Compositions targeting epidermal growth factor receptors and methods of making and using same

Abstract

The present invention relates to compositions targeting epidermal growth factor receptors and methods of making and using the same, and particularly provides antibody binding domains for the differentiation cluster 3 T cell receptor (CD3), antibody binding domains for the epidermal growth factor receptor (EGFR), cleavable linker sequences, and protease activatable bispecific fusion proteins, a T cell adaptor, such as a protease, can be activated, as well as uses and methods of treatment.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202480026174.7, filed on April 16, 2024, entitled "Composition Targeting Epidermal Growth Factor Receptor and Preparation and Use Method Thereof".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 459,828, filed April 17, 2023; and U.S. Provisional Patent Application Serial No. 63 / 463,273, filed May 1, 2023; the contents of which are hereby incorporated in their entirety. Technical Field

[0004] This invention relates to compositions targeting epidermal growth factor receptors, methods of their preparation and use, and particularly provides antibody-binding domains for differentiation cluster 3 T cell receptor (CD3), antibody-binding domains for epidermal growth factor receptor (EGFR), cleavable adaptor sequences, and protease-activated bispecific fusion proteins, such as protease-activated T cell adaptors, as well as uses and therapeutic methods. Background Technology

[0005] Epidermal growth factor receptor (EGFR), also known as ErbB1 and HER1, is a receptor tyrosine kinase involved in cell proliferation. Overexpression or aberrant activity of EGFR is associated with many cancers, making it an attractive target for therapeutic interventions. While approved therapies exist, their efficacy may be hampered by toxicity and / or low stability.

[0006] There is a long-standing and unmet need for therapeutic interventions for tumors expressing EGFR, including stable antibody-based therapeutics with improved therapeutic indices. Summary of the Invention

[0007] This disclosure specifically provides antigen-binding molecules with EGFR binding specificity, antigen-binding molecules with CD3 binding specificity, and bispecific antigen-binding molecules that bind both EGFR and CD3, for use in therapeutic settings requiring specific targeting of EGFR-expressing cells and T-cell-mediated killing. The aspects disclosed herein address a long-standing unmet need for EGFR-targeting cancer therapeutics, including T-cell adaptors (TCEs) with an increased therapeutic index. The aspects of this disclosure also address a long-standing unmet need for therapeutic interventions for immune-cold tumors expressing EGFR (e.g., solid tumors).

[0008] In one aspect, this disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain that specifically binds to the epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to the cluster 3 T cell receptor (CD3), wherein the first antigen-binding domain comprises: a VH domain comprising the CDR1 amino acid sequence of GGSVSSGDYYWT (SEQ ID NO: 562), the CDR2 amino acid sequence of HIYYSGNTNYNPSLKS (SEQ ID NO: 563), and the CDR3 amino acid sequence of DRVTGAFDI (SEQ ID NO: 564); and at least one of the following: a proline (P) residue at position 40 of FR2 (alternatively referred to as amino acid residue 42 relative to SEQ ID NO: 450), a valine (V) residue at position 67 of FR3 (alternatively referred to as amino acid residue 69 relative to SEQ ID NO: 450), and a valine (V) residue at position 71 of FR3 (alternatively referred to as amino acid residue 69 relative to SEQ ID NO: 450). NO:450, alternatively referred to as amino acid residue 73), asparagine (N) residue at position 76 of FR3 (alternatively referred to as amino acid residue 78 relative to SEQ ID NO:450), valine (V) residue at position 89 of FR3 (alternatively referred to as amino acid residue 94 relative to SEQ ID NO:450), alanine (A) residue at position 93 of FR3 (alternatively referred to as amino acid residue 98 relative to SEQ ID NO:450), and / or leucine (L) residue at position 108 of FR4 (alternatively referred to as amino acid residue 114 relative to SEQ ID NO:450), wherein the FR number is based on Kabat; and the VL domain comprising the CDR1 amino acid sequence of QASQDISNYLN (SEQ ID NO: 565), the CDR2 amino acid sequence of DASNLET (SEQ ID NO: 566), and the CDR3 amino acid sequence of QHFDHLPLA (SEQ ID NO: 566). 567); and wherein the chimeric polypeptide further comprises a masking polypeptide that binds to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the masking polypeptide and the bispecific antibody domain, such that the masking polypeptide can reduce the binding of the bispecific antibody domain to CD3 or EGFR, and wherein the protease-cleavable release segment can be cleaved by at least one protease present in the tumor.

[0009] In some embodiments, the VH domain comprises an asparagine (N) residue at position 76 of FR3. In some embodiments, the VH domain comprises an alanine (A) residue at position 93 of FR3. In some embodiments, the VH domain comprises a valine (V) residue at position 67 of FR3, a valine (V) residue at position 71 of FR3, an asparagine (N) residue at position 76 of FR3, a valine (V) residue at position 89 of FR3, and an alanine (A) residue at position 93 of FR3. In some embodiments, the VH domain comprises a proline (P) residue at position 40 of FR2, a valine (V) residue at position 67 of FR3, a valine (V) residue at position 71 of FR3, an asparagine (N) residue at position 76 of FR3, a valine (V) residue at position 89 of FR3, an alanine (A) residue at position 93 of FR3, and a leucine (L) residue at position 108 of FR4.

[0010] In some embodiments, the VL domain comprises at least one of the following: a tyrosine (Y) residue at position 87 of FR3 (represented by amino acid residue 87 relative to SEQ ID NO: 451) and / or a glutamine (Q) residue at position 100 of FR4 (represented by amino acid residue 100 relative to SEQ ID NO: 451), wherein the FR number is based on Kabat. In some embodiments, the VL domain comprises a tyrosine (Y) residue at position 87 of FR3 and a glutamine (Q) residue at position 100 of FR4.

[0011] In some embodiments, the VH domain comprises the amino acid sequence QVQLQX1X2GX3GLX4KPSETLSLTCX5VX6GGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS, where X1 corresponds to E or Q; X2 corresponds to S or W; X3 corresponds to P or A; X4 corresponds to V or L; X5 corresponds to T or A; and X6 corresponds to S or Y (SEQ ID NO: 576); and the VL domain comprises X1IX2X3TQSPX4X5LSX6SX7GX8RX9TX 10 X 11 CQASQDISNYLNWYQQKPGX 12 APX 13 LLIYDASNLETGX 14 PX 15 RFSGSGSGTDFTX16TISX17 LX 18 PEDX 19 AX 20 The amino acid sequence of YYCQHFDHLPLAFGQGTKVEIK, where X1 corresponds to D or E; X2 corresponds to Q or V; X3 corresponds to M or L; X4 corresponds to S, G, or A; X5 corresponds to S or T; X6 corresponds to L or A; X7 corresponds to P or V; X8 corresponds to D or E; X9 corresponds to V or A; X 10 Corresponding to I or L; X 11 Corresponding to T or S; X 12 Corresponding to K or Q; X 13 Corresponding to K or R; X 14 Corresponding to V or I; X 15 Corresponding to S, D, or A; X 16 Corresponding to F or L; X 17 Corresponding to S or R; X 18 Corresponding to Q or E; X 19 Corresponding to I or F; and X 20 Corresponding to T or V (SEQ ID NO: 577).

[0012] In one aspect, this disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain that specifically binds to the epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to the cluster 3 T cell receptor (CD3), wherein the chimeric polypeptide further comprises a masking polypeptide that binds to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the masking polypeptide and the bispecific antibody domain, such that the masking polypeptide can reduce the binding of the bispecific antibody domain to CD3 or EGFR, wherein the protease-cleavable release segment cannot be cleaved by podin in human plasma, or wherein the rate at which podin cleaves the protease-cleavable release segment in human plasma is less than about 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by podin.

[0013] In one aspect, this disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain that specifically binds to the epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to the cluster 3 T cell receptor (CD3), wherein the chimeric polypeptide has a melting temperature (Tm) greater than 62°C and / or a thermostability ratio greater than 0.5 at 62°C; wherein the chimeric polypeptide further comprises a masking polypeptide that binds to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the masking polypeptide and the bispecific antibody domain, such that the masking polypeptide can reduce the binding of the bispecific antibody domain to CD3 or EGFR, and wherein the protease-cleavable release segment can be cleaved by at least one protease present in a tumor.

[0014] In some implementations, Tm is determined by differential scanning fluorescence (DSF).

[0015] In some implementations, the thermal stability ratio is determined by: i) incubating the input amount of chimeric peptide at 62°C for 30 minutes to denature a portion of the input amount of chimeric peptide; ii) measuring the amount of monomeric chimeric peptide remaining after step i); and iii) dividing the amount of monomeric chimeric peptide by the input amount of chimeric peptide to produce the thermal stability ratio.

[0016] In some implementations, the amount of monomeric chimeric peptide is measured by mass spectrometry.

[0017] In one aspect, this disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain that specifically binds to cancer cell antigens and a second antigen-binding domain that binds to the cluster 3 T cell receptor (CD3), wherein the second antigen-binding domain comprises: a VH domain comprising the CDR1 amino acid sequence of GFTFSTYAMN (SEQ ID NO: 12), the CDR2 amino acid sequence of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and the CDR3 amino acid sequence of HENFGNSYVSWFAH (SEQ ID NO: 10); and a VL domain comprising the CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO: 1), the CDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 4), and the CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 10). 6) wherein the chimeric polypeptide further comprises a masking polypeptide that binds to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the masking polypeptide and the bispecific antibody domain, such that the masking polypeptide can reduce the binding of the bispecific antibody domain to CD3 or the cancer cell antigen, and wherein the protease-cleavable release segment can be cleaved by at least one protease present in the tumor.

[0018] In some embodiments, the second antigen-binding domain comprises: (i) a VL domain comprising the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127); and (ii) a VH domain comprising the amino acid sequence of EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126).

[0019] In some implementations, the cancer cell antigen is human α4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, EGFR, HER2, HER3, HER4, PD-L1, prostate-specific membrane antigen (PSMA), CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, or MUC16. βhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3; 9-O-acetyl-GD3, GM2, GloboH, fucose GM1, GD2, carbonic anhydrase IX, CD44v6, sound hedgehog factor (Shh), Wue-1, plasma cell antigen 1, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, prostate 6-transmembrane epithelial antigen (STEAP), mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6 Desmosome core protein 4, fetal acetylcholine receptor (fnAChR), CD25, cancer antigen 19-9 (CA19-9), cancer antigen 125 (CA-125), Müellerian inhibitory substance receptor type II (MISIIR), sialylated Tn antigen (sTN), fibroblast activation antigen (FAP), endothelial sialic acid protein (CD248), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMP, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1 or EphA2.

[0020] In some implementations, the cancer cell antigen is EGFR.

[0021] In some embodiments, the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side, the structural arrangement being defined as: (first antigen-binding domain)-(second antigen-binding domain)-(linker)-(masking polypeptide), (second antigen-binding domain)-(first antigen-binding domain)-(linker)-(masking polypeptide), (masking polypeptide)-(linker)-(first antigen-binding domain)-(second antigen-binding domain) or (masking polypeptide)-(linker)-(second antigen-binding domain)-(first antigen-binding domain), wherein each - is a covalently linked or polypeptide linker.

[0022] In some implementations, the masking peptide is an elongated non-natural peptide (ELNN).

[0023] In some implementations, the connector further includes spacers.

[0024] In some implementations, the protease-cleavable release segment is fused to a bispecific antibody domain via a spacer.

[0025] In some embodiments, the spacer is characterized in that: (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof; and (ii) it contains at least three types of amino acids selected from the group consisting of G, A, S, T, E, and P.

[0026] In some implementations, the spacer length is 9 to 14 amino acids.

[0027] In some implementations, the spacer contains at least four types of amino acids selected from the group consisting of G, A, S, T, E, and P.

[0028] In some implementations, the amino acids in the spacer are composed of A, E, G, S, P and / or T.

[0029] In some implementations, the spacer can be cleaved by non-mammal proteases.

[0030] In some implementations, the non-mammalian protease is Glu-C.

[0031] In some embodiments, the spacer contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the sequences listed in Table C.

[0032] In some embodiments, the spacer contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with GTSESATPES or GTATPESGPG.

[0033] In some embodiments, the protease-cleavable release segment comprises an amino acid sequence containing the sequence EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is any amino acid other than N. In some embodiments, X is S.

[0034] In some embodiments, the chimeric polypeptide includes a first masking polypeptide that binds to a first antigen-binding domain via a first linker, wherein the first linker includes a first protease-cleavable release segment (RS1) capable of being cleaved by at least one protease present in the tumor; and a second masking polypeptide that binds to a second antigen-binding domain via a second linker, wherein the second linker includes a second protease-cleavable release segment (RS2) capable of being cleaved by at least one protease present in the tumor.

[0035] In some embodiments, the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side, defined as: (mask 1)-(linker 1)-(first antigen-binding domain)-(second antigen-binding domain)-(linker 2)-(mask 2), (mask 1)-(linker 1)-(second antigen-binding domain)-(first antigen-binding domain)-(linker 2)-(mask 2), (mask 2)-(linker 2)-(first antigen-binding domain)-(second antigen-binding domain)-(linker 1)-(mask 1), or (mask 2)-(linker 2)-(second antigen-binding domain)-(first antigen-binding domain)-(linker 1)-(mask 1), wherein each – is individually a covalent bond or polypeptide linker.

[0036] In some implementations, the first masking peptide is a first ELNN (ELNN1), and the second masking peptide is a second ELNN (ELNN2).

[0037] In some embodiments, the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side, defined as: (ELNN1)-(linker 1)-(first antigen-binding domain)-(second antigen-binding domain)-(linker 2)-(ELNN2), (ELNN1)-(linker 1)-(second antigen-binding domain)-(first antigen-binding domain)-(linker 2)-(ELNN2), (ELNN2)-(linker 2)-(first antigen-binding domain)-(second antigen-binding domain)-(linker 1)-(ELNN1), or (ELNN2)-(linker 2)-(second antigen-binding domain)-(first antigen-binding domain)-(linker 1)-(ELNN1), wherein each – is individually a covalent bond or polypeptide linker.

[0038] In some implementations, connector 1 further includes a first spacer (spacer 1).

[0039] In some implementations, the connector 2 further includes a second spacer (spacer 2).

[0040] In some implementations, RS1 is fused to the bispecific antibody domain via spacer 1 and / or RS2 is fused to the bispecific antibody domain via spacer 2.

[0041] In some embodiments, the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side, defined as: (ELNN1)-(RS1)-(spacer 1)-(first antigen-binding domain)-(second antigen-binding domain)-(spacer 2)-(RS2)-(ELNN2), (ELNN1)-(RS1)-(spacer 1)-(second antigen-binding domain)-(first antigen-binding domain)-(spacer 2)-(RS2)-(ELNN2), (ELNN2)-(RS2)-(spacer 2)-(first antigen-binding domain)-(second antigen-binding domain)-(spacer 1)-(RS1)-(ELNN1), or (ELNN2)-(RS2)-(spacer 2)-(second antigen-binding domain)-(first antigen-binding domain)-(spacer 1)-(RS1)-(ELNN1), wherein each - is individually a covalent bond or polypeptide linker.

[0042] In some embodiments, spacer 1 and / or spacer 2 are characterized in that: (i) at least 90% of their amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof; and (ii) they contain at least three types of amino acids selected from the group consisting of G, A, S, T, E, and P.

[0043] In some implementations, spacer 1 and / or spacer 2 are 9 to 14 amino acids in length.

[0044] In some embodiments, spacer 1 and / or spacer 2 contain at least four types of amino acids selected from the group consisting of G, A, S, T, E and P.

[0045] In some embodiments, the amino acids of spacer 1 and / or spacer 2 are composed of A, E, G, S, P and / or T.

[0046] In some embodiments, spacer 1 and / or spacer 2 contain amino acid sequences that have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the sequences listed in Table C.

[0047] In some embodiments, spacer 1 and / or spacer 2 contain an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with GTSESATPES or GTATPESGPG.

[0048] In some embodiments, the amino acid sequence of the first ELNN is between 250 and 350 amino acids in length, and the amino acid sequence of the second ELNN is between 500 and 600 amino acids in length.

[0049] In some implementations, the amino acid sequence of the first ELNN is 294 amino acids long, and the amino acid sequence of the second ELNN is 582 amino acids long.

[0050] In some embodiments, RS1 and / or RS2 comprise an amino acid sequence containing the sequence EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is any amino acid other than N. In some embodiments, X is S.

[0051] In one aspect, this disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain having binding specificity to a cancer cell antigen and a second antigen-binding domain having binding specificity to an effector cell antigen expressed on an effector cell, wherein the chimeric polypeptide further comprises a first ELNN that binds to the first antigen-binding domain via a first linker, the first linker comprising a first protease-cleavable release segment (RS1) located between the first ELNN and the first antigen-binding domain, such that the first ELNN can reduce the binding of the first antigen-binding domain to the cancer cell antigen. The binding of the first antigen-binding domain to the second antigen-binding domain via a second linker, wherein the RS1 is cleavable by at least one protease present in the tumor, wherein the chimeric polypeptide further comprises a second ELNN that binds to the second antigen-binding domain via a second linker, the second linker comprising a second protease-cleavable release segment (RS2) located between the second ELNN and the second antigen-binding domain, such that the second ELNN reduces the binding of the first antigen-binding domain to the effector cell antigen, wherein the RS2 is cleavable by at least one protease present in the tumor, wherein the first ELNN has a shorter amino acid sequence than the second ELNN, and wherein the cancer cell antigen is EGFR.

[0052] In some embodiments, the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side, defined as: (ELNN1)-(linker 1)-(first antigen-binding domain)-(second antigen-binding domain)-(linker 2)-(ELNN2), (ELNN1)-(linker 1)-(second antigen-binding domain)-(first antigen-binding domain)-(linker 2)-(ELNN2), (ELNN2)-(linker 2)-(first antigen-binding domain)-(second antigen-binding domain)-(linker 1)-(ELNN1), or (ELNN2)-(linker 2)-(second antigen-binding domain)-(first antigen-binding domain)-(linker 1)-(ELNN1), wherein each – is individually a covalent bond or polypeptide linker.

[0053] In some implementations, connector 1 further includes a first spacer (spacer 1).

[0054] In some implementations, the connector 2 further includes a second spacer (spacer 2).

[0055] In some implementations, RS1 is fused to the bispecific antibody domain via spacer 1 and / or RS2 is fused to the bispecific antibody domain via spacer 2.

[0056] In some embodiments, the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side, the structural arrangement being defined as: (ELNN1)-(RS1)-(spacer 1)-(first antigen-binding domain)-(second antigen-binding domain)-(spacer 2)-(RS2)-(ELNN2), (ELNN1)-(RS1)-(spacer 1)-(second antigen-binding domain)-(first antigen-binding domain)-(spacer 2)-(RS2)-(ELNN2), (ELNN2)-(RS2)-(spacer 2)-(first antigen-binding domain)-(second antigen-binding domain)-(spacer 1)-(RS1)-(ELNN1), or (ELNN2)-(RS2)-(spacer 2)-(second antigen-binding domain)-(first antigen-binding domain)-(spacer 1)-(RS1)-(ELNN1), wherein each - is individually a covalent bond or polypeptide linker.

[0057] In one aspect, this disclosure provides a chimeric polypeptide comprising a bispecific antibody domain, the chimeric polypeptide comprising the following formulas from the N-terminal side to the C-terminal side: Formula 1: (mask 1)-(RS1)-(spacer 1)-(first antigen-binding domain)-[antibody domain linker]-(second antigen-binding domain); Formula 2: (first antigen-binding domain)-[antibody domain linker]-(second antigen-binding domain)-(spacer 2)-(RS2)-(mask 2); Formula 3: (mask 1)-(RS1)-(spacer 1)-(first antigen-binding domain)-[antibody domain linker]-(second antigen-binding domain)-(spacer 2)-(RS2)-(mask 2), wherein the first antigen-binding domain is effective against cancer cells. The cell antigen has binding specificity; the second antigen-binding domain has binding specificity for effector cell antigens expressed on effector cells; each individually contains a covalently linked or polypeptide linker; the masking agent 1 is a polypeptide capable of reducing the binding of the first antigen-binding domain to its target; the masking agent 2 is a polypeptide capable of reducing the binding of the second antigen-binding domain to its target; if the chimeric polypeptide comprises formula 1, then the spacer 1 consists of A, E, G, S, P and / or T residues; if the chimeric polypeptide comprises formula 2, then the spacer 2 consists of A, E, G, S, P and / or T residues; and if the chimeric polypeptide comprises formula 3, then the spacer 1 and / or the spacer 2 consists of A, E, G, S, P and / or T residues; and wherein the cancer cell antigen is EGFR.

[0058] In some embodiments, each - is individually covalently linked. In some embodiments, each - is individually a covalent bond. In some embodiments, each - is a peptide bond. In some embodiments, each - is individually a polypeptide linker of no more than 5 amino acids.

[0059] In some embodiments, the second antigen-binding domain is specific for binding to human CD3 and cynomolgus monkey CD3. In some embodiments, the second antigen-binding domain is specific for binding to human CD3. In some embodiments, the effector cell antigen is the cluster 3 T cell receptor (CD3). In some embodiments, CD3 is CD3ε, CD3δ, CD3γ, or CD3ζ. In some embodiments, CD3 is CD3ε.

[0060] In some implementations, mask 1 is a first ELNN and mask 2 is a second ELNN.

[0061] In some embodiments, the spacer 1 and / or the spacer 2 are characterized in that: (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof; and (ii) it contains at least three types of amino acids selected from the group consisting of G, A, S, T, E, and P.

[0062] In some embodiments, the spacer 1 and / or the spacer 2 has a length of 9 to 14 amino acids.

[0063] In some embodiments, spacer 1 and / or spacer 2 contain at least four types of amino acids selected from the group consisting of G, A, S, T, E, and P.

[0064] In some embodiments, the amino acids of spacer 1 and / or spacer 2 are composed of A, E, G, S, P and / or T.

[0065] In some implementations, spacer 1 and / or spacer 2 can be cleaved by non-mammal proteases.

[0066] The chimeric polypeptide according to claim 71, wherein the non-mammalian protease is Glu-C.

[0067] In some embodiments, spacer 1 and / or spacer 2 contain amino acid sequences that have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the sequences listed in Table C.

[0068] In some embodiments, spacer 1 and / or spacer 2 contain an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with GTSESATPES or GTATPESGPG.

