous region to the vector backbone. Both PCR products were cloned into the digested backbone using a Gibson cloning kit (SGI-DNA, Carlsbad, CA) to assemble pAH0011.

[0339] pJB0163 was digested with BsaI and BstEII to remove the coding sequences for the anti-CD3 and anti-EGFR binding domains. The PCR products encoding the anti-HER2 light and heavy chains were amplified using primers containing an 18 bp 5' homologous region to the N-terminal backbone DNA and a 21 bp 3' homologous region to the second PCR product. Using pJB0205 as a template, a second PCR product encoding the anti-CD3 scFv sequence variant (CD3.23) was amplified using primers containing an 18 bp 5' homologous region to the N-terminal first PCR product and a 23 bp 3' homologous region to the vector backbone. The two PCR products were cloned into the digested backbone using a Gibson assembly kit (SGI-DNA, Carlsbad, CA) to assemble pAH0013.

[0340] To generate pJB0244 and pJB0245, pAH0011 and pAH0013 were digested with BsaI and BsrDI to remove the coding sequences for the anti-Her2 (Her2.1) light and heavy chains. PCR products encoding the anti-Her2 (Her2.2) light and heavy chains were amplified using primers comprising a 25bp 5' homologous region at the 3' end of the respective vector backbone at the N-terminus and a 25bp 3' homologous region at the 5' end of the vector backbone. The PCR product of pJB0244 was cloned into the digested pAH0011 backbone to assemble pJB0244 using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA). pJB0244 encodes a 6xHIS affinity tag (SEQ ID NO: 1150), AE292 XTEN, RSR2295, an anti-HER2-anti-CD3 bispecific tandem scFv, RSR2295, and AE868 XTEN868 with a C-tag affinity tag, under the control of the PhoA promoter and STII secretion leader sequence. It has the DNA sequence and encoded amino acid sequence provided in Table 9. The PCR product of pJB0245 was cloned into the pAH0013 backbone to generate pJB0245. pJB0245 encodes a 6xHIS affinity tag (SEQ ID NO:1150), AE292 XTEN, RSR3058 release region, anti-HER2-anti-CD3 bispecific tandem scFv, RSR3058, and AE868 XTEN with a C-tag affinity tag under the control of the PhoA promoter and STII secretion leader sequence.

[0341] To introduce novel CD3 scFv variants with altered isoelectric point and removal of potential aggregation sites in the amino acid sequence, pJB0244 was digested with BsaI and BbvCI to remove both HER2 and CD3 scFv. DNA fragments encoding anti-EGFR scFv variants paired with CD3.33 were synthesized, including 40 bp homology at both the 5' and 3' ends with the digested vector to facilitate Gibson DNA assembly. Plasmids pJB0358-pJB0372 were assembled with 6xHIS affinity tags (SEQ ID NO: 1150), AE292 XTEN, RSR2295, and a single 15 anti-EGFR scFv variants paired with anti-CD3 scFv, RSR2295, and AE868 XTEN with a C-tag affinity tag.

[0342] pJB0368 and pJB0373 were initially digested with BtsI to remove anti-CD3 scFv, resulting in pAH0025 and pAH0026. A DNA fragment encoding anti-CD3.32 scFv was ordered, flanked by a 40 bp homologous region identical to the digested backbone. These fragments were introduced into pJB0368 and pJB0373 via Gibson assembly to generate plasmids encoding a 6xHIS affinity tag (SEQ ID NO: 1150), AE292 XTEN, RSR2295, an anti-EGFR-anti-CD3 bispecific tandem scFv, RSR2295, and an AE868 XTEN with a C-tag affinity tag, which was constructed with two different anti-EGFR binding domains, EGFR.23 and EGFR.2, to produce pAH0025 and pAH0026 constructs having the DNA sequences and encoded amino acid sequences provided in Table 9.

[0343] To generate a bispecific antigen-binding polypeptide construct with a shortened C-terminal XTEN, pJB0244 was digested with BtsI and EcoRI to remove the C-terminal XTEN and C-tag. A PCR fragment encoding the AE584XTEN sequence and the C-tag was amplified from pJB0244. A second fragment encoding the vector backbone was synthesized, exhibiting 40 bp homology following the EcoRI site, with a 34-base tail overlapping the first fragment. These two fragments were cloned into the digested pJB0244 backbone using a 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, AE584XTEN, and a C-tag affinity tag. To generate pJB0355, a PCR fragment encoding the AE293XTEN sequence and a C-tag was amplified from pJB0244. This fragment, along with the second fragment described above, was cloned into a digested pJB0244 backbone using a Gibson assembly kit to generate plasmid pJB0355, which encodes a 6xHIS affinity tag (SEQ ID NO: 1150), XTEN292, RSR2295, an anti-Her2-anti-CD3 bispecific tandem scFv, RSR2295, AE300 XTEN, and a C-tag affinity tag (DNA and amino acid sequences are shown in Table 9). Non-cleavable variants of pJB0354 and pJB0355 (pJB0377 and pJB0378, respectively) were also constructed, replacing RSR2295 with the sequence EAGRSANHTPAGLTGP (SEQ ID NO: 88).