[0069] In some implementations, the amino acid sequence of the first ELNN is at least 100 amino acids shorter than that of the second ELNN.

[0070] In some implementations, the amino acid sequence of the first ELNN is at least 200 amino acids shorter than that of the second ELNN.

[0071] In some implementations, the amino acid sequence of the first ELNN is at least 250 amino acids shorter than that of the second ELNN.

[0072] In some embodiments, the amino acid sequence of the first ELNN is between 250 and 350 amino acids in length, and the amino acid sequence of the second ELNN is between 500 and 600 amino acids in length.

[0073] In some implementations, the amino acid sequence of the first ELNN is 294 amino acids long, and the amino acid sequence of the second ELNN is 582 amino acids long.

[0074] In some embodiments, the first antigen-binding domain comprises a first antibody or an antigen-binding fragment thereof, and the second antigen-binding domain comprises a second antibody or an antigen-binding fragment thereof.

[0075] In some implementations, the first antigen-binding domain is Fab, scFv, or ISVD.

[0076] In some implementations, the second antigen-binding domain is Fab, scFV, or ISVD.

[0077] In some implementations, ISVD is a VHH domain.

[0078] In some implementations, the first antigen-binding domain is scFV.

[0079] In some implementations, the second antigen-binding domain is scFV.

[0080] In some implementations, an antibody domain linker exists between the first antigen-binding domain and the second antigen-binding domain.

[0081] In some implementations, the antibody domain adapter comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the sequences listed in Table A or Table B.

[0082] In some implementations, the antibody domain linker consists of G amino residues and S amino residues.

[0083] In some implementations, the length of the antibody domain linker is 6 to 12 residues.

[0084] In some implementations, the antibody domain linker contains the amino acid sequence GGGGS or GGGGSGGGS.

[0085] In some embodiments, the first antigen-binding domain and / or the second antigen-binding domain includes an scFv, which includes a VL domain, a VH domain, and a linker between the VL domain and the VH domain, wherein the linker is composed of A, E, G, S, P, and / or T residues.

[0086] In some embodiments, the linker is characterized in that: (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof; and (ii) it contains at least three types of amino acids selected from the group consisting of G, A, S, T, E, and P.

[0087] In some implementations, the linker between the VL domain and the VH domain is 25 to 35 amino acids in length.

[0088] In some implementations, the linker between the VL domain and the VH domain contains at least four types of amino acids selected from the group consisting of G, A, S, T, E, and P.

[0089] In some implementations, the amino acid linker between the VL and VH domains consists of A, E, G, S, P, and / or T.

[0090] In some implementations, the linker between the VL and VH domains can be cleaved by non-mammal proteases.

[0091] In some implementations, the non-mammalian protease is Glu-C.

[0092] In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0093] In some embodiments, the second antigen-binding domain comprises the following CDRs: a VL domain CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with RSSX1GAVTX2SNYAN, wherein X1 corresponds to T or N, and X2 corresponds to T or S; and a VL domain CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with GTNKRAP. Identity; VL domain CDR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with ALWYX4NLWV, where X4 corresponds to S or P; VH domain CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with GFTFX8TYAMN, where X8 corresponds to S or N; VH domain CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with RIRX 10 KX 11 NX 12 YATYYADSVKX 13 Having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or 100% identity, where X 10 Corresponding to T or S, X 11 Corresponding to R or Y, X 12 Corresponding to D or N, and X 13 Corresponding to G or D; VH domain CDR3, its amino acid sequence is the same as HX. 14 NFGNSYVSWFAX 15 Having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or 100% identity, where X 14 Corresponding to E or G, and X 15 Corresponding to H or Y.

[0094] In some embodiments, the second antigen-binding domain comprises: a VH domain comprising the CDR1 amino acid sequence of GFTFSTYAMN (SEQ ID NO: 12), the CDR2 amino acid sequence of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and the CDR3 amino acid sequence of HENFGNSYVSWFAH (SEQ ID NO: 10); and a VL domain comprising the CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO: 1), the CDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 4), and the CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 6).

[0095] In some embodiments, the second antigen-binding domain comprises: a VH domain containing the amino acid sequence of EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126); and a VL domain containing the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127).

[0096] In some embodiments, the first antigen-binding domain comprises the following CDRs: VL domain CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with QASQDISNYLN; VL domain CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with DASNLET; and VL domain CDR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with QHFDHLPLA. 00% identity; VH domain CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with GGSVSSGDYYWT; VH domain CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with HIYYSGNTNYNPSLKS; and VH domain CDR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with DRVTGAFDI.

[0097] In some embodiments, the VH domain comprises at least one of the following: a proline (P) residue at position 40 of FR2, a valine (V) residue at position 67 of FR3, a valine (V) residue at position 71 of FR3, an asparagine (N) residue at position 76 of FR3, a valine (V) residue at position 89 of FR3, an alanine (A) residue at position 93 of FR3, and / or a leucine (L) residue at position 108 of FR4, wherein the FR numbers are based on Kabat. In some embodiments, the VH domain comprises an asparagine (N) residue at position 76 of FR3. In some embodiments, the VH domain comprises an alanine (A) residue at position 93 of FR3. In some embodiments, the VH domain comprises a valine (V) residue at position 67 of FR3, a valine (V) residue at position 71 of FR3, an asparagine (N) residue at position 76 of FR3, a valine (V) residue at position 89 of FR3, and an alanine (A) residue at position 93 of FR3. In some embodiments, the VH domain comprises a proline (P) residue at position 40 of FR2, a valine (V) residue at position 67 of FR3, a valine (V) residue at position 71 of FR3, an asparagine (N) residue at position 76 of FR3, a valine (V) residue at position 89 of FR3, an alanine (A) residue at position 93 of FR3, and a leucine (L) residue at position 108 of FR4.

[0098] In some embodiments, the VL domain comprises at least one of the following: a tyrosine (Y) residue at position 87 of FR3 and / or a glutamine (Q) residue at position 100 of FR4, wherein the FR numbering is based on Kabat. In some embodiments, the VL domain comprises a tyrosine (Y) residue at position 87 of FR3 and a glutamine (Q) residue at position 100 of FR4.

[0099] In some embodiments, the first antigen-binding domain comprises a VH domain containing the amino acid sequence of SEQ ID NO: 576 and a VL domain containing the amino acid sequence of SEQ ID NO: 577.

[0100] In some embodiments, the first antigen-binding domain comprises: i) a VH domain comprising the amino acid sequence of SEQ ID NO: 468 and a VL domain comprising the amino acid sequence of SEQ ID NO: 469; ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 466 and a VL domain comprising the amino acid sequence of SEQ ID NO: 467; iii) a VH domain comprising the amino acid sequence of SEQ ID NO: 490 and a VL domain comprising the amino acid sequence of SEQ ID NO: 491; iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 492 and a VL domain comprising the amino acid sequence of SEQ ID NO: 493; v) a VH domain comprising the amino acid sequence of SEQ ID NO: 514 and a VL domain comprising the amino acid sequence of SEQ ID NO: 515; vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 516 and a VL domain comprising the amino acid sequence of SEQ ID NO: 517; vii) a VH domain comprising the amino acid sequence of SEQ ID NO: 468 and a VL domain comprising the amino acid sequence of SEQ ID NO: 469; The VH domain of the amino acid sequence 538 and the VL domain of the amino acid sequence containing SEQ ID NO: 539; or viii) the VH domain of the amino acid sequence containing SEQ ID NO: 540 and the VL domain of the amino acid sequence containing SEQ ID NO: 541.

[0101] In some implementations, the VL domain is at the N-terminus of the VH domain. In some implementations, the VL domain is at the C-terminus of the VH domain.

[0102] In some embodiments, the second antigen-binding domain comprises an scFV containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the following: (SEQ ID NO: 128).

[0103] In some embodiments, the first antigen-binding domain comprises an scFV containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the following: (SEQ ID NO: 449).

[0104] In some embodiments, RS includes a protease cleavage site that can be cleaved by at least one of the proteases listed in Table 6.

[0105] In some embodiments, RS comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the sequences listed in Table 7a.

[0106] In some implementations, RS can be cut by uPA, ST14, MMP2, MMP7, MMP9 and MMP14.

[0107] In some implementations, RS cannot be cleaved by pod protein.

[0108] In some implementations, RS cannot be cleaved by pod proteins in human blood, plasma, or serum.

[0109] In some implementations, the RS is not cut after incubating with about 1 nM or less of pod protein for about 20 hours.

[0110] In some implementations, the RS is inedible after being incubated with about 1 nM or less of pod protein in human blood, plasma, or serum for about 20 hours.

[0111] In some implementations, the rate at which pod protein cleaves RS in human plasma is less than about 50% of the rate at which pod protein cleaves RSR-2295 (EAGRSANHTPAGLTGP).

[0112] In some implementations, the rate at which pod protein cleaves RS in human plasma is less than about 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by pod protein.

[0113] In some implementations, the rate at which pod protein cleaves RS in human plasma is less than about 10% of the rate at which pod protein cleaves RSR-2295 (EAGRSANHTPAGLTGP).

[0114] In some implementations, the rate at which pod protein cleaves RS in human plasma is less than about 5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by pod protein.

[0115] In some implementations, the rate at which pod protein cleaves RS in human plasma is less than about 2.5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by pod protein.

[0116] In some embodiments, RS1 and / or RS2 contain a protease that can be cleaved by at least one of the proteases listed in Table 6.

[0117] In some embodiments, RS1 and / or RS2 contain amino acid sequences that have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the sequences listed in Table 7a.

[0118] In some implementations, RS1 and / or RS2 can be cut by uPA, ST14, MMP2, MMP7, MMP9 and MMP14.

[0119] In some implementations, RS1 and / or RS2 cannot be cleaved by pod proteins.

[0120] In some implementations, RS1 and / or RS2 cannot be cleaved by pod proteins in human blood, plasma, or serum.

[0121] In some implementations, RS1 and / or RS2 are not cut after being incubated with about 1 nM or less of pod protein for about 20 hours.

[0122] In some implementations, RS1 and / or RS2 are not cut after being incubated with about 1 nM or less of pod protein in human blood, plasma, or serum for about 20 hours.

[0123] In some implementations, the rate at which pod protein cleaves RS1 and / or RS2 in human plasma is less than about 50% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by pod protein.

[0124] In some implementations, the rate at which pod protein cleaves RS1 and / or RS2 in human plasma is less than about 25% of the rate at which pod protein cleaves RSR-2295 (EAGRSANHTPAGLTGP).

[0125] In some implementations, the rate at which pod protein cleaves RS1 and / or RS2 in human plasma is less than about 10% of the rate at which pod protein cleaves RSR-2295 (EAGRSANHTPAGLTGP).

[0126] In some implementations, the rate at which pod protein cleaves RS1 and / or RS2 in human plasma is less than about 5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by pod protein.

[0127] In some implementations, the rate at which pod protein cleaves RS1 and / or RS2 in human plasma is less than about 2.5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by pod protein.

[0128] In some embodiments, RS1 comprises a protease-cleavable amino acid sequence containing the sequence EAGRSANHTPAGLTGP (SEQ ID NO: 7627), where X is any amino acid other than N.

[0129] In some embodiments, RS2 comprises a protease-cleavable amino acid sequence containing the sequence EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is any amino acid other than N.

[0130] In some embodiments, RS1 and / or RS2 contain a protease-cleavable amino acid sequence containing the sequence EAGRSASHTPAGLTGP (SEQ ID NO:7628).

[0131] In some implementations, RS1 and RS2 are the same.

[0132] In some implementations, RS1 and RS2 are different.

[0133] In some embodiments, the first ELNN and the second ELNN are each characterized individually as follows: (i) at least 90% of the amino acids in each of the first ELNN and the second ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof; and (ii) each contains at least three types of amino acids selected from the group consisting of G, A, S, T, E, and P.

[0134] In some embodiments, the first ELNN and the second ELNN are further characterized individually in that: (i) each contains at least 100 amino acid residues; and (ii) each contains a plurality of non-overlapping sequence motifs, each of the plurality of non-overlapping sequence motifs having a length of 9 to 14 amino acids, wherein the plurality of non-overlapping sequence motifs comprises a set of non-overlapping sequence motifs, wherein each of the set of non-overlapping sequence motifs is repeated at least twice in the ELNN.

[0135] In some implementations, the plurality of non-overlapping sequence motifs includes at least one non-overlapping sequence motif that appears only once within the ELNN.

[0136] In some implementations, the non-overlapping sequence motif comprises one or any combination of the sequence motifs listed in Table 1.

[0137] In some implementations, the non-overlapping sequence motif comprises at least 2, 3, or 4 sequence motifs listed in Table 1.

[0138] In some implementations, the non-overlapping sequence motif comprises any one or any combination of GTSTEPSEGSAP, GTSESATPESGP, GSGPGTSESATP, GSEPATSGSETP, GSPAGSPTSTEE, and GTSPSATPESGP.

[0139] In some implementations, the first ELNN and the second ELNN each contain at least four types of amino acids selected from the group consisting of G, A, S, T, E, and P.

[0140] In some implementations, the amino acids of the first ELNN and the second ELNN are each composed of A, E, G, S, P and / or T.

[0141] In some embodiments, the amino acid sequence of the first ELNN is at least 100 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is at least 200 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is at least 250 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the length of the amino acid sequence of the first ELNN is between 250 and 350 amino acids, and the length of the amino acid sequence of the second ELNN is between 500 and 600 amino acids. In some embodiments, the length of the amino acid sequence of the first ELNN is 294 amino acids, and the length of the amino acid sequence of the second ELNN is 582 amino acids.

[0142] In some implementations, the first ELNN and / or the second ELNN contain at least 85% of the same amino acid sequence as the amino acid sequences listed in Table 3a or Table 3b.

[0143] In some embodiments, the first ELNN comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the following: .

[0144] In some embodiments, the second ELNN comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the following: .

[0145] In some implementations, the chimeric polypeptide comprises one or more barcode fragments.

[0146] In some implementations, the chimeric polypeptide comprises two or more barcode fragments.

[0147] In some implementations, each barcode segment is different from every other barcode segment.

[0148] In some implementations, each barcode fragment is distinct in both sequence and molecular weight from all other peptide fragments that can be released from the chimeric polypeptide after complete digestion by a non-mammalian protease.

[0149] In some implementations, the non-mammalian protease is Glu-C.

[0150] In some embodiments, the chimeric polypeptide contains a Glu-C cleavage site containing one of the following amino acid sequences: ATPESGPG, SGSETPGT, and GTSESATP.

[0151] In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences: or Each "." represents a Glu-C cleavage site, and n is any integer from 0 to 50.

[0152] In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences: or , where each "." is a Glu-C cleavage site, and n is any integer from 0 to 30.

[0153] In some implementations, n is any integer from 1 to 20. In some implementations, n is any integer from 5 to 15. In some implementations, n is any integer from 3 to 7. In some implementations, n is any integer from 5 to 10. In some implementations, n is 9. In some implementations, n is 4.

[0154] In some implementation schemes, X n Is PTGGTSAT, PGSGPGT, PGTTPGTT, PGTPPTST, PGTSPSAT, PGTGSAGT, PGTGGAGT, PGTSPGAT, PGTSGSGT, PGTSSAST, PGTGAGTT, PGTGSTST, GSEPATSG, APGTSTEP, PGTAGSGT, PGTSSGGT, PGTAGPAT, PGTPGTGT, PGTGGPTT, or PGTGSGST.

[0155] In some implementation schemes, X n Is TGTS, SGP, TTPG, TPPT, TSPS, TGSA, TGGA, TSPG, TSGS, TSSA, TGAG, TGST, EPAT, GTST, TAGS, TSSG, TAGP, TPGT, TGGP or TGSG.

[0156] In some implementations, neither the N-terminal nor the C-terminal amino acids of the chimeric polypeptide are included in the barcode fragment.

[0157] In some embodiments, the chimeric polypeptide comprises an ELNN having a non-overlapping sequence motif that appears only once within the ELNN, wherein the ELNN further comprises a barcode fragment containing at least a portion of the non-overlapping sequence motif that appears only once within the ELNN.

[0158] In some embodiments, the chimeric polypeptide comprises a first ELNN having a first barcode fragment and a second ELNN having a second barcode fragment, wherein neither the first nor the second barcode fragment contains a glutamic acid immediately adjacent to another glutamic acid (if present) in the ELNN containing the barcode fragment.

[0159] In some implementations, at least one of the barcode segments contains glutamic acid at its C-terminus.

[0160] In some embodiments, at least one of the barcode fragments has an N-terminal amino acid that is immediately following glutamic acid in the chimeric polypeptide.

[0161] In some implementations, the glutamic acid preceding the N-terminal amino acid in a barcode fragment is not adjacent to another glutamic acid.

[0162] In some implementations, at least one of the barcode segments does not contain a second glutamic acid at a position other than the C-terminus of the barcode segment, unless the second glutamic acid is immediately followed by proline.

[0163] In some embodiments, the chimeric polypeptide comprises a single polypeptide chain, wherein the chimeric polypeptide includes a barcode fragment located within the polypeptide chain at a position 10 to 200 amino acids or 10 to 125 amino acids from the N-terminus or C-terminus of the chimeric polypeptide.

[0164] In some embodiments, the first ELNN is located on the N-terminal side of the bispecific antibody domain, and the first barcode fragment is located within 200, 150, 100, or 50 amino acids at the N-terminus of the chimeric polypeptide.

[0165] In some embodiments, the second ELNN is located on the C-terminal side of the bispecific antibody domain, and wherein the second barcode fragment is located within 200, 150, 100, or 50 amino acids at the C-terminus of the chimeric polypeptide.

[0166] In some embodiments, at least one of the barcode segments has a length of at least 4 amino acids. In some embodiments, at least one of the barcode segments has a length of 4 to 20, 5 to 15, 6 to 12, or 7 to 10 amino acids.

[0167] In some implementations, each masking polypeptide includes a barcode fragment listed in Table 2 or disclosed in Table 3a.

[0168] In some embodiments, the chimeric polypeptide comprises a barcode fragment containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with SGPGSGPGTSE or SGPGTSPSATPE.

[0169] In some embodiments, the chimeric polypeptide comprises a barcode fragment containing at least 95% of the same amino acid sequence as SGPGSGPGTSE and a barcode fragment containing at least 95% of the same amino acid sequence as SGPGTSPSATPE.

[0170] In some implementations, the barcode fragment consists of A, E, G, S, P and / or T residues.

[0171] In some implementations, the barcode fragment is part of the masking peptide.

[0172] In some implementations, the masking peptide is a first ELNN or a second ELNN.

[0173] In one aspect, this disclosure provides a chimeric polypeptide comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the following: (SEQ ID NO: 1000).

[0174] In some embodiments, the chimeric polypeptide comprises the following amino acid sequence: (SEQ ID NO: 1000).

[0175] In one aspect, this disclosure provides a pharmaceutical composition comprising the chimeric polypeptide described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid or frozen form. In some embodiments, the pharmaceutical composition is formulated as a reconstituted lyophilized powder or cake prior to application.

[0176] In one aspect, this disclosure provides an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device includes a syringe.

[0177] In one aspect, this disclosure provides a polynucleotide sequence that encodes the chimeric polypeptide described herein.

[0178] In one aspect, this disclosure provides an expression vector comprising a multinucleotide sequence encoding the chimeric polypeptide described herein.

[0179] In one aspect, this disclosure provides a host cell comprising an expression vector containing a multinucleotide sequence encoding the chimeric polypeptide described herein.

[0180] In one aspect, this disclosure provides a method for generating the chimeric polypeptide described herein. In some embodiments, the method further includes isolating the chimeric polypeptide from a host cell.

[0181] In one aspect, this disclosure provides a method for treating cancer in a subject in need, the method comprising administering to the subject an effective amount of the chimeric polypeptide described herein.

[0182] In some implementations, cancer includes solid tumors.

[0183] In some implementations, cancer is carcinoma, sarcoma, or melanoma.

[0184] In some implementations, cancer cells express EGFR.

[0185] In some implementations, cancer cells overexpress EGFR.

[0186] In some implementations, the cancer comprises cells that express at least 3,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; 100,000; or 200,000 EGFR proteins per cell on average.

[0187] In some implementations, cancer includes cells with one or more oncogenic mutations in the EGFR gene.

[0188] In some implementations, cancer includes cells with EGFR gene amplification.

[0189] In some implementations, the cells contain 2 to 5 times, 2 to 10 times, 2 to 15 times, 2 to 30 times, 2 to 50 times, 3 to 5 times, 3 to 10 times, 3 to 15 times, 3 to 30 times, 3 to 50 times, 5 to 10 times, 5 to 15 times, 5 to 30 times, or 5 to 50 times more EGFR gene copies than non-cancerous cells of the same tissue type.

[0190] In some implementation schemes, the cancer is lung cancer, colorectal cancer, head and neck cancer, breast cancer, pancreatic cancer, brain cancer, liver cancer, kidney cancer, ovarian cancer, prostate cancer, esophageal cancer, cervical cancer, or bladder cancer.

[0191] In some implementations, the cancer is lung cancer.

[0192] In some implementation schemes, lung cancer is non-small cell lung cancer.

[0193] In some implementations, the cancer is colorectal cancer.

[0194] In some implementations, the cancer is squamous cell carcinoma of the head and neck.

[0195] In some implementations, the cancer is breast cancer.

[0196] In some implementations, the cancer is triple-negative breast cancer.

[0197] In some implementations, the cancer is brain cancer.

[0198] In some implementations, brain cancer is glioblastoma.

[0199] In some implementations, the method further includes administering a checkpoint inhibitor to the subject.

[0200] In some implementations, the checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0201] In some implementations, the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0202] In some implementations, the checkpoint inhibitor is pembrolizumab or cimiplimab.

[0203] In one aspect, this disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to EGFR, the antibody or antigen-binding fragment comprising: a VH domain comprising the CDR1 amino acid sequence of GGSVSSGDYYWT (SEQ ID NO: 562), the CDR2 amino acid sequence of HIYYSGNTNYNPSLKS (SEQ ID NO: 563), and the CDR3 amino acid sequence of DRVTGAFDI (SEQ ID NO: 564). 564); and at least one of the following: a proline (P) residue at position 40 of FR2, a valine (V) residue at position 67 of FR3, a valine (V) residue at position 71 of FR3, an asparagine (N) residue at position 76 of FR3, a valine (V) residue at position 89 of FR3, an alanine (A) residue at position 93 of FR3 and / or a leucine (L) residue at position 108 of FR4, wherein the FR numbers are based on Kabat; and a VL domain comprising the CDR1 amino acid sequence of QASQDISNYLN (SEQ ID NO: 565), the CDR2 amino acid sequence of DASNLET (SEQ ID NO: 566), and the CDR3 amino acid sequence of QHFDHLPLA (SEQ ID NO: 567).

[0204] In some embodiments, the VH domain comprises an asparagine (N) residue at position 76 of FR3. In some embodiments, the VH domain comprises an alanine (A) residue at position 93 of FR3. In some embodiments, the VH domain comprises a valine (V) residue at position 67 of FR3, a valine (V) residue at position 71 of FR3, an asparagine (N) residue at position 76 of FR3, a valine (V) residue at position 89 of FR3, and an alanine (A) residue at position 93 of FR3. In some embodiments, the VH domain comprises a proline (P) residue at position 40 of FR2, a valine (V) residue at position 67 of FR3, a valine (V) residue at position 71 of FR3, an asparagine (N) residue at position 76 of FR3, a valine (V) residue at position 89 of FR3, an alanine (A) residue at position 93 of FR3, and a leucine (L) residue at position 108 of FR4.

[0205] In some embodiments, the VL domain comprises at least one of the following: a tyrosine (Y) residue at position 87 of FR3 and / or a glutamine (Q) residue at position 100 of FR4, wherein the FR numbering is based on Kabat. In some embodiments, the VL domain comprises a tyrosine (Y) residue at position 87 of FR3 and a glutamine (Q) residue at position 100 of FR4.

[0206] In some embodiments, the antibody or antigen-binding fragment comprises a VH domain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with SEQ ID NO: 576; and a VL domain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with SEQ ID NO: 577.

[0207] In some embodiments, the antibody comprises: i) a VH domain comprising the amino acid sequence of SEQ ID NO: 468 and a VL domain comprising the amino acid sequence of SEQ ID NO: 469; ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 466 and a VL domain comprising the amino acid sequence of SEQ ID NO: 467; hi) a VH domain comprising the amino acid sequence of SEQ ID NO: 490 and a VL domain comprising the amino acid sequence of SEQ ID NO: 491; iv) a VH domain comprising the amino acid sequence of SEQ ID NO: 492 and a VL domain comprising the amino acid sequence of SEQ ID NO: 493; v) a VH domain comprising the amino acid sequence of SEQ ID NO: 514 and a VL domain comprising the amino acid sequence of SEQ ID NO: 515; vi) a VH domain comprising the amino acid sequence of SEQ ID NO: 516 and a VL domain comprising the amino acid sequence of SEQ ID NO: 517; vii) a VH domain comprising the amino acid sequence of SEQ ID NO: 468 and a VL domain comprising the amino acid sequence of SEQ ID NO: 469; The VH domain of the amino acid sequence 538 and the VL domain containing the amino acid sequence of SEQ ID NO: 539; or viii) the VH domain containing the amino acid sequence of SEQ ID NO: 540 and the VL domain containing the amino acid sequence of SEQ ID NO: 541.

[0208] In one aspect, the present invention provides an anti-CD3 antibody or an antigen-binding fragment thereof, the anti-CD3 antibody or the antigen-binding fragment thereof comprising the following CDRs: a VH domain comprising the CDR1 amino acid sequence of GFTFSTYAMN (SEQ ID NO: 12), the CDR2 amino acid sequence of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and the CDR3 amino acid sequence of HENFGNSYVSWFAH (SEQ ID NO: 10); and a VL domain comprising the CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO: 1), the CDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 4), and the CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 6).

[0209] In some embodiments, the VL domain contains the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127); and the VH domain contains the amino acid sequence of EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126).

[0210] The various features of this disclosure are 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 and accompanying drawings, which illustrate illustrative embodiments utilizing the principles of the invention. Attached Figure Description

[0211] Figure 1 A schematic diagram of an exemplary EGFR-targeted paTCE is depicted.

[0212] Figure 2 A schematic diagram depicting the proposed mechanism of action of the exemplary paTCE of this disclosure is provided.

[0213] Figure 3 Depicting from such Figure 1 The diagram shows the exemplary paTCEs: the fully unmasked paTCE (uTCE) and the single-masked metabolites paTCE (1x-N) and paTCE (1x-C).

[0214] Figure 4 A schematic diagram of antibody framework screening is depicted. To identify anti-EGFR antigen-binding fragments with improved properties, the CDR of the donor anti-EGFR antibody panitumumab was grafted in a combinatorial manner into the framework region from an approved monoclonal antibody therapy. patCE libraries containing anti-EGFR antigen-binding fragments were screened for stability, expression, and binding.

[0215] Figures 5A to 5D Results from screening for the following EGFR-targeting pATCEs were described: thermal stability ( Figure 5A ), combined with affinity ( Figure 5B ) and the thermal stability ratio, which represents the amount of thermally stable monomers remaining at 62°C relative to the amount of protein input. Figure 5C ). Figure 5D A simulated band-like structure of an anti-EGFR antibody fragment associated with EGFR was depicted.

[0216] Figure 6 The PTE score assessment of anti-CD3 antibody fragments using the internal PTE algorithm v22 is described.

[0217] Figure 7A The amino acid sequences of RSR-2295 and RSR-3213 were compared and the proteases capable of cleaving them were described. Figure 7B In vitro protease digestion of paTCE using either RSR-2295 or RSR-3213 was depicted. The RSR-3213 sequence was modified to significantly reduce the cleavage of pod proteins.

[0218] exist Figures 8A to 8D middle, Figure 8A and Figure 8B The relative plasma stability of paTCE using RSR-2295 or RSR-3213, measured on day 0 and day 7, was depicted. Figure 8A In this study, RSR-2295 uses an SCy5.5 fluorophore, while RSR-3213 uses an Scy7.5 fluorophore. Figure 8B In this study, RSR-2295 uses the Scy7.5 fluorophore, while RSR-3213 uses the Scy5.5 fluorophore. Figure 8C The observed in vivo cleavability of tumor homogenates from three different mouse tumor models is depicted. For each group of bars (i.e., %1x-C, %1x-N, %uTCE), each bar from left to right represents B1, B2, B3, B4, A1, A2, A3, A4, 43-1, 43-2, 43-3, and 43-4. B1 to B4 represent four different mice from the first tumor model (NCI-N87). A1 to A4 represent four different mice from the second tumor model (HT-29). 43-1 to 43-4 represent four different mice from the third tumor model (HT-55). Figure 8D The total amount of the three metabolites plus paTCE (paTCE, 1x-N, 1x-C, and uTCE) was depicted when RSR-2295 or RSR-3213 was used.

[0219] Figure 9The relative tumor uptake of paTCE using RSR-2295 or RSR-3213 was depicted. The plasma:tumor ratio was calculated in three different mouse tumor models (four mice per tumor model). For each of the three different tumor models, there was "Mouse 1", for each of the three different tumor models, there was "Mouse 2", for each of the three different tumor models, there was "Mouse 3", and for each of the three different tumor models, there was "Mouse 4".

[0220] Figures 10A to 10C An exemplary donor cell HT-29 ( Figure 10A ), MDA-MB-231 ( Figure 10B ) and A-431 ( Figure 10C ) cytotoxicity curve

[0221] Figure 11A and Figure 11D An in vitro cytokine induction assay was depicted from a representative HT-29 donor. IFNγ (…) was shown. Figure 11A ), TNFα ( Figure 11B ), IL-6 ( Figure 11C ) and IL-10 ( Figure 11D The inducement of ).

[0222] Figure 12 Depicting CD4 + T cells and CD8 + Expression of CD69, CD25 and PD-1 in T cells.

[0223] Figure 13 The in vitro plasma stability of AMX-525 was characterized from samples from healthy human donors, human cancer donors (8 pancreas, 2 head and neck, 4 ovaries), healthy cynomolgus monkeys, healthy mice, and tumor-bearing mice (implanted with HT-29 CDX).

[0224] Figure 14 Tumor growth curves were plotted in mice carrying HT-29 tumors.

[0225] Figure 15 Tumor growth curves were plotted in mice carrying LoVo tumors.

[0226] Figure 16 Depicting CD8 in tumor tissue from a LoVo xenograft mouse model + Immunohistochemical (IHC) images of T cells and corresponding quantification.

[0227] Figure 17 Depicting CD4 in tumor tissue from a LoVo xenograft mouse model +IHC images of T cells and corresponding quantification.

[0228] Figure 18 IHC images and corresponding quantifications of PD-L1 expression in tumor tissues from a LoVo xenograft mouse model were depicted.

[0229] Figure 19 Tumor growth curves were plotted in mice carrying MDA-MB-231 tumors.

[0230] Figure 20 The efficacy of AMX-525 was depicted, as indicated by tumor growth curves in mice carrying SK-OV-3 ovarian tumors. SK-OV-3 tumor cells were subcutaneously inoculated into NSG-MHC I / II DKO mice (day 0), implanted into PBMCs (day 18), and treated with the test product shown on the days indicated by the arrows.

[0231] Figure 21 The efficacy of the combination of AMX-525 and the anti-PD-1 antibody pembrolizumab was depicted, as shown by tumor growth curves in mice carrying SK-OV-3 ovarian tumors. SK-OV-3 tumor cells were subcutaneously inoculated into NSG-MHC I / IIDKO mice (day 0), implanted into PBMCs (day 18), and treated with the test product shown on the days indicated by the arrows. Detailed Implementation

[0232] In cancer treatment, there is a significant unmet need for EGFR-targeted bispecific therapies that are effective against solid tumors, particularly those residing in an immune-cold microenvironment. While TCEs have shown efficacy in inducing remission in some cancers, their extreme potency and on-target detumescent toxicity in healthy tissues have prevented the development of a wide range of therapeutic agents.

[0233] Unbound by any scientific theory, TCEs form a bridge between T cells and tumor cells, activating T cell-mediated tumor cell killing and further initiating a cytokine amplification cascade. This cytokine amplification cascade can promote further killing of tumor cells and potentially provide long-term immunity. T cells activated by TCEs release perforin / granzymes in a manner independent of antigen-MHC recognition. This generates a dual response: direct tumor cell death and amplification of tumor killing by initiating a robust cytokine response from tumor cells. Direct tumor cell death leads to the release of tumor antigens. The cytokine response may include, in particular, increased interferon-γ, which stimulates CD8 T cell activity and antigen presentation by APCs; increased IL2, leading to increased proliferation of activated T cells; and increased CXCL9 and CXCL10 responses, which increase T cell recruitment. The release of tumor antigens and the initiation of the cytokine response together lead to the activation of an endogenous T cell response, potentially causing epitope diffusion to induce long-term immunity.

[0234] One toxicity challenge of TCEs stems from the fact that many tumor targets are also expressed to some extent in healthy tissues, and normal cells can also produce cytokine responses, leading to cytokine release syndrome (CRS). These two robust responses of TCE-activated T cells in healthy tissues often result in an overall lack of acceptable therapeutic index for these agents.

[0235] This disclosure provides protease-activated TCEs (paTCEs) that address unmet needs and are superior to conventional antibody therapeutics or bispecific antibody therapeutics that are active at injection time in one or more aspects, including enhanced terminal half-life, improved stability, targeted delivery, and / or improved treatment ratio, while reducing toxicity to healthy tissues.

[0236] This article includes compounds, compositions, and methods for overcoming the shortcomings of existing TCEs by providing EGFR-targeting paTCEs (hereinafter referred to as EGFR-paTCE and exemplified by AMX-525).

[0237] AMX-525 contains the amino acid sequence shown in SEQ ID NO: 1000. Without being bound by any scientific theory, it should be understood that the paTCE described herein utilizes dysregulated protease activity present in tumor and healthy tissues, thereby enabling an expanded therapeutic index. The core of the paTCE contains antigen-binding domains; one targets CD3, and the other targets EGFR. In exemplary embodiments, the two antigen-binding domains can be two different antibody forms (such as, for example, single-chain antibody fragments (scFv) and VHH), or the same antibody form (such as, for example, scFv). Many different antibody fragments or forms can be used.

[0238] In some embodiments, the EGFR-targeting patCE comprises a first portion as an EGFR-binding scFv and a second portion as a CD3-binding scFv. One or more (e.g., two) unstructured peptide masks are attached to the core. In some embodiments, these unstructured peptide masks spatially reduce target binding to tumor targets and / or CD3 and also prolong the protein half-life. In some embodiments, the unstructured peptide mask is an elongated non-natural peptide (ELNN).

[0239] In some implementations, the properties of ELNNs also minimize the possibility of immunogenicity because their lack of a stable tertiary structure is unfavorable for antibody binding, and the absence of hydrophobic, aromatic, and positively charged residues that act as anchoring residues for peptide MHC II binding reduces the likelihood of T cell epitopes.

[0240] In some embodiments, one or more protease cleavage sites at the base of the ELNN enable proteolytic activation of the paTCE in the tumor microenvironment, thereby releasing a smaller, highly effective TCE capable of redirecting cytotoxic T cells to kill tumor cells expressing the target. In some embodiments, in healthy tissue where protease activity is tightly regulated, the paTCE remains primarily inactive, thus expanding the therapeutic index compared to unmasked TCEs.

[0241] In some implementations, the short half-life of the unmasked TCE form further broadens the therapeutic index, in addition to local activation, while providing T-cell immune enhancement for solid tumor eradication. In some implementations, the release site used in paTCE can be cleaved across a wide range of tumors by proteases that are collectively involved in every cancer marker (growth; survival and death; angiogenesis; invasion and metastasis; inflammation; and immune evasion). Thus, by utilizing enhanced protease activity, the TCE activity of paTCE is localized to the tumor; this enhanced protease activity is upregulated at all stages of cancer and tumor development, but is tightly regulated in healthy tissue.

[0242] the term

[0243] As used herein, unless otherwise stated, the following terms have the meanings that pertain to them.

[0244] As used in this specification and claims, the singular forms “an” and “the” include plural references unless the context clearly specifies otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof, unless the context clearly specifies otherwise.

[0245] Furthermore, the term “and / or” as used herein should be considered as each of two specified features or components being disclosed with or without the other. Therefore, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “B or C”; “A and C”; “A and B”; “B and C”; “A” (alone); “B” (alone); and “C” (alone).

[0246] It should be understood that the term "comprising" is used to describe aspects wherever it is used in this document; other similar aspects described as "consisting of" and / or "substantially consisting of" are also provided.

[0247] Numerical ranges include values ​​within defined ranges. Unless otherwise stated, amino acid sequences are written from left to right, from amino to carboxyl. The headings provided herein are not intended to limit any aspect of this disclosure. Therefore, the terms defined below are to be more fully defined by reference to the entire contents of this specification.

[0248] The term "about" is used herein to mean approximately, roughly, approximately, or within a range of... When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical value. Generally, the term "about" can modify a numerical value to be a variation (higher or lower) above or below the stated value, for example, by 10%. In some embodiments, the term indicates a deviation from the stated numerical value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, "about" indicates a deviation from the stated numerical value of ±10%. In some embodiments, "about" indicates a deviation from the stated numerical value of ±5%. In some embodiments, "about" indicates a deviation from the stated numerical value of ±4%. In some embodiments, "about" indicates a deviation from the stated numerical value of ±3%. In some embodiments, "about" indicates a deviation from the shown value of ±2%. In some embodiments, "about" indicates a deviation from the shown value of ±1%. In some embodiments, "about" indicates a deviation from the shown value of ±0.9%. In some embodiments, "about" indicates a deviation from the shown value of ±0.8%. In some embodiments, "about" indicates a deviation from the shown value of ±0.7%. In some embodiments, "about" indicates a deviation from the shown value of ±0.6%. In some embodiments, "about" indicates a deviation from the shown value of ±0.5%. In some embodiments, "about" indicates a deviation from the shown value of ±0.4%. In some embodiments, "about" indicates a deviation from the shown value of ±0.3%. In some embodiments, "about" indicates a deviation from the shown value of ±0.1%. In some embodiments, "about" indicates a deviation from the shown value of ±0.05%. In some embodiments, "about" indicates a deviation from the shown value of ±0.01%.

[0249] For naturally occurring compounds, the term "isolated" refers to a compound that is not in its native state (i.e., a polypeptide or polynucleotide) (e.g., free to varying degrees from components of naturally occurring associated compounds in nature). A specific level of purification is not required. For example, isolated polypeptides can be simply removed from their native or natural environment. For the purposes of this disclosure, recombinant polypeptides and proteins expressed in host cells are considered isolated, such as native or recombinant polypeptides that have been isolated, fractionated, or partially or substantially purified by any suitable technique. "Isolated" and "isolated" can also indicate the degree of separation from the original source or surrounding environment, depending on the context.

[0250] The term "polypeptide" refers to any polymer of two or more amino acids. Therefore, the terms peptide, dipeptide, tripeptide, oligopeptide, protein, amino acid chain, or any other term used to refer to a chain of two or more amino acids are included within the definition of "polypeptide." The term "polypeptide" also covers amino acid polymers that have been modified (e.g., through post-translational modification), for example, through disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeled component. Depending on the context, the term "polypeptide" can also be used to refer to proteins consisting of two or more polymers of two or more amino acids.

[0251] "Host cell" includes a single cell containing exogenous polynucleotides (e.g., in a culture). Host cells may include the offspring of a single host cell. Due to naturally occurring or genetically engineered variations, the offspring may not necessarily be identical to the original parent cell (in terms of morphology or the genome of total DNA complements).

[0252] A "fusion" or "chimeric" polypeptide or protein comprises a first polypeptide moiety linked to a second polypeptide moiety that is not naturally linked to the first polypeptide moiety. In some embodiments, these moieties may typically be present in separate proteins and aggregated together in a fusion polypeptide; they may typically be present in the same protein but arranged in a new configuration in the fusion polypeptide; or the moieties may aggregate together from different sources. In some embodiments, a fusion protein or chimeric protein comprises two or more moieties that are not naturally occurring (e.g., generated, designed, or otherwise produced by humans, such as binding domains, masking agents, linkers, barcodes, and other polypeptides provided herein). Chimeric proteins can be generated, for example, through chemical synthesis or through recombinant expression (e.g., including the generation and translation of polynucleotides in which peptide regions are encoded in a desired relationship).

[0253] The terms “conjugated,” “connected,” “fused,” and “fusion” are used interchangeably in this document, depending on the context. These terms can refer to the covalent bonding of two or more chemical (e.g., polypeptide) elements or components together by any means, including chemical conjugation or recombination.

[0254] As is known in the art, “sequence identity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of the second polypeptide. Similarly, “sequence identity” between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide with the sequence of the second polynucleotide. The terms “identity %”, “identity %”, or similar terms are intended to specifically refer to the percentage of identical nucleotides or amino acids (where applicable) in the optimal alignment between the sequences to be compared. This percentage can be purely statistical, and the differences between the two sequences can, but are not necessarily, randomly distributed across the entire length of the sequences to be compared. The comparison of two sequences is typically performed after optimal alignment, relative to segments or “comparison windows”, in order to identify local regions of the corresponding sequences. For example, the best alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482; the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444; or with the aid of computer programs using algorithms (GAP, BESTFIT, FASTA, BLASTP, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Drive, Madison, Wis.). In some implementations, the BLASTN or BLASTP algorithms are used to determine the percentage of identity between two sequences. These algorithms are available on the website of the National Center for Biotechnology Information (NCBi) (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some implementations, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word length set to 28; (hi) a maximum match in the query range set to 0; (iv) a match / non-match score set to 1, -2; (v) a gap cost set to linear; and (vi) the use of filters for low-complexity regions.In some implementations, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word length set to 3; (iii) a maximum match in the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to exist: 11, expansion: 1; and (vi) conditional composition score matrix adjustment. When discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison, WI53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the optimal homologous region between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to this disclosure, parameters are, of course, set such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and allows for a homology gap of up to 5% of the total number of amino acids in the reference sequence.

[0255] As used herein, the terms “masking peptide,” “masking,” and “masking motif” refer to peptides that, in the context of fusion proteins (such as chimeric peptides) provided herein, reduce the binding of antigen-binding domains (e.g., antibodies) to target antigens. Exemplary masking peptides include, but are not limited to, the ELNN peptide described herein. Additional masking peptides include albumin, peptides composed of proline, serine, and alanine, coiled-coil domains, albumin-binding domains, Fc domains, and binding domains specific to conserved regions of antibody variable domains. Lucchi et al. (ACS Cent Sci. 2021 May 26; 7(5): 724–738) further describe masking peptides in detail.

[0256] As used herein, the terms "ELNN peptide" and "ELNN" are synonymous and refer to an elongated peptide comprising a substantially non-repetitive sequence (e.g., a peptide motif) that is not naturally occurring, consisting primarily of small hydrophilic amino acids, wherein the sequence has a low degree of secondary or tertiary structure or no secondary or tertiary structure under physiological conditions. ELNN peptides include unstructured hydrophilic peptides comprising a repeating motif of six natural amino acids (G, A, P, E, S, and / or T). In some embodiments, ELNN peptides comprise multiple motifs of the six natural amino acids (G, A, P, E, S, T), wherein these motifs are identical or comprise combinations of different motifs. In some embodiments, when an ELNN peptide is linked to a protein (including T-cell adaptors as disclosed herein), it may confer certain desired pharmacokinetic, physicochemical, and pharmaceutical properties. Such desired properties may include, but are not limited to, enhanced pharmacokinetic parameters and solubility characteristics, and an improved therapeutic index. ELNN peptides are known in the art, and are related to the peptide known as XTEN. ® Non-restrictive descriptions of peptides and examples thereof are available in Schellenberger et al., (2009) NatBiotechnol 27(12): 1186-90; Brandl et al., (2020) Journal of Controlled Release 327:186-197; and Radonet et al., (2021) Advanced Functional Materials 31,2101633 (pp. 1-33), the full contents of which are incorporated herein by reference.