[0344] To generate a protein with shortened N-terminal and C-terminal XTENs, three PCR products were amplified. The first PCR product consisted of an N-terminal His tag and AE144_7AXTEN amplified from pCW1199. The second PCR product consisted of an N-terminal release site 2295, an anti-HER2-anti-CD3 bispecific tandem scFv, and 286 amino acids of the C-terminal release site 2295 and XTEN sequence. These two fragments were cloned into a backbone generated by PCR amplification via Gibson assembly to form pJB0380, which included the last 17 XTEN amino acids at its 5' end (including 30 bp homology with the second PCR product and the STII signal peptide, 6xHis tag (SEQ ID NO: 1150)) and 5 XTEN residues at its 3' end (including 39 bp homology with 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, AE293XTEN, and a C-tag affinity tag (DNA and amino acid sequences are in Table 9). An uncleavable variant of pJB0380 (pJB0379) was also constructed, replacing RSR2295 with the sequence EAGRSANHTPAGLTGP (SEQ ID NO: 88). Constructs with CD3.24, CD3.30, CD3.31, CD3.33 scFvs and an antigen-binding fragment for the target cell markers described herein were prepared using the same method in any combination or orientation (i.e., AF1-AF2 or AF2-AF1 with an N-terminal to C-terminal orientation).

[0345] Table 9: DNA and amino acid sequences of the constructs

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579]

[0580]

[0581]

[0582]

[0583]

[0584]

[0585]

[0586]

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640]

[0641]

[0642]

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692]

[0693]

[0694]

[0695]

[0696]

[0697]

[0698]

[0699]

[0700]

[0701]

[0702]

[0703]

[0704]

[0705]

[0706]

[0707]

[0708]

[0709]

[0710]

[0711]

[0712]

[0713] Table 10: Amino acid sequences of the constructs

[0714]

[0715]

[0716]

[0717]

[0718]

[0719]

[0720]

[0721]

[0722]

[0723]

[0724]

[0725]

[0726]

[0727]

[0728]

[0729]

[0730]

[0731]

[0732]

[0733]

[0734]

[0735]

[0736]

[0737]

[0738]

[0739]

[0740]

[0741]

[0742]

[0743]

[0744]

[0745]

[0746]

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753]

[0754]

[0755]

[0756]

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773]

[0774]

[0775]

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787]

[0788]

[0789]

[0790]

[0791]

[0792]

[0793]

[0794]

[0795]

[0796]

[0797]

[0798]

[0799]

[0800]

[0801]

[0802]

[0803]

[0804]

[0805]

[0806]

[0807]

[0808]

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817]

[0818]

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825] Example 2: Evaluation of CD3 scFv sequence variants compared to parental CD3 scFv

[0826] The aim of the experiment was to evaluate four CD3 sequence variants to determine whether the variants had enhanced performance compared to the CD3.9 parent scFv.

[0827] 1. Deconvolution temperature (T) m The determination of )

[0828] The melting temperature of each scFv variant was measured to determine its thermal stability. Briefly, a uniform amount of scFv was aliquoted into PCR tubes in 200 μL of 1% BSA-PBST. The tubes were incubated for one hour at several different temperatures (50 °C, 51.4 °C, 53.7 °C, 57.3 °C, 61.7 °C, 65.5 °C, and 68 °C). 50 μL of each sample was added to a PCR tube coated with... ELISA plates containing the target antigen (Creative Biomart) or BSA (reference for resolving viscosity). The wells of the ELISA plates are pre-filled with 1% BSA-PBST (50 μl / well). Incubate the plates at room temperature for 1 hour. Wash the plates three times with water containing 0.05% Tween to remove unbound scFv. Detect bound scFv by adding anti-YOL antibody (Thermo Scientific #MA180189) (1:500 dilution in 1% BSA-PBST (0.05%)). This antibody detects the porcine α-tubulin motif at the linker between the heavy and light chains. Incubate the samples at room temperature for 1 hour. Wash the plates three times with water containing 0.05% Tween to remove unbound scFv. Anti-YOL antibodies were detected by adding anti-rat-HRP antibody (Thermo Scientific #31470) (1:7500 diluted in 1% BSA-PBST (0.05%)) (100 μl / well) and incubating at room temperature for 1 hour. The plates were washed three times with water containing 0.05% Tween to remove unbound antibodies. The plates were developed using TMB (3,3',5,5'-tetramethylbenzidine) substrate (100 μl / well, 6 min 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 was plotted for each temperature. The melting temperature was determined as the EC50 for each sample, i.e., the temperature at which scFv binding decreased to 50% of the maximum signal. The results are listed in Table 11.