[0257] In some embodiments, the repeatability of the ELNN sequence refers to 3-mer repeatability and can be measured by a computer program or algorithm or by other means known in the art. In some embodiments, the 3-mer repeatability of the ELNN can be assessed by determining the number of occurrences of overlapping 3-mer sequences within the polypeptide. For example, a polypeptide of 200 amino acid residues has 198 overlapping 3-amino acid sequences (3-mers), but the number of unique 3-mer sequences will depend on the amount of repeatability within the sequence. In some embodiments, a score reflecting the degree of 3-mer repeatability throughout the polypeptide sequence (hereinafter referred to as the “subsequence score”) can be generated. In this context, the “subsequence score” means the sum of the occurrences of each unique 3-mer frame in the 200 consecutive amino acid sequences of the polypeptide divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequences. An example of such a subsequence score derived from the first 200 amino acids of repeating and non-repeating polypeptides is presented in Example 73 of International Patent Application Publication No. WO 2010 / 091122 A1, which is incorporated herein by reference in its entirety.

[0258] In some implementations, and in the context of ELNN, "substantially non-repetitive sequence" means an ELNN sequence in which (1) there are few or no four identical amino acids in the rows of the ELNN sequence, and in which (2) the ELNN has a subsequence fraction of 12 or 10 or less (as defined in the preceding paragraph of this document), or the sequence motifs constituting the polypeptide sequence do not follow a fixed pattern from the N-terminus to the C-terminus.

[0259] The term "single-chain variable fragment" (scFV) corresponds to an antigen-binding domain consisting of at least one heavy chain variable domain (VH) linked to at least one light chain variable domain (VL). The VH and VL can be linked by any linker recognized in the art, including but not limited to SESATPESGPGTSPGATPESGPGTSESATP. In some embodiments, the scFV comprises a VH domain and a VL domain from its N-terminus to its C-terminus. In other embodiments, the scFV comprises a VL domain and a VH domain from its N-terminus to its C-terminus. Tandem scFVs, such as bivalent scFVs (di-scFVs), are scFVs comprising a plurality of tandemly linked scFVs. A bivalent scFV comprises two VH domains and two VL domains, each scFV having the same or different (e.g., bispecific) target specificity. In some embodiments, the scFVs described herein are monovalent or bivalent scFVs.

[0260] The term "immunoglobulin single variable domain" (ISVD) defines an immunoglobulin molecule in which the antigen-binding site is located on and formed by a single immunoglobulin domain. This distinguishes the ISVD from "regular" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, bivalent scFv), where two immunoglobulin domains, specifically two variable domains, interact to form the antigen-binding site. Typically, in regular immunoglobulins, the heavy chain variable domain (VH) and light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL contribute to the antigen-binding site formation; a total of six CDRs are involved in antigen-binding site formation. In contrast, in ISVD, only three CDRs from the single domain contribute to antigen-binding site formation.

[0261] Given the above definition, the antigen-binding domain of conventional 4-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules; known in the art) or the antigen-binding domain of Fab fragments, F(ab')2 fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments) or biantibodies (all known in the art) derived from such conventional 4-chain antibodies are generally not considered as immunoglobulin monovariable domains, because in these cases, binding to the corresponding epitope of the antigen usually does not occur through a single immunoglobulin domain, but rather through a pair of associated immunoglobulin domains (such as a light chain variable domain and a heavy chain variable domain), i.e., through a pair of VH-VL immunoglobulin domains that jointly bind the epitope of the corresponding antigen.

[0262] In contrast, a single variable domain (VH) of an immunoglobulin can specifically bind to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of a single variable domain is formed by a single VH, a single VHH, or a single VL domain.

[0263] Therefore, a single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0264] Immunoglobulin single variable domains (ISVDs) can be, for example, heavy chain ISVDs, such as VH, VHH, including camelified VH or humanized VHH. In some embodiments, the heavy chain ISVD is a VHH, including camelified VH or humanized VHH. The heavy chain ISVD can be derived from conventional four-chain antibodies or from heavy chain antibodies.

[0265] For example, an immunoglobulin monovariable domain can be a monovariable antibody (or an amino acid sequence suitable for use as a monovariable antibody), "dAb" or dAb (or an amino acid sequence suitable for use as a dAb); other monovariable domains, or any suitable fragment of any of them.

[0266] In some implementations, the immunoglobulin single variable domain can be NANOBODY. ® The molecule or its suitable antigen-binding fragment. NANOBODY ® It is a registered trademark of Ablynx NV.

[0267] The “VHH domain,” also known as VHH, VHH region, VHH antibody fragment, and VHH antibody, was initially described as a variable domain of antigen-binding immunoglobulin in “heavy chain antibodies” (i.e., “antibodies lacking light chains”; Hamers-Casterman et al. Nature 363: 446-448, 1993). The term “VHH domain” was chosen to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (referred to herein as “VH domain,” “VH region,” and “VH”) and the light chain variable domains present in conventional 4-chain antibodies (referred herein as “VL domain,” “VL region,” and “VL”). For a further description of VHH, refer to Muyldermans’ review article (Reviews in Molecular Biotechnology 74: 277-302, 2001).

[0268] A “vector” is a nucleic acid molecule that transfers an inserted nucleic acid molecule into a host cell and / or between host cells. In some embodiments, the vector replicates itself in a suitable host. The term includes vectors primarily used for inserting DNA or RNA into cells, replication of vectors primarily used for replicating DNA or RNA, and expression vectors for transcription and / or translation of DNA or RNA. Vectors that provide more than one of the above functions are also included. An “expression vector” is a polynucleotide that, when introduced into a suitable host cell, can be used to transcribe mRNA to translate it into a polypeptide. In some embodiments, an “expression system” is a suitable host cell containing an expression vector that can be used to produce a desired expression product.

[0269] The terms “treatment / treating” and “improvement” are used interchangeably herein. These terms refer to methods used to achieve beneficial or desired outcomes, including but not limited to therapeutic benefits. A “therapeutic benefit” means the eradication or improvement of the underlying barrier being treated. In some embodiments, a therapeutic benefit is achieved by eradicating or improving one or more physiological symptoms associated with an underlying disease condition, resulting in an observed improvement in the subject, although the subject may still have the underlying barrier. In some embodiments, a therapeutic benefit includes slowing or stopping the growth of one or more tumors. In some embodiments, a therapeutic benefit includes reducing the size of one or more tumors. In some embodiments, a therapeutic benefit includes eradicating one or more tumors from the subject. In some embodiments, a therapeutic benefit includes achieving the death of cancer cells.

[0270] As used herein, the term "therapeuticly effective amount" refers to the amount of a bioactive agent (such as the fusion protein described herein, for example, as part of a pharmaceutical 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. This effect does not need to be absolutely beneficial. A disease or condition can refer to a disorder or disease, such as cancer or symptoms of cancer.

[0271] Antigen-binding domains, cleavage sequences, barcode fragments, and fusion peptides

[0272] This disclosure provides, in particular, novel and useful anti-EGFR antibodies, novel and useful anti-CD3 antibodies, cleavage sequences, barcode fragments, and fusion proteins comprising them. This document includes fusion peptides comprising (i) one or more masking peptides (such as ELNN), (ii) a bispecific antibody (BsAb, e.g., TCE) linked to the masking peptide, and (iii) one or more protease-cleavable release segments (RS), wherein the RS is located between the masking peptide and the BsAb.

[0273] In some implementations, the anti-EGFR antibodies provided herein include a VH domain comprising the following sequence: QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS (SEQ ID NO: 468), and a VL domain containing the following sequence: DIQMTQSPSSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQGTKVEIK (SEQ ID NO: 469).

[0274] In some embodiments, the anti-CD3 antibody provided herein comprises a VH domain containing a CDR of the VH domain having the following sequence: EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126) and / or VL domains, wherein the VL domain contains a CDR of VL domains having the following sequence: ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127).

[0275] BsAbs comprising, for example, anti-EGFR antibodies and / or anti-CD3 antibodies disclosed herein are also provided. In some embodiments, the bispecific antibody comprises the VH and VL regions of the anti-EGFR antibody region disclosed herein. In some embodiments, the BsAb comprises the VH and VL regions of the anti-CD3 antibody disclosed herein. In some embodiments, the BsAb comprises an anti-EGFR scFV region containing the VH and VL pairs disclosed herein and an anti-CD3 scFV region containing the VH and VL pairs disclosed herein. In some embodiments, the BsAb is a TCE.

[0276] In some embodiments, the fusion peptide comprises a first ELNN (such as the ELNN described herein). In some embodiments, the peptide further comprises a second ELNN (such as the ELNN described herein). In some embodiments, the peptide comprises an ELNN (“N-terminal ELNN”) at or near its N-terminus. In some embodiments, the peptide comprises an ELNN (“C-terminal ELNN”) at or near its C-terminus. In some embodiments, the peptide comprises both an N-terminal ELNN and a C-terminal ELNN.

[0277] In some embodiments, the fusion polypeptide comprises a BsAb, and a first ELNN is attached to the N-terminus of the BsAb via a first RS, and a second ELNN is attached to the C-terminus of the BsAb via a second RS. In some embodiments, each RS is cleavable by a protease mentioned herein. In some embodiments, each RS comprises the RS sequence disclosed herein. In some embodiments, the fusion polypeptide is paTCE.

[0278] This document includes polypeptide sequences that can be used, for example, to link one polypeptide moiety within a fusion protein to another polypeptide moiety. For example, useful adapters are provided that can be cleaved by a variety of proteases but not by podin. In some embodiments, such adapters can be used outside the context of antibodies, such as those described herein.

[0279] In some embodiments, the fusion polypeptide (e.g., one or more ELNNs of paTCE and / or another portion of the fusion polypeptide, such as a linker or spacer sequence) may comprise one or more barcode fragments (e.g., as described herein), which the fusion polypeptide may release (e.g., configured to release) upon protease cleavage or digestion of the fusion polypeptide (e.g., paTCE). In some embodiments, the protease is a non-mammalian protease. In some embodiments, each barcode fragment is sequence- and molecular-differentiated from all other peptide fragments (including all other barcode fragments, if present) that can be released from the polypeptide after complete digestion by the protease, thereby making it unique and its presence detectable by techniques such as mass spectrometry.

[0280] Extended recombinant peptide (ELNN)

[0281] Chain length and amino acid composition

[0282] In some embodiments, the ELNN contains at least 100 or at least 150 amino acids. In some embodiments, the ELNN is 100 to 3000 or 150 to 3000 amino acids in length. In some embodiments, the ELNN is 100 to 1000 or 150 to 1000 amino acids in length. In some embodiments, the ELNN is at least (about) 100, at least (about) 150, at least (about) 200, at least (about) 250, at least (about) 300, at least (about) 350, at least (about) 400, at least (about) 450, at least (about) 500, at least (about) 550, at least (about) 600, at least (about) 650, at least (about) 700, at least (about) 750, at least (about) 800. At least 850, at least 900, at least 950, at least 1,000, at least 1,100, at least 1,200, at least 1,300, at least 1,400, at least 1,500, at least 1,600, at least 1,700, at least 1,800, at least 1,900, or at least 2,000 amino acids. In some implementations, the length of the ELNN is at most (approximately) 100, at most (approximately) 150, at most (approximately) 200, at most (approximately) 250, at most (approximately) 300, at most (approximately) 350, at most (approximately) 400, at most (approximately) 450, at most (approximately) 500, at most (approximately) 550, at most (approximately) 600, at most (approximately) 650, at most (approximately) 700, at most (approximately) 750, at most (approximately) 800. Up to (approximately) 850, up to (approximately) 900, up to (approximately) 950, up to (approximately) 1,000, up to (approximately) 1,100, up to (approximately) 1,200, up to (approximately) 1,300, up to (approximately) 1,400, up to (approximately) 1,500, up to (approximately) 1,600, up to (approximately) 1,700, up to (approximately) 1,800, up to (approximately) 1,900 or up to (approximately) 2,000 amino acids. In some implementations, the ELNN has a length of approximately 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 amino acids, or a range between any two of the aforementioned values.In some embodiments, at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P). In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P). In some embodiments, the ELNN contains at least three different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, the ELNN contains at least four different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, the ELNN contains at least five different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, the ELNN is composed of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, the ELNN comprises G, A, S, T, E, or P amino acids. In some embodiments, the ELNN (e.g., ELNN1, ELNN2, etc.) is characterized by: (i) comprising at least 100 or at least 150 amino acids; (ii) at least 90% of the amino acid residues of the ELNN being glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P); and (hi) comprising at least four different types of amino acids from G, A, S, T, E, or P. As used herein, the term "glutamate" is synonymous with "glutamic acid" and refers to glutamic acid residues, regardless of whether the side chain carboxyl group is deprotonated. In some embodiments, the ELNN-containing fusion polypeptide comprises a first ELNN and a second ELNN. In some implementations, the total number of amino acids in the first ELNN and the total number of amino acids in the second ELNN is at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids.

[0283] Non-overlapping sequence motifs

[0284] In some embodiments, the ELNN comprises or is formed by multiple non-overlapping sequence motifs. In some embodiments, at least one of the non-overlapping sequence motifs is repeated (or repeated at least twice in the ELNN). In some embodiments, the ELNN comprises at least one other non-overlapping sequence motif (or found only once in the ELNN). In some embodiments, the multiple non-overlapping sequence motifs comprise (a) a set of (repeated) non-overlapping sequence motifs, wherein each non-overlapping sequence motif in the set is repeated at least twice in the ELNN; and (b) a non-overlapping (non-repeated) sequence motif that appears (or is found) only once in the ELNN. In some embodiments, each non-overlapping sequence motif is 9 to 14 (or 10 to 14, or 11 to 13) amino acids in length. In some embodiments, each non-overlapping sequence motif is 12 amino acids in length. In some embodiments, the plurality of non-overlapping sequence motifs comprises a set of non-overlapping (repetitive) sequence motifs, wherein each non-overlapping sequence motif in the set of non-overlapping sequence motifs (1) is repeated at least twice in ELNN; and (2) is between 9 and 14 amino acids in length. In some embodiments, the set of (repetitive) non-overlapping sequence motifs comprises 12-mer sequence motifs identified herein by SEQ ID NO: 179 to SEQ ID NO: 200 and SEQ ID NO: 1715 to SEQ ID NO: 1722 in Table 1. In some embodiments, the set of (repetitive) non-overlapping sequence motifs comprises 12-mer sequence motifs identified herein by SEQ ID NO: 186 to SEQ ID NO: 189 in Table 1. In some embodiments, the set of (repetitive) non-overlapping sequence motifs comprises at least two, at least three, or all four 12-mer sequence motifs of SEQ ID NO: 186 to SEQ ID NO: 189 in Table 1. In some embodiments, the ELNN further includes sequences other than the 12-mer sequence motifs shown in Table 1. In some embodiments, the ELNN includes sequences not shown in Table 1, such as ASSATPESGP, GSGPGTSESATP, or GTSESATP. In some embodiments, the ELNN includes sequences not shown in Table 1, such as ATPESGP, GTSPSATPESGP, or GTSESAGEPEA. In some embodiments, the ELNN includes barcode sequences.

[0285] Table 1. Exemplary 12-merchant sequence motifs used to construct ELNN

[0286]

[0287]

[0288] Unstructured peptide confirmation

[0289] In various embodiments, one or more ELNN components of the fusion protein have an unstructured conformation under physiological conditions, independent of the length of the polymer (e.g., elongation). For example, ELNNs are characterized by large conformational freedom of the peptide backbone. In some embodiments, ELNNs are characterized by the absence of long-range interactions, as determined by NMR. In some embodiments, this disclosure provides ELNNs with structures similar to denatured sequences that are substantially lacking in secondary structure under physiological conditions. In some embodiments, ELNNs may be substantially free of secondary structure under physiological conditions. As used in this context, "substantially free" means that less than 50% of the ELNN amino acid residues contribute to secondary structure, as measured or determined by the means described herein. As used in this context, "substantially free" means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the ELNN amino acid residues do not contribute to secondary structure, as measured or determined by the means described herein.

[0290] Various methods have been established in the art to identify the presence or absence of secondary and tertiary structures in a given polypeptide. In some embodiments, the ELNN secondary structure can be measured spectrophotometrically, for example by circular dichroism spectroscopy in the far-ultraviolet (far-UV) spectral region (190 nm to 250 nm). Secondary structural elements, such as α-helices and β-sheets, each produce characteristic shapes and magnitudes of the CD spectrum. The secondary structure of a polypeptide sequence can also be predicted by certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, PY, et al. (1974) Biochemistry, 13: 222-45) and the Gamer-Osghuthorpe-Robson (“GOR”) algorithm (Gamier J, Gibrat JF, Robson B. (1996), GOR method for predicting protein secondary structure from amino acids sequence. Methods Enzymol 266:540-553), as described in U.S. Patent Application Publication No. 20030228309A1 (the entire contents of which are incorporated herein by reference). For a given sequence, the algorithm can predict whether there are some secondary structures or no secondary structures at all, expressed as the total number and / or percentage of sequence residues forming, for example, α-helices or β-sheets, or predict the percentage of sequence residues that lead to random coil formation (which lacks secondary structures).

[0291] In some embodiments, the ELNN used in the fusion protein composition may have an α-helix percentage ranging from 0% to less than about 5%, as determined by the Chou-Fasman algorithm. In some embodiments, the ELNN of the fusion protein composition may have a β-sheet percentage ranging from 0% to less than about 5%, as determined by the Chou-Fasman algorithm. In some embodiments, the ELNN of the fusion protein composition may have both an α-helix percentage and a β-sheet percentage ranging from 0% to less than about 5%, as determined by the Chou-Fasman algorithm. In some embodiments, the ELNN of the fusion protein composition will have an α-helix percentage and a β-sheet percentage of less than about 2%. In some embodiments, the ELNN of the fusion protein composition may have a high random coil percentage, as determined by the GOR algorithm. In some implementations, the ELNN may have at least about 80%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% random curl, as determined by the GOR algorithm.

[0292] Net charge

[0293] In some embodiments, the ELNN peptide may have unstructured features conferred by incorporating amino acid residues with net charges and / or reducing the proportion of hydrophobic amino acids in the ELNN sequence. The total net charge and net charge density can be controlled, for example, by varying the content of charged amino acids in the ELNN. In some embodiments, the net charge density of the ELNN in the composition may be greater than +0.1 charge / residue or less than -0.1 charge / residue. In some embodiments, the net charge of the ELNN may be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more.

[0294] Since most tissues and surfaces in humans or animals have a net negative charge, ELNNs can optionally be designed to have a net negative charge to minimize non-specific interactions between the ELNN-containing composition and various surfaces, such as blood vessels, healthy tissues, or various receptors. Unbound by any particular theory, ELNNs can adopt an open conformation due to the electrostatic repulsion between individual amino acids of the ELNN peptide, each carrying a high net negative charge and distributed along the sequence of the ELNN peptide. This distribution of net negative charge along the extended sequence length of the ELNN can lead to an unstructured conformation, which in turn can result in an efficient increase in the hydrodynamic radius. Thus, in some embodiments, the ELNN contains glutamic acid such that glutamic acid is present at about 8%, 10%, 15%, 20%, 25%, or even about 30% of the amino acids in the sequence. The ELNNs of the compositions disclosed herein typically do not have positively charged amino acids or have a low content of positively charged amino acids. In some embodiments, the ELNN may have less than about 10% positively charged amino acid residues, or less than about 7%, or less than about 5%, or less than about 2% positively charged amino acid residues. However, this disclosure contemplates polypeptides in which a limited number of positively charged amino acids (such as lysine) can be incorporated into the ELNN, for example, to allow conjugation between the ε-amine of lysine and reactive groups on the peptide, linker bridges, or reactive groups on the drug or small molecule to be conjugated to the ELNN backbone.

[0295] In some embodiments, the ELNN may contain charged residues separated by other residues, such as serine or glycine, which can lead to better expression or purification behavior. Based on net charge, the ELNN of the subject composition may have an isoelectric point (pI) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In some embodiments, the ELNN will have an isoelectric point between 1.5 and 4.5. In some embodiments, the ELNN incorporated into the patCE fusion protein carries a net negative charge under physiological conditions, which contributes to reduced unstructured conformation and binding of the ELNN component to mammalian proteins and tissues.

[0296] Because hydrophobic amino acids can impart a polypeptide structure, in some embodiments, the content of hydrophobic amino acids in the ELNN is less than 5%, or less than 2%, or less than 1%. In some embodiments, the ELNN does not contain hydrophobic amino acids. In some embodiments, the methionine and tryptophan amino acid content in the ELNN component of the patCE fusion protein is less than 5%, or less than 2%, and most preferably less than 1%. In some embodiments, the ELNN has a sequence having less than 10% positively charged amino acid residues, or less than about 7%, or less than about 5%, or less than about 2% positively charged amino acid residues, the sum of methionine and tryptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 10% of the total ELNN sequence. In some embodiments, the ELNN does not contain methionine or tryptophan residues.

[0297] Increased hydrodynamic radius

[0298] In some embodiments, the ELNN may have a high hydrodynamic radius, conferring a correspondingly increased apparent molecular weight to the paTCE fusion protein incorporating the ELNN. The linking of the ELNN to a BsAb (e.g., TCE) sequence can produce a paTCE composition that has an increased hydrodynamic radius, increased apparent molecular weight, and increased apparent molecular weight factor compared to a BsAb (e.g., TCE) not linked to the ELNN. For example, in some therapeutic applications requiring extended half-life, incorporating one or more ELNNs with a high hydrodynamic radius into a fusion protein containing a BsAb (e.g., TCE) can effectively expand the hydrodynamic radius of the fusion protein beyond a glomerular pore size of approximately 3 to 5 nm (corresponding to an apparent molecular weight of approximately 70 kDa) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv. Drug Deliv. Rev. 55:1261-1277), thereby resulting in a reduction in renal clearance of circulating proteins. In some embodiments, the hydrodynamic radius of a protein is determined by its molecular weight and its structure, including shape and compactness. Unbound by any particular theory, ELNNs can adopt an open conformation due to electrostatic repulsion between individual charges of the peptide or the inherent flexibility conferred by the lack of specific amino acids in the sequence that have the potential to confer secondary structure. In some embodiments, the open, extended, and unstructured conformations of ELNN peptides have a larger proportion of hydrodynamic radii compared to peptides with comparable sequence lengths and / or molecular weights (such as typical globular proteins) having secondary and / or tertiary structures. Methods for determining the hydrodynamic radius are well known in the art, such as the use of size exclusion chromatography (SEC), as described in U.S. Patents 6,406,632 and 7,294,513. In some embodiments, increasing the ELNN length results in a proportional increase in the parameters of the hydrodynamic radius, apparent molecular weight, and apparent molecular weight factor, thereby allowing the paTCE to be adjusted to the desired characteristic cut-off apparent molecular weight or hydrodynamic radius. Therefore, in some embodiments, the paTCE fusion protein can be configured with an ELNN such that the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or at least about 15 nm. In some embodiments, the large hydrodynamic radius imparted by the ELNN in the paTCE fusion protein can lead to a decrease in the renal clearance of the resulting fusion protein, thereby resulting in a corresponding increase in terminal half-life, an increase in mean residence time, and / or a decrease in renal clearance.

[0299] In some implementations, one ELNN (or multiple ELNNs, such as two ELNNs) having a selected length and sequence may be selectively incorporated into paTCE to produce a fusion protein that, under physiological conditions, will have an apparent molecular weight of at least about 150 kDa, or at least about 300 kDa, or at least about 400 kDa, or at least about 500 kDa, or at least about 600 kDa, or at least about 700 kDa, or at least about 800 kDa, or at least about 900 kDa, or at least about 1000 kDa, or at least about 1200 kDa, or at least about 1500 kDa, or at least about 1800 kDa, or at least about 2000 kDa, or at least about 2300 kDa or higher. In some embodiments, an ELNN (or multiple ELNNs, such as two ELNNs) having a selected length and sequence may be selectively linked to a BsAb (e.g., TCE) to produce a paTCE fusion protein having an apparent molecular weight factor of at least 3, alternatively at least 4, alternatively at least 5, alternatively at least 6, alternatively at least 7, alternatively at least 8, alternatively at least 9, alternatively at least 10, alternatively at least 15, or at least 20 or greater under physiological conditions. In some embodiments, the paTCE fusion protein has an apparent molecular weight factor of about 4 to about 20, or about 6 to about 15, or about 8 to about 12, or about 9 to about 10 relative to the actual molecular weight of the fusion protein under physiological conditions. In some embodiments, the fusion peptide exhibits an apparent molecular weight factor greater than about 6 under physiological conditions.

[0300] Increased terminal half-life

[0301] In some embodiments, the fusion peptide containing an ELNN (such as paTCE) has a terminal half-life that is at least two times, three times, four times, or five times longer than that of the corresponding bioactive peptide not linked to an ELNN. In some embodiments, the (fusion) peptide has a terminal half-life that is at least twice as long as that of the bioactive peptide not linked to an ELNN.

[0302] In some implementations, when administered to subjects at a comparable dose, administration of a therapeutically effective amount of the paTCE fusion protein to a subject in need results in at least two-fold, at least three-fold, at least four-fold, or at least five-fold or more time spent within the therapeutic window of the fusion protein compared to a corresponding BsAb (e.g., TCE) not connected to the ELNN.

[0303] In some embodiments, the TCE released from paTCE upon protease cleavage comprises one or more short polypeptides (e.g., of about 30, 25, 20, 15, 14, 13, 12, 11, 10 or fewer amino acids) that do not contain amino acids other than G, A, P, E, S and / or T. For example, short polypeptides that do not contain amino acids other than G, A, P, E, S and / or T may be incorporated into one or more spacer or linker sequences of the TCE and / or a portion of one or more spacers or linkers that remain a portion of the TCE after cleavage. In some embodiments, the TCE released from paTCE contains GTSESATPES on the N-terminal side of the TCE (e.g., the nearest amino acid in the sequence is within 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid position of the N-terminal amino acid, or the sequence contains an N-terminus). In some embodiments, the TCE released from the paTCE contains GTATPESGPG on the C-terminal side of the TCE (e.g., the nearest amino acid in the sequence is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid position of the N-terminal amino acid, or the sequence contains an N-terminus). In some embodiments, the TCE contains an internal linker (e.g., between the VL and VH regions of the scFV) containing a polypeptide sequence that does not have amino acids other than G, A, P, E, S, and / or T, such as SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0304] low immunogenicity

[0305] In some embodiments, this disclosure provides compositions in which ELNNs have a low degree of immunogenicity or are substantially non-immunogenic. Several factors can contribute to the low immunogenicity of ELNNs, such as substantially non-repetitive sequences, unstructured conformations, high solubility, low degree or absence of self-aggregation, low degree or absence of intra-sequence proteolytic sites, and low degree or absence of epitopes in ELNNs.

[0306] Those skilled in the art will understand that, in general, polypeptides having highly repetitive short amino acid sequences (e.g., sequences of 200 amino acid length containing an average of 20 or more repeats of a finite set of 3-mers or 4-mers) and / or having continuously repeating amino acid residues (e.g., sequences of 5-mers or 6-mers having the same amino acid residues) tend to aggregate or form higher-order structures or form contacts, thereby resulting in crystalline or pseudocrystalline structures.

[0307] In some implementations, the ELNN sequence is substantially non-repetitive, wherein (1) the ELNN sequence does not have three consecutive amino acids of the same amino acid type, unless the amino acid is serine, in which case no more than three consecutive amino acids may be serine residues; and wherein (2) the ELNN does not contain 3-amino acid sequences (3-mers) that appear more than 16, more than 14, more than 12, or more than 10 times within a sequence at least 200 amino acids long (e.g., the entire span of an ELNN at least amino acid long). Without being bound by any scientific theory, such substantially non-repetitive sequences have a lower tendency to aggregate, and therefore, it is possible to design long-sequence ELNNs with relatively low frequencies of charged amino acids, where charged amino acids are more likely to aggregate if the sequence or amino acid residues are more repetitive.

[0308] Conformational epitopes can form from regions on the surface of proteins, consisting of multiple discontinuous amino acid sequences of a protein antigen. Unbound by any scientific theory, the precise folding of proteins can make these sequences well-defined, stable spatial conformations or epitopes that can be recognized as “foreign” by the host’s humoral immune system, leading to the production of antibodies against the protein and / or triggering a cell-mediated immune response. In the latter case, the individual’s immune response to the protein is heavily influenced by T-cell epitope recognition, a function specific to the individual’s HLA-DR allotype peptide binding. Binding of MHC class II peptide complexes to homologous T-cell receptors on the surface of T cells, as well as cross-binding with certain other co-receptors (such as CD4 molecules), can induce an activated state within T cells. Activation can lead to the release of cytokines, further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a complete cellular immune response.

[0309] Unbound by any scientific theory, the ability of a peptide to bind a given MHC class II molecule to be presented on the surface of an APC (antigen-presenting cell) can depend on many factors; most notably its primary sequence. In some embodiments, a lower degree of immunogenicity can be achieved by designing an ELNN that counteracts antigen processing in antigen-presenting cells and / or selecting sequences that do not bind well to MHC receptors. In some embodiments, ELNN-containing fusion proteins have substantially non-repetitive ELNN peptides designed to reduce binding to MHC II receptors and avoid forming epitopes or antibody binding to T-cell receptors, thereby resulting in a lower degree of immunogenicity. Unbound by any scientific theory, the avoidance of the immunogenic portion is a direct result of the conformational flexibility of ELNNs; that is, the lack of secondary structure due to the selection and sequence of amino acid residues. For example, sequences with a low tendency to adjust to a compact folded conformation in aqueous solution or under physiological conditions that may generate conformational epitopes are of particular interest. When using standard treatment practices and administration, the administration of fusion proteins containing ELNN generally does not result in the formation of neutralizing antibodies against ELNN and may also reduce the immunogenicity of BsAb (e.g., TCE) fusion couples in paTCE compositions.

[0310] In some embodiments, the ELNN used for the subject fusion protein may be substantially free of epitopes recognized by human T cells. The elimination of such epitopes for the purpose of producing less immunogenic proteins has been previously disclosed; see, for example, WO 98 / 52976, WO 02 / 079232, and WO 00 / 3317, which are incorporated herein by reference. The assay of human T cell epitopes has been described (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Of particular interest are peptide sequences that can be oligomerized without producing T cell epitopes or non-human sequences. This can be achieved by testing for the presence of T-cell epitopes in direct repetitions of these sequences and the appearance of 6- to 15-mers, particularly non-human 9-mer sequences, and then modifying the ELNN sequence to eliminate or disrupt the epitope sequence. In some embodiments, the ELNN is substantially non-immunogenic by limiting the number of epitopes in the ELNN that are predicted to bind to the MHC receptor. With a decrease in the number of epitopes capable of binding to MHC receptors, there is a potential accompanying decrease in T cell activation and T cell helper function, a decrease in B cell activation or upregulation, and a decrease in antibody production. Low-level predicted T cell epitopes can be determined using epitope prediction algorithms, such as, for example, TPEITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61), as illustrated in Example 74 of International Patent Application Publication No. WO 2010 / 144502 A2, which is incorporated herein by reference in its entirety. Aspects of TPITOPE scores for a given peptide framework within a protein are disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555. These scores range from at least 20 log, from about 10 to about -10 (corresponding to 10e...). 10 K D up to 10e -10 K D (binding restrictions), and can be reduced by avoiding hydrophobic amino acids, such as M, I, L, V, or F, that can act as anchoring residues during peptide display on the MHC. In some embodiments, the ELNN component incorporated into the paTCE does not have the predicted T cell epitope when the TEPIETOPE fraction is about -5 or greater, or -6 or greater, or -7 or greater, or -8 or greater, or -9 or greater. As used herein, a fraction of "-9 or greater" will encompass TEPIETOPE fractions from 10 to -9, including end values, but will not encompass fractions of -10, as -10 is less than -9.

[0311] In some embodiments, by restricting known proteolytic sites from the ELNN sequence, reducing the processing of ELNN into small peptides capable of binding MHC II receptors, ELNNs (including those incorporated into the subject paTCE fusion protein) can be made substantially non-immunogenic. In some embodiments, by using sequences substantially lacking secondary structure, conferring resistance to many proteases due to the high entropy of the structure, ELNN sequences can be made substantially non-immunogenic. Therefore, the reduction of TPITOPE fraction and the elimination of known proteolytic sites from ELNNs can render ELNN compositions (including ELNNs of paTCE fusion protein compositions) substantially unbindable by mammalian receptors (including those of the immune system). In some embodiments, the ELNN of the paTCE fusion protein may have a K+ >100 nM. D The binding of [a substance] to mammalian receptors, or greater than 500 nM K. D or greater than 1µM K D It binds to peptide receptors on the surface of mammalian cells or in circulation.

[0312] Furthermore, the substantially non-repetitive sequences and corresponding epitopes of such embodiments of ELNN lack the ability to restrict B cells from binding to or being activated by ELNN. In some embodiments, although ELNN can contact many different B cells along its extended sequence, each individual B cell may only contact a single ELNN once or a small number of times. Therefore, ELNN may generally have a much lower tendency to stimulate B cell proliferation and thus stimulate an immune response. In some embodiments, paTCE may have reduced immunogenicity compared to the corresponding BsAb (e.g., TCE) not fused to the masking peptide such as ELNN. In some embodiments, administration of up to three parenteral doses of paTCE to a mammal may result in detectable anti-paTCE IgG at a 1:100 serum dilution, but undetectable at a 1:1000 dilution. In some embodiments, administration of up to three parenteral doses of paTCE to a mammal may result in detectable anti-BsAb (e.g., TCE) IgG at a 1:100 serum dilution, but undetectable at a 1:1000 dilution. In some embodiments, administration of up to three parenteral doses of paTCE to a mammal may result in detectable anti-ELNN IgG at a 1:100 serum dilution, but undetectable at a 1:1000 dilution. In some embodiments, the mammal may be, for example, a mouse, rat, rabbit, cynomolgus monkey, or human. In some embodiments, the mammal is a human.

[0313] Compared to sequences with fewer non-repetitive sequences (such as sequences with three identical consecutive amino acids), some ELNNs with substantially non-repetitive sequences have the additional characteristic that non-repetitive ELNNs form a weaker contact with antibodies (e.g., monovalent interaction), resulting in a lower likelihood of immune clearance, allowing the paTCE composition to remain in circulation for an extended period of time.

[0314] In some implementations, bioactive peptides containing ELNNs (such as BsAbs, e.g., TCEs) are less immunogenic than fusion peptides not linked to any ELNNs, wherein immunogenicity is determined by measuring the production of IgG antibodies that selectively bind to the bioactive peptides after administering a suitable dose to a subject.

[0315] Barcode fragment

[0316] In some embodiments, the polypeptide (e.g., a fusion polypeptide or a portion thereof, such as ELNN) comprises one or more barcode fragments (e.g., a first barcode fragment, a second barcode fragment, or a third barcode fragment) that can be released from the polypeptide upon digestion by a protease. In some embodiments, the protease is a non-mammalian protease. In some embodiments, the protease is a prokaryotic protease. As used herein, the term "barcode fragment" (or "barcode" or "barcode sequence") can refer to a polypeptide moiety cleavably fused within a polypeptide, or a resulting peptide fragment released from the polypeptide.

[0317] In some embodiments, the barcode fragment (1) is part of the ELNN, which includes at least a portion of a (non-repetitive, non-overlapping) sequence motif that appears (or is found) only once within the ELNN; and (2) is different in sequence and molecular weight from all other peptide fragments that can be released from the peptide after protease cleavage or complete digestion of the peptide.

[0318] In some embodiments, the barcode fragment does not contain an N-terminal or C-terminal amino acid of the fusion peptide. As described herein, in some embodiments, the barcode fragment is releasable (e.g., configured to be releasable) after Glu-C digestion of the fusion peptide. In some embodiments, the barcode fragment is in the ELNN and does not contain a glutamate immediately adjacent to another glutamate residue in the ELNN (if present). In some embodiments, the barcode fragment has a glutamate residue at its C-terminus. Those skilled in the art will understand that when cleavably fused within a peptide (such as an ELNN), the C-terminus of the barcode fragment can refer to the "last" (or C-terminal) amino acid residue within the barcode fragment, even if other non-barcode amino acid residues are located at the C-terminus of the barcode fragment within the peptide (e.g., the ELNN). In some embodiments, the barcode fragment has an N-terminal amino acid immediately following the glutamate residue. In some embodiments, the glutamate residue preceding the N-terminal amino acid is not immediately adjacent to another glutamate residue. In some embodiments, the barcode fragment does not contain a (second) glutamic acid residue at any position other than the C-terminus of the barcode fragment, unless the glutamic acid is immediately followed by a proline residue. In some embodiments, the barcode fragment is located at a distance of 10 to 150 or 10 to 125 amino acids from the N-terminus or C-terminus of the polypeptide. In some embodiments, the barcode fragment is located within 300, 280, 260, 250, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40, 36, 30, 24, 20, 12, or 10 amino acids from the N-terminus of the polypeptide, or at such position, or within a range between the foregoing. In some embodiments, the barcode fragment is located within 200, 150, 100, or 50 amino acids from the N-terminus of the polypeptide. In some embodiments, the barcode fragment is located between 10 and 200, 30 and 200, 40 and 150, or 50 and 100 amino acids from the N-terminus of the polypeptide. In some embodiments, the barcode fragment is located within 300, 280, 260, 250, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40, 36, 30, 24, 20, 12, or 10 amino acids from the C-terminus of the polypeptide, or at such location, or within a range between the foregoing. In some implementations, the barcode fragment is located within 200, 150, 100, or 50 amino acids at the C-terminus of the polypeptide.In some embodiments, the barcode fragment is located at a position between 10 and 200, 30 and 200, 40 and 150, or 50 and 100 amino acids from the C-terminus of the polypeptide. In some embodiments, the barcode fragment (BAR) is characterized by: (i) not containing a glutamic acid immediately adjacent to another glutamic acid in ELNN (if present); (ii) having a glutamic acid residue at the C-terminus; (iii) having an N-terminal amino acid immediately following the glutamic acid residue; and (iv) being located at a distance from the N-terminus or C-terminus of the polypeptide, wherein the length of such distance is 10 to 150 amino acids or 10 to 125 amino acids. In some embodiments, the barcode fragment is in the ELNN and (i) does not contain an N-terminal or C-terminal amino acid of the peptide; (ii) does not contain a glutamic acid immediately adjacent to another glutamic acid in the ELNN; (iii) has a glutamic acid at its C-terminus; (iv) has an N-terminal amino acid immediately following the glutamic acid residue; and (v) is located at a distance from the N-terminus or C-terminus of the peptide, wherein the length of such distance is 10 to 150 or 10 to 125 amino acids. In some embodiments, the glutamic acid residue preceding the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, the barcode fragment does not contain a glutamic acid residue at any position other than the C-terminus of the barcode fragment, unless the glutamic acid is immediately followed by a proline. Depending on the context and when referring to placement within a peptide sequence, the term "distance" can refer to the number of amino acid residues from the N-terminus of the peptide to the N-terminal amino acid residue of the barcode fragment or from the C-terminus of the peptide to the C-terminal amino acid residue of the barcode fragment. In some embodiments, for a barcoded ELNN fused with a bioactive peptide, at least one barcode fragment (or at least two, or three barcode fragments) contained in the barcoded ELNN is located at a distance of at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids from the bioactive peptide. In some embodiments, the length of the barcode fragment is at least 4, 5, 6, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids, or within any of the foregoing values. In some implementations, the length of the barcode segment is between 4 and 20, 5 and 15, 6 and 12, or 7 and 10 amino acids.In some implementations, the barcode fragment contains the amino acid sequence identified herein by SEQ ID NO: 68 to SEQ ID NO: 79 and SEQ ID NO: 1010 to SEQ ID NO: 1027 in Table 2.

[0319] Table 2. Exemplary barcode fragments that can be released after Glu-C digestion

[0320]

[0321] In some embodiments, each barcode fragment is distinct in both sequence and molecular weight from all other peptide fragments that can be released from the chimeric polypeptide described herein after complete digestion of the chimeric polypeptide by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

[0322] In some embodiments, the chimeric polypeptides disclosed herein contain a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG, SGSETPGT, and GTSESATP.

[0323] In some embodiments, the chimeric polypeptide disclosed herein comprises at least one of the following amino acid sequences: PE.GSX n PE.SG, PE.GSX n SE.GG, PE.GSX n SE.TG, PE.GSX n SE.SA, PE.SGX n PE.SG, PE.SGX n SE.GG, PE.SGX n SE.TG, PE.SGX n SE.SA and PE.TPX n PE.SG, PE.TPX n SE.GG, PE.TPX n SE.TG, PE.TPX n SE.SA, where each "." is a Glu-C cleavage site, and n is any integer from 0 to 50. In some embodiments, the chimeric polypeptide disclosed herein comprises at least one of the following amino acid sequences: PE.SGX n PE.SG, PE.GSX n SE.GG, PE.TPX n SE.TG, PE.SGX nSE.SA. In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is any integer from 5 to 15. In some embodiments, X n Is SPGGTGTSATPE, SPGGSGPGTSE, SPGTTPGTTPE, SPGTPPTSTPE, SPGGTSPSATPE, SPGTGSAGTPE, SPGTGGAGTPE, SPGTSPGATPE, SPGTSGSGTPE, SPGGTSSASTPE, SGPGTGAGTTPE, SGPGTGSTSTPE, TPGSEPATSGSE, GSAPGTSTEPSE, SGPGTAGSGTPE, SGPGTSSGGTPE, SGPGTAGPATPE, SGPGTPGTGTPE, SGPGTGGPTTPE, or SGPGTGSGSTPE.

[0324] In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences:

[0325]

[0326] EPSE.SATPX n GTPE.GSAP or EPSE.SATPX n EPSE.SATP, where each "." is a Glu-C cleavage site, and n is any integer from 0 to 50. In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences:

[0327] TTPE.SGPGX n TTPE.SGPG or STPE.SGPGX n STPE.SGPG, where each "." is a Glu-C cleavage site, and n is any integer from 0 to 30. In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 3 to 7. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is 4. In some embodiments, n is any integer from 5 to 15. In some embodiments, where X nIt is PGTGTSAT, PGSGPGT, PGTTPGTT, PGTPPTST, PGTSPSAT, PGTGSAGT, PGTGGAGT, PGTSPGAT, PGTSGSGT, PGTSSAST, PGTGAGTT, PGTGSTST, GSEPATSG, APGTSTEP, PGTAGSGT, PGTSSGGT, PGTAGPAT, PGTPGTGT, PGTGGPTT, or PGTGSGST. In some implementations, X n Is TGTS, SGP, TTPG, TPPT, TSPS, TGSA, TGGA, TSPG, TSGS, TSSA, TGAG, TGST, EPAT, GTST, TAGS, TSSG, TAGP, TPGT, TGGP or TGSG.

[0328] In some embodiments, the barcodes are designed to have improved analytical properties. In some embodiments, such barcodes can be released with a relatively moderate concentration of non-mammalian proteases such as Glu-C. This facilitates better detection, for example by LC / MS, and also allows for the measurement of peptides generated from cleavable linkers, thereby allowing for the measurement of cleavage products using, for example, LC / MS.

[0329] In some embodiments of the fusion protein comprising an ELNN, the fusion protein has a single polypeptide chain containing a barcode fragment located within the polypeptide chain at a position 10 to 200 amino acids or 10 to 125 amino acids from the N-terminus or C-terminus of the polypeptide chain. In some embodiments, the fusion protein (such as paTCE) comprises a first ELNN and a second ELNN, the first ELNN being located on the N-terminal side of the bispecific antibody domain, and the first barcode fragment being located within 200, 150, 100, or 50 amino acids from the N-terminus of the fusion protein. In some embodiments, the second ELNN is located on the C-terminal side of the bispecific antibody domain, and the second barcode fragment is located within 200, 150, 100, or 50 amino acids from the C-terminus of the chimeric polypeptide.

[0330] In some embodiments, the ELNN further comprises one or more additional barcode fragments, each of which is different in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide after complete digestion by the protease. In some embodiments, the barcoded ELNN comprises only one barcode fragment. In some embodiments, the barcoded ELNN comprises a set of barcode fragments, which includes a first barcode fragment, such as those described herein. In some embodiments, the set of barcode fragments includes a second barcode fragment (or additional barcode fragments), such as those described herein. In some embodiments, the set of barcode fragments includes a third barcode fragment, such as those described herein.

[0331] A set of barcode fragments fused within an N-terminal ELNN may be referred to as a set of N-terminal barcodes (“N-terminal group”). A set of barcode fragments fused within a C-terminal ELNN may be referred to as a set of C-terminal barcodes (“C-terminal group”). In some embodiments, the N-terminal group comprises a first barcode fragment and a second barcode fragment. In some embodiments, the N-terminal group further comprises a third barcode fragment. In some embodiments, the C-terminal group comprises a first barcode fragment and a second barcode fragment. In some embodiments, the C-terminal group further comprises a third barcode fragment. In some embodiments, the polypeptide comprises a set of barcode fragments comprising a first barcode fragment, a further (second) barcode fragment, and at least one additional barcode fragment, wherein each barcode fragment in the set of barcode fragments (1) is part of a second ELNN and (2) is different in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide after complete digestion by the protease.