[0829] Results: The assay results confirmed that CD3 scFv 3.23 and 3.24 had a Tm 5°C higher than the parent CD3.9, while CD3.25 and CD3.26 (sequences shown in Table 12) scFv had a Tm 5°C higher than the parent CD3.9. m Comparable to the parent CD3.9.

[0830] 2. Determination of binding affinity with CD3

[0831] Binding affinity of each scFv was measured using a ForteBio BLItz instrument. Dilution series of each scFv were prepared in PBS (300 μL / tube), with 1:1 dilution steps from 1000 nM to 62.5 nM for CD3.24–26, and 1:1 dilution steps from 400 nM to 25 nM for CD3.23. Biotinylated scFvs were then... The antigen (CreativeBiomart) was diluted to a final concentration of 30 μg / ml in PBS. The streptoantibiotic biosensor (ForteBio) was activated in PBS for 10 minutes. For measurement, the streptoantibiotic biosensor was applied to the BLItz instrument. A tube containing 300 μL of PBS was transferred to the BLItz instrument for 30 seconds. The biotinylated... Tubes containing 30 μg / ml, 300 μL / tube were transferred to the BLItz instrument to measure antigen capture by the sensor for 120 seconds. Tubes containing 300 μL PBS were transferred to the BLItz instrument for 30 seconds to measure the baseline signal. Tubes containing the test scFv (30 μg / ml, 300 μL / tube) were transferred to the BLItz instrument to measure scFv binding to the antigen-loaded biosensor for 120 seconds. Tubes containing 300 μL PBS were transferred to the BLItz instrument for 120 seconds to measure scFv dissociation from the antigen-loaded biosensor. This protocol was repeated for each scFv dilution. The KD of each antibody was determined using BLI software (ForteBio). The results are shown in Table 11, which shows the melting temperature and binding affinity of the CD3-binding variants, demonstrating that variants such as CD3.23 have reduced binding affinity to CD3.

[0832] The binding affinity of the bivalent anti-HER2, anti-CD3 XTENation binding agent AC2275 (see Example 24) to the targets (HER2 and CD3) was measured using a ForteBio Octet Red instrument. This assay was performed in PBSTB buffer (10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride, 0.5% BSA, 0.005% Tween-20). To bind human HER2 or cynomolgus monkey HERa2, a series of dilutions of each analyte was prepared in PBSTB buffer (500 μL / tube) in a 1:1 dilution series from 64 nM to 1 nM. To bind human CD3 or cynomolgus monkey CD3, a series of dilutions of each analyte was prepared in PBSTB buffer (500 μL / tube) in a 1:1 dilution series from 1010 nM to 16 nM. The targets were diluted in PBSTB to a final concentration of 33 μg / ml. The anti-human Fc biosensor (ForteBio) was activated in PBSTB buffer for 10 minutes. For measurement, a set of anti-human Fc biosensors was placed on a sensor holder and transferred to an Octet Red instrument. A 96-well non-binding opaque plate containing 200 μL PBSTB buffer, glycine buffer, target, and analyte was transferred to the Octet Red instrument. The biosensor was transferred to PBSTB buffer for 600 seconds for equilibration. For activation, the biosensor was transferred to 10 mM glycine buffer, pH 1.5 for 10 seconds, followed by a transfer to PBSTB buffer for 10 seconds. This activation step was repeated two more times. The biosensor was transferred to the target well for 100 seconds for loading. The biosensor was transferred to PBSTB...

Claims

1. An antigen-binding fragment that specifically binds to CD3, wherein the antigen-binding fragment comprises a light chain variable region (VL) containing light chain complementarity-determining regions (CDR-L) CDR-L1, CDR-L2, and CDR-L3 and a heavy chain variable region (VH) containing heavy chain complementarity-determining regions (CDR-H) CDR-H1, CDR-H2, and CDR-H3, wherein: The CDR-H1 consists of the amino acid sequence of SEQ ID NO: 8; The CDR-H2 consists of the amino acid sequence of SEQ ID NO: 9; The CDR-H3 consists of the amino acid sequence of SEQ ID NO: 10; The CDR-L1 consists of the amino acid sequence of SEQ ID NO: 1; The CDR-L2 consists of the amino acid sequence of SEQ ID NO: 4; and The CDR-L3 consists of the amino acid sequence of SEQ ID NO:

6.

2. The antigen-binding fragment according to claim 1, 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.

3. The antigen-binding fragment according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibodies, and single-chain variable fragments (scFv).

4. A pharmaceutical composition comprising an antigen-binding fragment according to any one of claims 1-3 and one or more pharmaceutically suitable excipients.

5. An isolated nucleic acid, said nucleic acid (a) encoding an antigen-binding fragment according to any one of claims 1-3; or (b) being a complement of (a).

6. An expression vector comprising the nucleic acid according to claim 5 and a recombinant regulatory sequence operatively linked to said nucleic acid.

7. An isolated host cell comprising the expression vector according to claim 6.

Images