[0332] This document includes a mixture comprising multiple polypeptides of different lengths; the mixture comprises a first group of polypeptides and a second group of polypeptides. In some embodiments, each polypeptide in the first group comprises a barcode fragment that (a) can be released from the polypeptide by digestion with a protease, and (b) has a sequence and molecular weight different from all other fragments that can be released from the first group of polypeptides. In some embodiments, the second group of polypeptides lacks the barcode fragment of the first group of polypeptides (e.g., due to truncation). In some embodiments, the first group of polypeptides and the second group of polypeptides each comprise a reference fragment that (a) is common to both the first group of polypeptides and the second group of polypeptides, and (b) can be released by digestion with a protease. In some embodiments, the ratio of the first group of polypeptides to the polypeptide containing the reference fragment is greater than 0.70. In some embodiments, the ratio of the first group of polypeptides to the polypeptide containing the reference fragment is greater than 0.80, 0.90, 0.95, or 0.98. In some embodiments, the reference fragment appears no more than once in each polypeptide of the first group of polypeptides and the second group of polypeptides. In some embodiments, the protease is a protease that cleaves at the C-terminus of a glutamate residue. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not a trypsin. In some embodiments, the polypeptides of different lengths comprise polypeptides containing at least one ELNN, such as any polypeptide described herein. In some embodiments, the first group of polypeptides comprises a full-length polypeptide, wherein the barcode fragment is a portion of the full-length polypeptide. In some embodiments, the full-length polypeptide is a (fusion) polypeptide, such as any polypeptide described above or anywhere else herein. In some embodiments, the polypeptides of different lengths in the mixture are distinguished from each other due to N-terminal truncation, C-terminal truncation, or both N-terminal and C-terminal truncation of the full-length polypeptide. In some embodiments, the first group of polypeptides and the second group of polypeptides may differ in one or more pharmacological properties.

[0333] This disclosure also provides a method for evaluating the relative amounts of a first group of peptides and a second group of peptides in a mixture comprising peptides of different lengths, wherein (1) each peptide in the first group of peptides shares a barcode fragment that appears once and only once in the peptide, and (2) each peptide in the second group of peptides lacks a barcode fragment shared by the first group of peptides, wherein each individual peptide of both the first group of peptides and the second group of peptides contains a reference fragment. In some embodiments, these methods include contacting the mixture with a protease to generate a plurality of proteolytic fragments resulting from cleavage of the first group of peptides and the second group of peptides, wherein the plurality of proteolytic fragments contains a plurality of reference fragments and a plurality of barcode fragments. In some embodiments, these methods may further include determining a ratio of the amount of barcode fragments to the amount of reference fragments to evaluate the relative amounts of the first group of peptides and the second group of peptides. In some embodiments, the barcode fragment appears no more than once in each peptide of the first group of peptides. In some embodiments, the reference fragment appears no more than once in each peptide of the first group of peptides and the second group of peptides. In some embodiments, the plurality of proteolytic fragments comprises a plurality of reference fragments and a plurality of barcode fragments. In some embodiments, the protease cleaves the first group of polypeptides and the second group of polypeptides (or polypeptides of different lengths) at the C-terminus of glutamate residues, which are not followed by proline residues. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not a trypsin. In some embodiments, the step of determining the ratio of the amount of barcode fragment to the amount of reference fragment includes identifying the barcode fragment and the reference fragment from the mixture after contacting the mixture with the protease. In some embodiments, the barcode fragment and the reference fragment are identified based on their respective masses. In some embodiments, the barcode fragment and the reference fragment are identified via mass spectrometry.

[0334] In some embodiments, the barcode fragment and the reference fragment are identified by liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the step of determining the ratio of the barcode fragment to the reference fragment includes isobaric labeling. In some embodiments, the step of determining the ratio of the barcode fragment to the reference fragment includes adding one or both of an isotopically labeled reference fragment and an isotopically labeled barcode fragment to the mixture. In some embodiments, polypeptides of varying lengths comprise polypeptides containing at least one ELNN, as described above or anywhere else herein. In some embodiments, the ELNN is characterized by (i) containing at least 100 or at least 150 amino acids; (ii) at least 90% of the amino acid residues of the ELNN being glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P); and (iii) containing at least four different types of amino acids of the types G, A, S, T, E, or P. In some embodiments, the barcode fragment is part of the ELNN when present. In some embodiments, the mixture of polypeptides of different lengths comprises polypeptides of any polypeptide as described above or anywhere else herein. In some embodiments, the polypeptides of different lengths comprise full-length polypeptides and truncated fragments thereof. In some embodiments, the polypeptides of different lengths consist substantially of full-length polypeptides and truncated fragments thereof. In some embodiments, the polypeptides of different lengths in the mixture are distinguished from each other due to N-terminal truncation, C-terminal truncation, or both N-terminal and C-terminal truncation of the full-length polypeptide. In some embodiments, the full-length polypeptide is a polypeptide as described above or anywhere else herein. In some embodiments, the ratio of the amount of barcode fragment to reference fragment is greater than 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, or 0.99.

[0335] Peptide quantification based on isotope labeling

[0336] In some implementations, isotopic tagging can be used to determine the ratio of a barcode fragment to a reference fragment. Isotopic tagging is a mass spectrometry strategy used in quantitative proteomics, in which a peptide or protein (or a portion thereof) is tagged with various chemical groups that are isotopic (of the same mass) but differ in their distribution of heavy isotopes around their structure. In some implementations, these tags (often referred to as tandem mass tags) are designed such that the mass tag is cleaved at a specific linker region during high-energy collision-induced dissociation (CID) during tandem mass spectrometry, thereby producing reporter ions of different masses. Some of the most common isotopic tags are amine reactive tags.

[0337] Exemplary barcode-encoded ELNN peptide

[0338] This article includes ELNNs containing barcode fragments, which are part of the ELNN.

[0339] The amino acid sequences of exemplary barcoded ELNNs containing one barcode (e.g., SEQ ID NO: 8002 to SEQ ID NO: 8003, SEQ ID NO: 8005 to SEQ ID NO: 8009 and SEQ ID NO: 8013 to SEQ ID NO: 8022), two barcodes (e.g., SEQ ID NO: 8001, SEQ ID NO: 8004 and SEQ ID NO: 8012), or three barcodes (e.g., SEQ ID NO: 8011) are shown in Table 3a. In some implementations, in these exemplary barcode-encoded ELNNs, 12 (SEQ ID NO: 8001 to SEQ ID NO: 8003, SEQ ID NO: 8008 to SEQ ID NO: 8009, SEQ ID NO: 8011, SEQ ID NO: 8015 to SEQ ID NO: 8019 and SEQ ID NO: 8022) are fused to the C-terminus of a bioactive protein (such as TCE), and 10 (SEQ ID NO: 8004 to SEQ ID NO: 8007, SEQ ID NO: 8010, SEQ ID NO: 8012 to SEQ ID NO: 8014, SEQ ID NO: 8020 and SEQ ID NO: 8021) are fused to the N-terminus of the bioactive protein. In some implementations, the ELNN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with the sequences identified herein by SEQ ID NO: 8001 to SEQ ID NO: 8022 in Table 3a.

[0340] Table 3a. Exemplary Barcode-based ELNN

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354] In some embodiments, a barcoded ELNN can be obtained by inducing one or more mutations in an existing ELNN (such as any ELNN listed in Table 3b) according to one or more of the following criteria: minimizing sequence changes in the ELNN, minimizing changes in the amino acid composition of the ELNN, substantially maintaining the net charge of the ELNN, substantially maintaining (or improving) the low immunogenicity of the ELNN, and substantially maintaining (or improving) the pharmacokinetic properties of the ELNN. In some embodiments, the ELNN sequence has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NO: 601 to SEQ ID NO: 659 listed in Table 3b. In some embodiments, the ELNN sequence having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) but less than 100% sequence identity with any of the SEQ ID NO: 601 to SEQ ID NO: 659 listed in Table 3b is obtained by one or more mutations (e.g., less than 10, less than 8, less than 6, less than 5, less than 4, less than 3, less than 2 mutations) of the corresponding sequence from Table 3b. In some embodiments, the one or more mutations include the deletion of a glutamate residue, the insertion of a glutamate residue, the substitution of a glutamate residue, or the substitution of a glutamate residue, or any combination thereof. In some embodiments, when the ELNN sequence differs from any of SEQ ID NO: 601 to SEQ ID NO: 659 listed in Table 3b but has at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the ELNN sequence and the corresponding sequence in Table 3b involves the deletion, insertion, substitution, or replacement of a glutamate residue, or any combination thereof. In some such embodiments, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the ELNN sequence and the corresponding sequence in Table 3b involves the substitution or replacement of a glutamate residue, or both.

[0355] As used herein, “substitution of the first amino acid” means that the first amino acid residue is replaced by the second amino acid residue, resulting in the second amino acid residue occupying the substitution position in the obtained sequence. For example, “substitution of glutamic acid” means that the glutamic acid (E) residue is replaced by a non-glutamic acid residue (e.g., serine (S)).

[0356] Table 3b. Exemplary existing ELNNs for engineering into barcode-based ELNNs

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374] In some implementations, in order to construct the sequence of the barcoded ELNN, amino acid mutations are performed on the ELNNs of intermediate lengths of those in Table 3b and the ELNNs of longer lengths than those in Table 3b, such as those in which one or more 12-mer motifs of Table 1 are added to the N-terminus or C-terminus of the generic ELNNs in Table 3b.

[0375] Additional examples of existing ELNNs that can be used according to this disclosure are disclosed in U.S. Patent Publications No. 2010 / 0239554 A1, 2010 / 0323956 A1, 2011 / 0046060 A1, 2011 / 0046061 A1, 2011 / 0077199 A1, or 2011 / 0172146 A1, or International Patent Publications No. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO2011028229 A1, WO 2011028344 A2, WO 2014 / 011819 A2, or WO No. 2015 / 023891

[0376] In some embodiments, a barcoded ELNN (“N-terminal ELNN”) fused within the polypeptide chain adjacent to the N-terminus may be attached to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus to facilitate purification of the fusion polypeptide. In some embodiments, a barcoded ELNN (“C-terminal ELNN”) fused within the polypeptide chain at the C-terminus may contain or be attached to a sequence EPEA at the C-terminus to facilitate purification of the fusion polypeptide. In some embodiments, the fusion polypeptide comprises both an N-terminal barcoded ELNN and a C-terminal barcoded ELNN, wherein the N-terminal barcoded ELNN is attached at the N-terminus to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49); and wherein the C-terminal barcoded ELNN is attached at the C-terminus to a sequence EPEA, thereby facilitating the purification of the fusion polypeptide to a purity of, for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% by chromatographic methods known in the art (including but not limited to IMAC chromatography, C-tagXL affinity matrix, and other such methods).

[0377] The barcode fragments described herein can be cleavably fused within an ELNN and can be released from the ELNN (i.e., configured for release) after protease digestion of the polypeptide. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease cleaves at the C-terminus of glutamate residues that are not followed by a proline. In some embodiments, the barcode-coded ELNN (wherein the ELNN contains the barcode fragment) is designed to achieve high efficiency, precision, and accuracy of protease digestion. For example, in some embodiments, adjacent Glu-Glu (EE) residues in the ELNN sequence can produce different cleavage patterns during Glu-C digestion. Therefore, when the Glu-C protease is used for barcode release, the barcode-coded ELNN or the barcode fragment may not contain any Glu-Glu (EE) sequence. Additionally, if the dipeptide Glu-Pro (EP) sequence is present in the fusion polypeptide, it may not be cleaved by the Glu-C protease during the barcode release process.

[0378] Structural configurations that can activate TCE

[0379] In some embodiments, the fusion protein comprises a single BsAb in the form of TCE and a single ELNN. In some embodiments, such a fusion protein may have at least the following conformations, each arranged in the N-terminal to C-terminal direction: (TCE)-(ELNN); (ELNN)-(TCE); (TCE)-(linker)-(ELNN); and (ELNN)-(linker)-(TCE).

[0380] In some embodiments, the fusion protein comprises a C-terminal ELNN and optionally a linker between the ELNN and the TCE (such as the linkers described herein, e.g., those in Table C). In some embodiments, such a fusion protein may be represented by Formula I (described as N-terminus to C-terminus): (TCE)-(Connector)-(ELNN)(I),

[0381] Wherein the TCE is as described herein; the linker is a linker sequence containing between 1 and 50 amino acid residues (such as the linker sequence described herein, for example, the linker sequence in Table C), which may optionally contain a TCE release segment (e.g., as described herein); and the ELNN may be any ELNN described herein.

[0382] In some embodiments, the fusion protein comprises an N-terminal ELNN and optionally a linker between the ELNN and the TCE (such as the linker described herein, e.g., the linker in Table C). In some embodiments, such a fusion protein may be represented by Formula II (described as N-terminus to C-terminus): (ELNN)-(Connector)-(TCE)(II),

[0383] Wherein TCE is as described herein; the linker is a linker sequence containing between 1 and 50 amino acid residues (such as the linker sequence described herein, for example, the linker sequence in Table C), which may optionally contain a TCE release segment (e.g., as described herein); and the ELNN may be any ELNN described herein.

[0384] In some embodiments, the fusion protein comprises both N-terminal ELNN and C-terminal ELNN. In some embodiments, such a fusion protein can be represented by Formula III: (ELNN)-(Connector)-(TCE)-(Connector)-(ELNN)(III)

[0385] Wherein the TCE is as described herein; each linker is individually a linker sequence having between 1 and 50 amino acid residues (e.g., the linker sequence described herein, such as the linker sequence in Table C), which may optionally contain a TCE release segment (e.g., as described herein); and each ELNN may individually be any ELNN described herein.

[0386] This disclosure provides BsAbs (e.g., TCEs) that include one or more sequences disclosed herein in any of Tables 5a to 5f.

[0387] Of particular interest are BsAbs (e.g., TCEs) that seek to increase pharmacokinetic parameters, increase solubility, increase stability, masking activity, or some other enhanced drug property, or those BsAbs (e.g., TCEs) whose increased terminal half-life would improve efficacy and / or safety. Therefore, paTCE fusion protein compositions are prepared with various objectives in mind, including improving the therapeutic efficacy of TCEs by, for example, increasing in vivo exposure or the length of time TCEs remain within the therapeutic window when administered to a subject, compared to TCEs without any ELNNs.

[0388] It should be understood that, without departing from the spirit of this disclosure, various amino acid substitutions (especially conserved amino acid substitutions) can be made in the bispecific sequence to produce variants with respect to, for example, the biological activity or pharmacological properties of the TCE. Examples of conserved substitutions of amino acids in peptide sequences are shown in Table 4. Furthermore, variants may also include peptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the full-length native amino acid sequence of the TCE, the TCE retaining at least a portion of the biological activity of the native peptide.

[0389] In some embodiments, sequences that retain at least about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or more of activity compared to the corresponding original TCE sequence are considered suitable for inclusion in the subject paTCE. In some embodiments, it is found that a TCE retaining a suitable level of activity can be linked to one or more ELNN peptides that have at least about 80% sequence identity with the sequences from Tables 3a to 3b (e.g., at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity).

[0390] Table 4: Exemplary Conserved Amino Acid Substitutions

[0391]

[0392]

[0393] This disclosure provides ELNN-modified TCEs (such as paTCE) that target EGFR, wherein the TCE is a bispecific antibody (e.g., a bispecific TCE) that specifically binds to EGFR in a portion of the bispecific TCE and specifically binds to CD3 in another portion of the bispecific TCE.

[0394] In some implementations, the ELNN-based TCE comprises (1) a first portion comprising a first binding domain and a second binding domain, and (2) a second portion comprising a release segment, and (3) a third portion comprising an unstructured peptide mask (sometimes referred to herein as a masking portion).

[0395] In some implementations, the ELNN-TCE ​​includes the configuration of formula Ia (depicting the N-terminus to the C-terminus): (Part 1) - (Part 2) - (Part 3) (Ia)

[0396] The first part is a bispecific antibody domain comprising two antigen-binding domains as described above, wherein the first binding domain has a specific binding affinity for EGFR (e.g., as expressed on cancer cells), and the second binding domain has a specific binding affinity for CD3 (e.g., as expressed on effector cells); the second part comprises a release segment (RS) capable of being cleaved by mammalian proteases; and the third part is a masking portion for masking the biological properties of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment capable of being cleaved by proteases present in the tumor microenvironment.

[0397] In some embodiments, the first portion includes two binding domains, each containing VL and VH, wherein the first portion of the binding domains may be sequential (VL-VH)1-(VL-VH)2, where “1” and “2” represent the first binding domain and the second binding domain, respectively, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, wherein the paired binding domains are connected by a peptide linker (e.g., as described herein).

[0398] In some implementations, the domain that combines EGFR is scFv, which contains VH and VL.

[0399] In some embodiments, the first binding domain comprises the sequences provided in Tables 5a to 5f, wherein Tables 5a to 5e show sequences binding CD3 and Table 5f shows sequences binding EGFR; the RS sequence comprises the sequences provided in Tables 7a to 7b (e.g., as described herein); and the masking portion is an ELNN. In some embodiments, the masking portion is an ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence group shown in Tables 3a to 3b. In some embodiments, the composition is a recombinant fusion protein. In some embodiments, these portions are linked by chemical conjugation.

[0400] In some embodiments, the fusion protein comprises the conformation of formula IIa (described from the N-terminus to the C-terminus): (Part Three) - (Part Two) - (Part One) (IIa)

[0401] The first part is a bispecific antibody comprising two antigen-binding domains, wherein the first binding domain has a specific binding affinity for EGFR (e.g., as expressed on cancer cells), and the second binding domain has a specific binding affinity for CD3 (e.g., as expressed on effector cells); the second part includes a release segment (RS) capable of being cleaved by mammalian proteases; and the third part is a masking portion for masking the biological properties of the bispecific antibody domains. In some embodiments, the RS is a protease-cleavable release segment that is ubiquitously cleavable in the tumor microenvironment.

[0402] In some embodiments, the first portion includes two binding domains, each containing VL and VH, wherein the first portion of the binding domains may be sequential (VL-VH)1-(VL-VH)2, where “1” and “2” represent the first binding domain and the second binding domain, respectively, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, wherein the paired binding domains are connected by a peptide linker (e.g., as described herein).

[0403] In some implementations, the domain that combines EGFR is scFv, which contains VH and VL.

[0404] In some embodiments, the first binding domain comprises the sequences provided in Tables 5a to 6f, wherein Tables 5a to 5e show sequences binding CD3 and Table 5f shows sequences binding EGFR; the RS sequence comprises the sequences provided in Tables 7a to 7b (e.g., as described herein); and the masking portion is an ELNN. In some embodiments, the masking portion is an ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence group shown in Tables 3a to 3b. In some embodiments, the composition is a recombinant fusion protein. In some embodiments, these portions are linked by chemical conjugation.

[0405] In some embodiments, the paTCE composition comprises the configuration of formula Illa (described from the N-terminus to the C-terminus): (Part 5)-(Part 4)-(Part 1)-(Part 2)-(Part 3))(IIIa)

[0406] The first part is a bispecific antibody containing two antigen-binding domains, wherein the first binding domain has a specific binding affinity for EGFR (e.g., as expressed on cancer cells) and the second binding domain has a specific binding affinity for CD3 (e.g., as expressed on effector cells); the second part contains a release segment (RS) cleavable by mammalian proteases; the third part is a masking portion used to mask the biological properties of the bispecific antibody domains; the fourth part contains a release segment (RS) cleavable by mammalian proteases, and the fourth part may be the same as or different from the second part; and the fifth part is a masking portion that may be the same as or different from the third part.

[0407] In some embodiments, the first portion includes two binding domains, each containing VL and VH, wherein the first portion of the binding domains may be sequential (VL-VH)1-(VL-VH)2, where “1” and “2” represent the first binding domain and the second binding domain, respectively, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, wherein the paired binding domains are connected by a peptide linker (e.g., as described herein).

[0408] In some implementations, the domain that combines EGFR is scFv, which contains VH and VL.

[0409] In some embodiments, the first binding domain comprises the sequences provided in Tables 5a to 5f, where Tables 5a to 5e show sequences binding CD3 and Table 5f shows sequences binding EGFR; each RS sequence individually comprises the sequences provided in Tables 7a to 7b (e.g., as described herein); and each masking portion is individually an ELNN. In some embodiments, each masking portion is an ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence group shown in Tables 3a to 3b. In some embodiments, paTCE is a recombinant fusion protein. In some embodiments, one or more portions of paTCE are linked by chemical conjugation.

[0410] This document provides compositions that advantageously provide bispecific therapeutic agents targeting EGFR, which, once cleaved by proteases present in the target tissue or tissue that has become unhealthy due to disease, exhibit greater selectivity, a longer half-life, and result in less toxicity and fewer side effects, thus providing an improved therapeutic index compared to bispecific antibody compositions known in the art. Such compositions can be used to treat cancer. In some embodiments, when the paTCE approaches a target tissue or target cell carrying or secreting a protease capable of cleaving RS, the bispecific binding domain is released from the ELNN by the action of the protease, thereby removing the steric barrier and freeing the TCE to exert its pharmacological action. This property is particularly advantageous in treating immunocold tumors expressing EGFR. In some embodiments, the paTCE provided herein is activated in a target tissue, wherein the target tissue is a solid tumor of an organ or system.

[0411] Combined structural domain

[0412] In some embodiments, the binding domains provided herein comprise one or more full-length antibodies or one or more antigen-binding fragments thereof. Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides that comprise one or more portions of an antibody that specifically binds to an antigen. Any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody, can be used to derive antigen-binding fragments of the antibody, for example, from a complete antibody molecule. The terms binding domain and antibody domain are used interchangeably herein.

[0413] In some implementations, single-chain binding domains are used, such as, but not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single-domain antibodies, VHH, single-chain antibody molecules (scFv), and biantibodies, which are capable of binding ligands or receptors associated with antigens of effector cells and diseased tissues or cells (cancer, tumors, or other malignant tissues).

[0414] In some embodiments, the binding domain is a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain that specifically binds to a first target and a second antigen-binding domain that specifically binds to a second target. In some embodiments, the first antigen-binding domain is a first antigen-binding fragment (e.g., scFv or ISVD, such as VHH), and the second antigen-binding domain is a second antigen-binding fragment (e.g., scFv or ISVD, such as VHH).

[0415] In some embodiments, the antigen-binding fragment (AF) (e.g., the first antigen-binding fragment (AF1) and / or the second antigen-binding fragment (AF2)) can be (each independently) a chimeric, humanized, or human antigen-binding fragment. The antigen-binding fragment (AF) (e.g., the first antigen-binding fragment (AF1) and / or the second antigen-binding fragment (AF2)) can be (each independently) Fv, Fab, Fab', Fab'-SH, linear antibody, VHH, or scFv.

[0416] In some embodiments, one or two antigen-binding fragments (e.g., first and / or second antigen-binding fragments) may be configured as (Fab')2 or single-chain bispecific antibodies. In some embodiments, the bispecific antibody comprises a first binding domain specific for binding to cancer cell markers and a second binding domain specific for binding to effector cell antigens. In some embodiments, the binding domain for the tumor cell target is a variable domain of a T-cell receptor engineered to bind to an MHC loaded with a peptide fragment of a protein overexpressed by tumor cells.

[0417] In some embodiments, the paTCE is designed to provide a wide therapeutic window, taking into account the location of the target cathepsin and the presence of the same protease in healthy tissue not intended to be targeted, as well as the presence of the target ligand in healthy tissue but a greater presence of the ligand in unhealthy target tissue. The "therapeutic window" refers to the difference between the minimum effective dose and the maximum tolerated dose of a given therapeutic composition. In some embodiments, to help achieve a wide therapeutic window for the TCE, the binding domain of the TCE is shielded by proximity to one or more masking (e.g., ELNN) portions, such that the binding affinity of the intact composition for one or both ligands is reduced compared to a composition cleaved by a mammalian protease, thereby releasing the first portion from the shielding effect of the masking portion.

[0418] In some embodiments, the complete antigen recognition and binding site comprises a dimer of a heavy chain variable domain (VH) and a light chain variable domain (VL). Within each VH and VL chain are three complementarity-determining regions (CDRs) that interact to define the antigen binding site on the surface of the VH-VL dimer; the six CDRs of the binding domain confer antigen-binding specificity to the antibody or single-chain binding domain. The framework sequences flanking the CDRs have a tertiary structure that is substantially conserved in cross-species natural immunoglobulins, and framework residues (FRs) serve to hold the CDRs in their proper orientation. In some embodiments, the constant domain is not required for binding function but may contribute to stabilizing the VH-VL interaction. In some embodiments, the binding site may be a pair of VH-VL, VH-VH, or VL-VL domains of the same or different immunoglobulins; however, it is generally preferred to prepare a single-chain binding domain using the corresponding VH and VL chains from the parent antibody. In some embodiments, the order of the VH and VL domains within the polypeptide chain is not restricted, provided that the VH and VL domains are arranged such that the antigen-binding site can fold correctly. Therefore, in some embodiments, the single-chain binding domains containing VH and VL (e.g., in scFv) may have VH and VL arranged in a VL-VH or VL-VH configuration.

[0419] In some implementations, the V chain arrangement can be VH (cancer cell surface antigen) - VL (cancer cell surface antigen) - VL (effective cell antigen) - VH (effective cell antigen), VH (cancer cell surface antigen) - VL (cancer cell surface antigen) - VH (effective cell antigen) - VL (effective cell antigen), VL (cancer cell surface antigen) - VH (cancer cell surface antigen) - VL (effective cell antigen) - VH (effective cell antigen), VL (cancer cell surface antigen) - VH (cancer cell surface antigen) - VH (effective cell antigen) - VL (effective cell antigen), VHH (cancer cell surface antigen) - VL (effective cell antigen) - VH (effective cell antigen), VL (cancer cell surface antigen) - VH (cancer cell surface antigen) - VHH (effective cell antigen) or VH (cancer cell surface antigen) - VL (cancer cell surface antigen) - VHH (effective cell antigen).

[0420] In some implementations, the following sequence is possible: VH (effective cell antigen) - VL (effective cell antigen) - VL (cancer cell surface antigen) - VH (cancer cell surface antigen), VH (effective cell antigen) - VL (effective cell antigen) - VH (cancer cell surface antigen) - VL (cancer cell surface antigen), VL (effective cell antigen) - VH (effective cell antigen) - VL (cancer cell surface antigen) - VH (cancer cell surface antigen), VL (effective cell antigen) - VH (effective cell antigen) - VL (cancer cell surface antigen), VHH (effective cell antigen) - VL (cancer cell surface antigen) - VH (cancer cell surface antigen), VL (effective cell antigen) - VH (effective cell antigen) - VHH (cancer cell surface antigen) or VH (effective cell antigen) - VL (effective cell antigen) - VHH (cancer cell surface antigen).

[0421] As used herein, “…of the N-terminus” or “…of the C-terminus” and their grammatical variations indicate relative positions within the primary amino acid sequence, rather than placement at the absolute N-terminus or C-terminus of the bispecific single-chain antibody. Therefore, by way of non-limiting example, “located at the C-terminus of the second binding domain” means that the first binding is located on the carboxyl side of the second binding domain within the bispecific single-chain antibody, and does not preclude the possibility of additional sequences, such as linkers and / or ELNNs, His tags, or other compounds such as radioisotopes, located at the C-terminus of the bispecific single-chain antibody.

[0422] In some embodiments, the paTCE comprises a first portion containing a first binding domain and a second binding domain, wherein each binding domain is an scFv, and wherein each scFv contains a VL and a VH. In some embodiments, the first binding domain is a CD3-binding scFv, and the second binding domain is an EGFR-binding scFv. In some embodiments, the paTCE composition comprises a first portion containing a first binding domain and a second binding domain, wherein one of these binding domains is an scFv, and the other binding domain is a VHH. In some embodiments, the paTCE comprises a first portion containing a first binding domain and a second binding domain, wherein these binding domains are in a biantibody configuration, and one domain contains a VL region and a VH region, and the other domain contains a VL region and a VH region. Exemplary VH and VL of the CD3 binding domain are shown in Tables 5a to 5e. Exemplary VH and VL of the EGFR binding domain are shown in Table 5f.

[0423] In non-limiting examples, the TCE may contain a sequence exhibiting at least about 80% sequence identity with the antibody sequence identified herein, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the TCE contains a bispecific sequence (e.g., BsAb) containing a first binding domain and a second binding domain, wherein the first binding domain has a specific binding affinity for a tumor-specific marker or cancer cell antigen, and exhibits at least about 80% sequence identity with the paired VL and VH sequences of the anti-EGFR antibody disclosed herein in Table 5f, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or 100% sequence identity. 6%, 97%, 98%, 99% or 100% sequence identity; and wherein the second binding domain has a specific binding affinity for effector cells and exhibits at least about 80% sequence identity with the paired VL and VH sequences of the anti-CD3 antibody disclosed herein in any of Tables 5a to 5e, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0424] In some embodiments, the TCE may include a binding domain (e.g., VH and / or VL amino acid sequences) of or derived from an anti-CD3 antibody. Non-limiting examples of anti-CD3 antibodies include OKT3 (also known as muromonab) and the humanized anti-CD3 monoclonal antibody hOKT31 (Ala-Ala) (KC Herold et al., New England Journal of Medicine 346:1692-1698.2002), as well as fragments and derivatives thereof that selectively bind CD3. Additional examples are described in U.S. Patent Nos. 5,885,573, 6,491,916, and U.S. Patent Application Publication No. 2021 / 0054077-A1, the entire contents of which are incorporated herein by reference. Additional non-limiting examples of anti-CD3 antibody sequences include those of pertuxizumab (also known as AMG-212) and acapatamab (also known as AMG-160).

[0425] In some embodiments, the TCE may include a binding domain (e.g., VH and / or VL amino acid sequences) of or derived from an anti-EGFR antibody. Non-limiting examples of anti-EGFR antibody sequences include those of panitumumab and cetuximab.

[0426] This disclosure provides an antigen-binding domain that binds to EGFR. This disclosure provides EGFR-binding scFvs (e.g., scFvs having paired VH and VL as shown in Table 5f). This disclosure further provides nucleic acids encoding antigen-binding domains (e.g., scFvs) or polypeptides, as well as vectors, hosts, and methods for generating such antigen-binding domains or polypeptides. Multispecific polypeptides are also provided, comprising an EGFR-binding antigen-binding domain according to this disclosure and at least one CD3-binding domain, including paTCE. Methods for therapeutic use utilizing the antigen-binding domains or polypeptides according to this disclosure are also included.

[0427] Also provided are nucleic acid molecules encoding antigen-binding domains (e.g., scFv) or polypeptides disclosed herein, or vectors containing such nucleic acids.

[0428] This disclosure also relates to the transformation or transfection of non-human host or host cells with a nucleic acid or vector encoding an antigen-binding domain (e.g., scFv) or polypeptide disclosed herein.

[0429] This disclosure also relates to compositions comprising antigen-binding domains (e.g., scFv) or peptides disclosed herein, such as pharmaceutical compositions.

[0430] This document includes a method for generating antigen-binding domains (e.g., scFv) or peptides as disclosed herein, the method comprising the following steps: a. Expressing, in a host cell or host organism, or in another expression system, a nucleic acid sequence encoding an antigen-binding domain (e.g., scFv) or a polypeptide; optionally, followed by: b. Isolate and / or purify antigen-binding domains (e.g., scFv) or peptides.

[0431] This document provides compositions and peptides comprising antigen-binding domains (e.g., scFv) for use as pharmaceuticals. In some embodiments, these peptides or compositions are used to treat proliferative diseases. In some embodiments, the proliferative disease is cancer.

[0432] This disclosure also provides a treatment method comprising the step of administering to a subject in need a composition or peptide comprising an antigen-binding domain (e.g., scFv). In some embodiments, the treatment method is used to treat proliferative disorders. In some embodiments, the proliferative disorder is cancer.

[0433] This document includes compositions and peptides containing antigen-binding domains (e.g., scFv) for preparing a medicament. In some embodiments, the medicament is used to treat proliferative diseases. In some embodiments, the proliferative disease is cancer.

[0434] In some embodiments, the structure of the VH or VL of the antigen-binding domain (e.g., scFv) sequence may be considered to contain four frame regions (“FR”), referred to in the art and herein as “Frame Region 1” (“FR1”); “Frame Region 2” (“FR2”); “Frame Region 3” (“FR3”); and “Frame Region 4” (“FR4”), respectively; these frame regions are interrupted by three complementarity-determining regions (“CDR”), referred to in the art and herein as “Complementarity-determining Region 1” (“CDR1”); “Complementarity-determining Region 2” (“CDR2”); and “Complementarity-determining Region 3” (“CDR3”), respectively.

[0435] In some implementations, the techniques provided herein use antigen-binding domains (e.g., scFv) capable of binding to EGFR. In the context of this technique, “binding” to a target molecule has a common meaning in the art, as understood in the context of antibodies and their corresponding antigens.

[0436] As will be clear from the above and further description herein, the antigen-binding domains (e.g., scFv) of this technology can be used as “building blocks” to form polypeptides of this technology, for example, by appropriately combining them with other groups, residues, portions or binding units to form compounds or fusion proteins as described herein (such as, but not limited to, the bivalent / trivalent / tetravalent / multivalent polypeptides and bivalent / trivalent / tetravalent / multispecific polypeptides of this technology described herein), which combine one or more desired properties or biological functions within a single molecule.

[0437] The term "specificity" or "specific binding" refers to the number of different target molecules (such as antigens) from the same organism to which a particular binding unit (such as an antigen-binding domain (e.g., scFv)) can bind with sufficiently high affinity (see below). "Specificity" or "specific binding" is used interchangeably herein with "selectivity" or "selective binding." A binding unit, such as scFv, preferably binds specifically to its designated target.

[0438] The specificity / selectivity of binding units can be determined based on affinity. Affinity represents the strength or stability of molecular interactions. Affinity is typically determined by K... D It is given, and it is expressed in moles per liter (or M).

[0439] Affinity is a measure of the strength of binding between a portion of a molecule and its binding site on a target molecule: K D The lower the value, the stronger the binding strength between the target molecule and the target region.

[0440] Typically, the bonding units (such as scFv) used in this technology will be in the form of 10 -5 Up to 10 -12 mol / L or less, preferably 10 -7 Up to 10 -12 mol / L or less, and more preferably 10 -8 Up to 10 -12 moles / liter of K D Combined with its target.

[0441] In some implementations, greater than 10 -4 moles / liter of K D The value is considered nonspecific. In some implementations, values ​​less than 10 are acceptable. -4 moles / liter of K D The value is considered specific.

[0442] K is considered a specific biological interaction, such as the binding of antibody sequences to antigens. D Typically in the range of 10,000 nM or 10 μM to 0.001 nM or 1 μM or smaller.

[0443] Therefore, specific / selective binding can mean—using the same measurement method, such as SPR—binding units (or peptides containing them) at 10 -5 Up to 10 -12 K moles per liter or less D Values ​​that combine with EGFR and are greater than 10 -4 moles / liter of K D Values ​​are combined with different targets.

[0444] Specific binding to a specific target from a specific species does not preclude the binding unit from specifically binding to similar targets from different species. For example, specific binding to human EGFR does not preclude the binding unit (or the polypeptide containing it) from specifically binding to EGFR from cynomolgus monkeys.

[0445] The specific binding of the binding unit to its designated target can be determined by any suitable means known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay, as well as various variants of these known per se in the art; and other techniques mentioned herein.

[0446] The dissociation constant can be, for example, an actual or apparent dissociation constant, as will be clear to a person skilled in the art. The methods used to determine the dissociation constant will be clear to a person skilled in the art and include, for example, the techniques mentioned below.

[0447] The affinity of molecular interactions between two molecules can be measured using various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technology (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559). As used herein, the term “surface plasmon resonance” refers to an optical phenomenon that allows the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip, and another molecule flows through the immobilized molecule under flow conditions, generating k on k off Measured values, and thus K. D Value. This can be achieved, for example, using the well-known BIAcore. ® The system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ) was used. For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991 Biotechniques 11: 620-627), Johnson et al. (1995, J. Mol. Recognit. 8: 125-131) and Johnson et al. (1991, Anal. Biochem. 198: 268-277).

[0448] Another well-known biosensor technique for determining the affinity of biomolecular interactions is biomembrane interferometry (BLI) (see, for example, Abdiche et al. 2008, Anal. Biochem. 377: 209-217). As used herein, the term “biomembrane interferometry” or “BLI” refers to a label-free optical technique for analyzing the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a fixed protein layer (signal beam) on the biosensor tip. Changes in the number of molecules bound to the biosensor tip cause a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the surface of the biosensor tip. Because interactions can be measured in real time, association and dissociation rates, as well as affinity, can be determined. BLI can, for example, use the well-known Octet... ® The system (ForteBio, division of Pall Life Sciences, Menlo Park, USA) was used.

[0449] Alternatively, affinity can be determined using KinExA in a kinetic exclusion assay (KinExA) (see, for example, Drake et al. 2004, Anal. Biochem., 328: 35-43). ® The platform (Sapidyne Instruments Inc, Boise, USA) measures the binding affinity and kinetics of unmodified molecules. As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of an antibody / antigen complex is passed through a column containing beads pre-coated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is performed using a fluorescently labeled protein that binds to the antibody (or antigen).

[0450] GYROLAB ® Immunoassay systems provide a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).

[0451] In some embodiments, the patCE comprises a first binding domain and a second binding domain of the scFv. In some embodiments, the first scFv comprises VL and VH domains and specifically binds to effector cell antigens (such as CD3), and the second scFv specifically binds to cancer cell antigens (such as EGFR). In some embodiments, the scFv comprises six CDRs. In some embodiments, the scFv containing the VH and VL regions comprises amino acid sequences that are at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to or the same as those of the paired VL and VH sequences of the anti-CD3 antibodies identified in Table 5a. In some embodiments, the scFv comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions of the paired VL and VH sequences of the anti-CD3 antibodies identified in Table 5a. In some embodiments, the scFv is derived from an anti-EGFR antibody identified as an antibody shown in Table 5f. In some embodiments, the scFv comprises a VH region and a VL region containing amino acid sequences that are at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical or the same as the VH and VL sequences disclosed in Table 5f. In some embodiments, the VH and VL contain the CDR-1, CDR-2, and CDR-3 regions of the VH and VL sequences in Table 5f.

[0452] In some embodiments, the paTCE comprises a first binding domain that is also an scFv and a second binding domain that is also an scFv. In some embodiments, the scFv comprises a VL domain and a VH domain, respectively derived from monoclonal antibodies having binding specificity to tumor-specific markers or cancer cell antigens and effector cell antigens. In some embodiments, the first and second binding domains each comprise six CDRs, respectively derived from monoclonal antibodies having binding specificity to cancer cell markers (such as tumor-specific markers) and effector cell antigens. In some embodiments, the first and second binding domains of the first portion of the subject composition may have 3, 4, 5, or 6 CDRs within each binding domain. In some embodiments, the paTCE comprises a first and second binding domain, each comprising a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, wherein each region is derived from a monoclonal antibody capable of binding to tumor-specific markers or cancer cell antigens and effector cell antigens.

[0453] In some embodiments, the second binding domain comprises a VH region and a VL region derived from a monoclonal antibody capable of binding human CD3. In some embodiments, the second binding domain comprises an scFv containing a VH region and a VL region, wherein each VH region and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the paired VL and VH sequences of the anti-CD3 antibodies identified in Table 5a. In some embodiments, the second domain comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, wherein each region is derived from a monoclonal antibody identified herein as an antibody shown in Table 5a. In some embodiments, the VH domain and / or the VL domain may be configured as an scFv or a biantibody.

[0454] In some embodiments, paTCE comprises a first binding domain and a second binding domain that are also biantibodies. In some embodiments, the biantibody comprises a VL domain and a VH domain derived from monoclonal antibodies that have binding specificity to tumor-specific markers or cancer cell antigens and effector cell antigens, respectively.

[0455] In some embodiments, this disclosure provides a paTCE composition wherein the second binding domain of the biantibody comprises a VH region and a VL region, wherein the VH region and the VL region each exhibit at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the VL and VH sequences of the huUCHT1 antibody of Table 5a. In some embodiments, the second binding domain of the biantibody in the composition is derived from the anti-CD3 antibody described herein. In some embodiments, the anti-CD3 biantibody is linked to the anti-EGFR binding scFv sequence disclosed herein.

[0456] Methods for measuring the binding affinity and / or other biological activities of antigen-binding domains can be those disclosed herein or methods generally known in the art. For example, various suitable assays can be used to determine the binding affinity denoted as K. D The binding affinity of binding pairs (e.g., antibodies and antigens) is measured, including but not limited to radioactive binding assays, non-radioactive binding assays such as fluorescence resonance energy transfer and surface plasmon resonance (SPR, Biacore), as well as enzyme-linked immunosorbent assays (ELISA) and kinetic exclusion assays (KinExA). ®(or as described in the embodiments.) An increase or decrease in binding affinity, for example, the increased binding affinity of a TCE that has been cut to remove the masking portion compared to a paTCE with an attached masking portion, can be determined by measuring the binding affinity of the TCE to its target binding partner with and without the masking portion.

[0457] The half-life of a subject chimeric assembly can be measured using a variety of suitable methods. For example, the half-life of a substance can be determined by administering the substance to a subject and periodically sampling a biological sample (e.g., a biological fluid, such as blood or plasma or ascites) to determine the concentration and / or amount of the substance in the sample over time. The concentration of the substance in the biological sample can be determined using a variety of suitable methods, including enzyme-linked immunosorbent assay (ELISA), immunoblotting, and chromatographic techniques, including high-performance liquid chromatography and rapid protein liquid chromatography. In some cases, the substance can be labeled with a detectable tag (such as a radioactive tag or a fluorescent tag) that can be used to determine the concentration of the substance in a sample (e.g., a blood sample or plasma sample). Various pharmacokinetic parameters are then determined from the results, which can be done using software packages such as SoftMax Pro or by manual calculations known in the art.

[0458] Furthermore, the physicochemical properties of the paTCE composition can be measured to determine the extent of solubility, structure, and stability retention. Determination of the subject composition allows for the determination of the binding characteristics of the binding domains to the ligands, including affinity and binding constant (K). D k on and k off The half-life of the ligand-receptor complex dissociation, and the activity of the binding domain in inhibiting the biological activity of the isolated ligand compared to the free ligand (IC50). 50 Value). Term "EC" 50 "" refers to the concentration required to achieve half of the maximum biological response of an active substance, and is typically determined by ELISA or cell-based assays, including the methods described in the embodiments herein.

[0459] Anti-CD3 binding domain

[0460] Anti-CD3 antibodies, fragments thereof, and fusion proteins containing such antibodies and / or fragments are also provided.

[0461] In some embodiments, this disclosure provides a paTCE composition comprising a binding domain of a first portion having binding affinity for T cells. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody binding CD3. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody against CD3ε and / or CD3δ. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody against CD3ε. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody against CD3δ. Exemplary non-limiting examples of VL and VH sequences of monoclonal antibodies against CD3 are presented in Table 5a. In some embodiments, this disclosure provides a paTCE comprising a binding domain having binding affinity for CD3, the binding domain comprising the anti-CD3 VL and VH sequences shown in Table 5a. In some embodiments, this disclosure provides a paTCE comprising a binding domain of a first portion having binding affinity for CD3ε, the binding domain comprising the anti-CD3ε VL and VH sequences shown in Table 5a. In some embodiments, this disclosure provides a paTCE composition wherein the binding domain of the first portion comprises an scFv comprising a VH region and a VL region, wherein each VH region and VL region exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity with or identical to the paired VL and VH sequences of the huUCHT1 anti-CD3 antibody of Table 5a. In some embodiments, this disclosure provides a paTCE composition comprising a binding domain having binding affinity for CD3, the binding domain comprising CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 regions, each derived from the corresponding anti-CD3VL and VH sequences shown in Table 5a. In some embodiments, this disclosure provides a paTCE composition comprising a binding domain having binding affinity for CD3, the binding domain comprising the CDR-L1 region of RSSNGAVTSSNYAN (SEQ ID NO: 1), the CDR-L2 region of GTNKRAP (SEQ ID NO: 4), the CDR-L3 region of ALWYPNLWV (SEQ ID NO: 6), the CDR-H1 region of GFTFSTYAMN (SEQ ID NO: 12), the CDR-H2 region of RIRTKRNNYATYYADSVKG (SEQ ID NO: 13), and the CDR-H3 region of HENFGNSYVSWFAH (SEQ ID NO: 10).In some embodiments, this disclosure provides a paTCE composition comprising a binding domain having binding affinity for CD3, the binding domain comprising the CDR-L1 region of RSSNGAVTSSNYAN (SEQ ID NO: 1), the CDR-L2 region of GTNKRAP (SEQ ID NO: 4), the CDR-L3 region of ALWYPNLWV (SEQ ID NO: 6), the CDR-H1 region of GFTFSTYAMN (SEQ ID NO: 12), the CDR-H2 region of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and the CDR-H3 region of HENFGNSYVSWFAH (SEQ ID NO: 10).

[0462] The CD3 complex is a group of cell surface molecules that associate with the T-cell antigen receptor (TCR) and play a role in TCR expression on the cell surface and in the signal transduction cascade that occurs when peptide:MHC ligands bind to the TCR. Unbound by any scientific theory, generally, when an antigen binds to a T-cell receptor, CD3 sends a signal across the cell membrane into the cytoplasm inside the T cell. This triggers T-cell activation, which rapidly divides to produce new T cells that are sensitized to attack the specific antigen exposed to the TCR. The CD3 complex contains the CD3ε molecule and four other membrane-bound peptides (CD3-γ, CD3-δ, and / or CD3-ζ). In humans, CD3-ε is encoded by the CD3E gene on chromosome 11. The intracellular domain of each CD3 chain contains an activation motif (ITAM) based on the tyrosine residue of the immune receptor, which acts as a nucleation site for intracellular signal transduction mechanisms upon T-cell receptor binding.

[0463] Many therapeutic strategies modulate T-cell immunity by targeting TCR signaling, particularly anti-human CD3 monoclonal antibodies (mAbs) widely used in clinical immunosuppressive regimens. The CD3-specific mouse mAb OKT3 was the first mAb licensed for human use (Sgro, C. Side-effects of a monoclonal antibody, muromonab CD3 / orthoclone OKT3: bibliographic review. Toxicology 105:23-29, 1995), and is widely used clinically as an immunosuppressant in transplantation (Chatenoud, Clin. Transplant 7:422-430, (1993); Chatenooud, Nat. Rev. Immunol. 3:123-132 (2003); Kumar, Transplant. Proc. 30:1351-1352 (1998)), type 1 diabetes, and psoriasis. Importantly, anti-CD3 mAbs can induce partial T cell signaling and clonal unresponsiveness (Smith, JA, Nonmitogenic Anti-CD3 Monoclonal Antibodies Delivera Partial T Cell Receptor Signal and Induce Clonal Anergy J. Exp.Med.185:1413-1422 (1997)). OKT3 has been described in the literature as a T cell mitogen and an effective T cell killer (Wong, JT. The mechanism of anti-CD3 monoclonal antibodies. Mediation of cytolysis by inter-T cell bridging. Transplantation 50:683-689 (1990)). In particular, Wong's research demonstrated that target killing can be achieved by bridging CD3 T cells and target cells, and that FcR-mediated ADCC and complement fixation are not necessary for bivalent anti-CD3 MAB to lyse target cells.

[0464] OKT3 exhibits mitogenic and T-cell cytotoxic activity in a time-dependent manner; following early T-cell activation that leads to cytokine release, OKT3 subsequently blocks all known T-cell functions upon further administration. It is precisely because of this subsequent T-cell function blockade that OKT3 has been found to be widely used as an immunosuppressant in treatment regimens to reduce or even eliminate allogeneic graft rejection. Other antibodies specific to the CD3 molecule are disclosed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50. WO2005 / 118635 and WO2007 / 033230 describe anti-human monoclonal CD3ε antibodies. U.S. Patent 5,821,337 describes the VU and VH sequences of mouse anti-CD3 monoclonal Ab UCHT1 (muxCD3, Shalaby et al., J. Exp. Med. 175, 217-225 (1992) and a humanized variant of the antibody (hu UCHT1). U.S. Patent Application 20120034228 discloses a binding domain capable of binding to epitopes of human and non-chimpanzee primate CD3ε chains.

[0465] In some embodiments, the anti-CD3 antibody domain comprises a VH region or its CDR containing the sequence EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311), and a VL region or its CDR containing the sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361).

[0466] In some embodiments, the anti-CD3 antibody domain comprises a VH region or its CDR containing the sequence EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126), and a VL region or its CDR containing the sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127).

[0467] Table 5a: Anti-CD3 monoclonal antibodies and sequences

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475] In some embodiments, this disclosure relates to antigen-binding fragments (AFs) that have specific binding affinity for effector cell antigens.

[0476] Various AFs that bind to effector cell antigens, particularly CD3 on T cells, have specific utilities that pair with antigen-binding fragments that have binding affinity for EGFR antigens associated with diseased cells or tissues in combination form, in order to recruit and achieve effector cell-mediated cell killing of diseased cells or tissues.

[0477] The binding specificity with the antigen of interest can be determined by the complementarity-determining region (CDR) or CDR (such as the light chain CDR or heavy chain CDR). In many cases, binding specificity is determined by both the light chain CDR and the heavy chain CDR. A given combination of heavy chain CDR and light chain CDR provides a given binding pocket that confers greater affinity and / or specificity to effector cell antigens compared to other reference antigens. The resulting bispecific composition is bispecific, wherein each antigen-binding fragment has a specific binding affinity for its corresponding ligand. This bispecific composition binds to effector cell antigens on one hand and to antigens on diseased cells or tissues on the other, having a first antigen-binding fragment targeting EGFR linked to a second antigen-binding fragment with binding specificity to effector cell antigens via a short, flexible peptide linker.

[0478] It should be understood that in such compositions, AFs targeting EGFR in diseased tissue are used in combination with AFs targeting effector cell markers to bring effector cells into close proximity to cells in the diseased tissue, thereby achieving cell lysis of the diseased tissue cells. Furthermore, a first antigen fragment (AF1) and a second antigen fragment (AF2) are incorporated into a specially designed polypeptide containing a cleavable release segment and an ELNN segment to impart an inactive character to the composition. Upon approach to diseased tissue having a protease capable of cleaving the release segment at one or more sites within the release segment sequence, these compositions are activated by releasing fused AF1 and AF2 after cleaving the release segment.

[0479] In some embodiments, the AF2 of the subject composition has binding affinity for effector cell antigens expressed on the surface of T cells. In some embodiments, the AF2 of the subject composition has binding affinity for CD3. In some embodiments, the AF2 of the subject composition has binding affinity for members of the CD3 complex, including all known CD3 subunits of the CD3 complex in individual or independently combined forms; for example, CD3ε, CD3δ, CD3γ, and CD3ζ. In some embodiments, AF2 has binding affinity for CD3ε, CD3δ, CD3γ, or CD3ζ.

[0480] In some embodiments, the present invention provides an antigen-binding domain (e.g., an antibody or its antigen-binding fragment) that binds to the differentiation cluster 3 T cell receptor (CD3), the antigen-binding domain comprising the following CDRs: a VL domain CDR1 having an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with RSSX1GAVTX2SNYAN, wherein X1 corresponds to T or N, and X2 corresponds to T or S; and a VL domain CDR2 having an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with GTNKRAP. 99% or 100% identity with ALWYX4NLWV; VL domain CDR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with ALWYX4NLWV, where X4 corresponds to S or P; VH domain CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with GFTFX8TYAMN, where X8 corresponds to S or N; VH domain CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with RIRX. 10 KX 11 NX 12 YATYYADSVKX 13 Having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or 100% identity, where X 10 Corresponding to T or S, X 11 Corresponding to R or Y, X 12 Corresponding to D or N, and X 13 Corresponding to G or D; VH domain CDR3, its amino acid sequence is the same as HX. 14 NFGNSYVSWFAX 15 Having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or 100% identity, where X 14 Corresponding to E or G, and X 15 Corresponding to H or Y.

[0481] In some embodiments, the present invention provides an antigen-binding domain (e.g., an antibody or its antigen-binding fragment) that binds to differentiation cluster 3 T cells (CD3), the antigen-binding domain comprising the following CDRs: VL region CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with RSSNGAVTSSNYAN; VL region CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with GTNKRAP; VL region CDR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with ALWYPNLWV. 8% or 99% or 100% identity; VH region CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with GFTFSTYAMN; VH region CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with RIRTKRNDYATYYADSVKG; and VH region CDR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with HENFGNSYVSWFAH.

[0482] In some embodiments, the antigen-binding domain comprises the following FRs: VL region FR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with ELVVTQEPSLTVSPGGTVTLTC; VL region FR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with WVQQKPGQAPRGLIG. 9% or 100% identity; VL region FR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity with GTPARFSGSLLEGKAALTLSGVQPEDEAVYYC; VL region FR4, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with FGGGTKLTVL. Or 100% identity; VH region FR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity with EVQLVESGGGIVQPGGSLRLSCAAS; VH region FR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity with WVRQAPGKGLEWVG. The amino acid sequence of FR3 in the VH region is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical to that of FRTISRDDSKNTLYLQMNSLKTEDTAVYYCVR; and the amino acid sequence of FR4 in the VH region is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or 100% identical to that of WGQGTLVTVSS.

[0483] In some embodiments, this disclosure provides an antigen-binding domain (e.g., an antibody or an antigen-binding fragment thereof) that binds to CD3, the antigen-binding domain comprising: a VL region containing three VL CDRs, wherein the three VL CDRs comprise CDR1, CDR2, and CDR3 of the VL region containing the following amino acid sequences: ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127); and a VH region containing three VH CDRs, wherein these three VH CDRs contain CDR1, CDR2 and CDR3 of the VH region containing the following amino acid sequence: EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126).

[0484] In some embodiments, the present invention provides an antigen-binding domain (e.g., an antibody or its antigen-binding fragment) that binds to differentiation cluster 3 T cells (CD3), the antigen-binding domain comprising the following CDRs: VL region CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with RSSNGAVTSSNYAN; VL region CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with GTNKRAP; VL region CDR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with ALWYPNLWV. 8% or 99% or 100% identity; VH region CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with GFTFSTYAMN; VH region CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with RIRTKRNNYATYYADSVKG; and VH region CDR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with HENFGNSYVSWFAH.

[0485] In some embodiments, the antigen-binding domain comprises the following FRs: VL region FR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with ELVVTQEPSLTVSPGGTVTLTC; VL region FR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with WVQQKPGQAPRGLIG. 9% or 100% identity; VL region FR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity with GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC; VL region FR4, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with FGGGTKLTVL. Or 100% identity; VH region FR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity with EVQLVESGGGIVQPGGSLRLSCAAS; VH region FR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or 100% identity with WVRQAPGKGLEWVG. Sex; VH region FR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR; and VH region FR4, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with WGQGTLVTVSS.

[0486] In some embodiments, this disclosure provides an antigen-binding domain (e.g., an antibody or an antigen-binding fragment thereof) that binds to CD3, the antigen-binding domain comprising: a VL region containing three VL CDRs, wherein the three VL CDRs comprise CDR1, CDR2, and CDR3 of the VL region containing the following amino acid sequences: ELVVTQEPSLTVSPGGTVTLTCRSSX1GAVTX2SNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX3YYCALWYX4NLWVFGGGTKLTVL, where X1 corresponds to T or N, X2 corresponds to T or S, X3 corresponds to E or V, and X4 corresponds to S or P; and a VH region containing three VH CDRs, wherein these three VH CDRs contain CDR1, CDR2, and CDR3 of the VH region containing the following amino acid sequences: EVQLX5ESGGGX6VQPGGSLX7LSCAASGFTFX8TYAMNWVRQAPGKGLEWVX9RIRX 10 KX 11 NNYATYYADSVKX 12 RFTISRDDSKNTX 13 YLQMNX 14 LKTEDTAVYYCVRHX 15 NFGNSYVSWFAX 16 WGQGTLVTVSS, where X5 corresponds to V or L, X6 corresponds to I or L, X7 corresponds to R or K, X8 corresponds to S or N, X9 corresponds to G or A, X 10 Corresponding to T or S, X 11 Corresponding to R or Y, X 12 Corresponding to G or D, X 13 Corresponding to V or A, X 14 Corresponding to S or N, X 15 Corresponding to E or G, and X 16 Corresponding to H or Y.

[0487] In some embodiments, this disclosure provides an antigen-binding domain (e.g., an antibody or an antigen-binding fragment thereof) that binds to CD3, the antigen-binding domain comprising: a VL region containing three VL CDRs, wherein the three VL CDRs comprise CDR1, CDR2, and CDR3 of the VL region containing the following amino acid sequences: ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361); and a VH region containing three VH CDRs, wherein these three VH CDRs contain CDR1, CDR2, and CDR3 of the VH region containing the following amino acid sequences: EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311).

[0488] In some embodiments, this disclosure provides an antigen-binding domain (e.g., an antibody or an antigen-binding fragment thereof) that binds to CD3, the antigen-binding domain comprising a VL region amino acid sequence SEQ ID NO / VH region amino acid sequence SEQ ID NO selected from the group consisting of: 896 / 897; 902 / 903; 700 / 701; 702 / 703; 716 / 717; 718 / 719; 728 / 729; 736 / 737; 738 / 739; 740 / 741; 742 / 743; 744 / 745; 746 / 747; 748 / 749; 750 / 751; 752 / 753; 754 / 755; 756 / 75 7; 758 / 759; 760 / 761; 762 / 763; 764 / 765; 766 / 767; 774 / 775, 776 / 777; 790 / 791; 792 / 793; 798 / 799; 800 / 801; 806 / 807; 808 / 809; 814 / 815; 816 / 817; 822 / 823; 824 / 825; or 826 / 867.

[0489] In some embodiments, this disclosure provides antigen-binding fragments (e.g., AF1 or AF2) that bind to CD3 protein complexes, exhibiting enhanced stability compared to CD3-binding antibodies or antigen-binding fragments known in the art. In some embodiments, the CD3 antigen-binding fragments of this disclosure are designed to confer a higher degree of stability to chimeric bispecific antigen-binding fragment compositions into which they are integrated, resulting in improved expression and recovery of the fusion protein, increased shelf life, and enhanced stability upon administration to a subject. In some embodiments, the anti-CD3 AF of this disclosure exhibits a higher degree of thermostability compared to certain CD3-binding antibodies and antigen-binding fragments known in the art. In some embodiments, the anti-CD3 AF of this disclosure exhibits a higher degree of thermostability compared to SP34 or its antigen-binding fragment. In some embodiments, the anti-CD3 AF of this disclosure exhibits a higher degree of thermostability compared to CD3.9 and / or CD3.23 disclosed in PCT International Patent Application Publication No. WO2021263058, the entire contents of which are hereby incorporated by reference. In some embodiments, the anti-CD3 AF of this disclosure exhibits lower immunogenicity in humans compared to certain CD3-binding antibodies and antigen-binding fragments known in the art. In some embodiments, the immunogenicity of the anti-CD3 AF of this disclosure is lower than that of SP34 or its antigen-binding fragment. In some embodiments, the immunogenicity of the anti-CD3 AF of this disclosure is lower than that of CD3.9 and / or CD3.23 disclosed in PCT International Patent Application Publication No. WO2021263058, the entire contents of which are hereby incorporated herein by reference. In some embodiments, the immunogenicity of AFs is determined by immunogenicity prediction methods such as TELEPITOPAN (described in Zhang et al. PLoS One. 2012;7(2):e30483. doi: 10.1371 / journal.pone.0030483, PMID: 22383964, the entire contents of which are incorporated herein by reference) or NetMHCpan-4.1 and NetMHCiPlan-4.0 (each described in Reynisson et al., Nucleic Acids Res2020; 48(W1):W449-W454. doi: 10.1093 / nar / gkaa379„ PMID: 32406916, the entire contents of which are hereby incorporated herein by reference). In some embodiments, anti-CD3 is used as a component of the chimeric bispecific antigen-binding fragment composition to which they are integrated. AF exhibits favorable drug properties, including high thermal stability and low aggregation tendency, resulting in improved expression and recovery during manufacturing and storage, as well as promoting a long serum half-life.Biophysical properties, such as thermal stability, are often limited by antibody variable domains, which vary considerably in their inherent properties. High thermal stability is typically 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). In some implementations, thermal stability is measured by the “melting temperature” (T). m The melting temperature is determined by [method / method], defined as the temperature at which half of the molecules are denatured. The melting temperature of each heterodimer indicates its thermal stability. [The determination of T] m The in vitro determination of the heterodimer is known in the art, including 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), or as described in the following examples.

[0490] In some embodiments of the peptides disclosed herein, the antigen-binding fragment (e.g., AF1 or AF2) may exhibit higher thermal stability than the anti-CD3-binding fragment consisting of the sequence of SEQ ID NO: 206 (see Table 5e), as demonstrated in in vitro assays by the higher melting temperature (T0) of the first antigen-binding fragment relative to the anti-CD3-binding fragment. m As demonstrated by ); or after incorporating the first antigen-binding fragment into the test bispecific antigen-binding domain, the T of the test bispecific antigen-binding domain is... m The test bispecific antigen-binding domain is higher than the control bispecific antigen-binding domain, wherein the test bispecific antigen-binding domain includes a first antigen-binding fragment and a reference antigen-binding fragment binding antigens other than CD3; and wherein the control bispecific antigen-binding domain consists of an anti-CD3 binding fragment and a reference antigen-binding fragment, the anti-CD3 binding fragment consisting of the sequence of SEQ ID NO:206 (see Table 5e). In some embodiments, the melting temperature (T) of the first antigen-binding fragment is... m The T-cell anti-CD3 binding fragment composed of the sequence of SEQ ID NO: 206 is comparable to that of the CD3-binding fragment.m At least 2°C higher, or at least 3°C ​​higher, or at least 4°C higher, or at least 5°C higher (see Table 5e).

[0491] In some embodiments, the polypeptide of any of the subject composition embodiments described herein comprises an antigen-binding fragment (AF) that specifically binds to human CD3. The antigen-binding fragment (AF) can specifically bind to human CD3. In some embodiments, the antigen-binding fragment (AF) can bind to a CD3 complex subunit of a CD3ε, CD3δ, CD3γ, or CD3ζ unit identified herein as CD3. The antigen-binding fragment (AF) can bind to the CD3ε fragment of CD3. In some embodiments, the antigen-binding fragment (AF) can have a binding affinity (K0) 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 The constant specifically binds to human CD3, as determined in an in vitro antigen-binding assay containing human CD3 antigen. In some embodiments, the polypeptide of any of the subject composition embodiments described herein comprises an antigen-binding fragment (AF) with a binding affinity (K) weaker than about 10 nM, or about 50 nM, or about 100 nM, or about 150 nM, or about 200 nM, or about 250 nM, or about 300 nM, or about 350 nM, or weaker than about 400 nM. D It specifically binds to human CD3, as measured in in vitro antigen-binding assays. For clarity, K... D The antigen-binding fragment (AF) of 400 is compared to K D The antigen-binding fragment binds its ligand more weakly than a 10 nM antigen-binding fragment. In some embodiments, the polypeptide of any of the subject composition embodiments described herein comprises an antigen-binding fragment (AF) that specifically binds to human CD3 with a binding affinity at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times weaker than the antigen-binding fragments consisting of the amino acid sequences in Tables 5a to 5e, as indicated by the corresponding binding affinity (K) in an in vitro antigen binding assay. D The measurements were taken by [the relevant authority].

[0492] In some embodiments, this disclosure provides a bispecific polypeptide comprising an antigen-binding fragment (AF) that exhibits a binding affinity for CD3 that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or at least 1000 times weaker than that of the anti-EGFR AF embodiments described herein incorporated into the subject polypeptide (anti-CD3 AF), as indicated by the corresponding binding affinity (K0) in an in vitro antigen binding assay. D The measurements were taken by [the relevant authority].

[0493] The binding affinity of the subject composition to the target ligand can be determined, for example, using binding or competitive binding assays, such as the Biacore assay or ELISA assay using a chip to bind receptors or binding proteins 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.

[0494] In some embodiments, this disclosure provides an antigen-binding fragment (AF) (anti-CD3 AF) incorporated into a chimeric bispecific polypeptide composition that binds CD3, the polypeptide composition being engineered to have an isoelectric point (pI) that imparts enhanced stability to the composition compared to corresponding compositions comprising a CD3-binding antibody or antigen-binding fragment known in the art. In some embodiments, the polypeptide of any of the subject composition embodiments described herein comprises a CD3-binding AF (anti-CD3 AF), wherein the anti-CD3 AF exhibits a pI between 6.0 and 6.6 (inclusive). In some embodiments, the polypeptide of any of the subject composition embodiments described herein comprises a CD3-binding AF (anti-CD3 AF), wherein the anti-CD3 AF exhibits a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than that of a reference antigen-binding fragment (e.g., composed of the sequence shown in SEQ ID NO: 206 (see Table 5e)). In some embodiments, the peptide of any of the subject composition embodiments described herein comprises a CD3-binding AF (anti-EGFR AF) fused to another EGFR antigen-binding AF (anti-EGFR AF), wherein the anti-CD3 AF exhibits a pI 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 EGFR antigen-binding AF or its epitope. In some embodiments, the peptide of any of the subject composition embodiments described herein comprises a CD3-binding AF (anti-CD3 AF) fused to an EGFR antigen-binding AF (anti-EGFR AF), wherein the AF exhibits a pI within at least about 0.1 to about 1.5, or at least about 0.3 to about 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 to about 0.9 pH units of the pI of the anti-CD3 AF. Specifically, it is anticipated that such designs, with the pI of two of the antigen-binding fragments within such a range, will result in fusion antigen-binding fragments that will confer a higher degree of stability to the chimeric bispecific antigen-binding fragment composition into which they are integrated, leading to improved expression and enhanced recovery of the fusion protein in a soluble, non-aggregated form, increased shelf life of the formulated chimeric bispecific peptide composition, and enhanced stability when the composition is administered to a subject. In some embodiments, having two AFs (anti-CD3 AF and anti-EGFR AF) within a relatively narrow pI range allows selection of buffers or other solutions in which both AFs (anti-CD3 AF and anti-EGFR AF) are stable, thereby promoting the overall stability of the composition. In some embodiments, the antigen-binding fragments (AFs) may exhibit an isoelectric point (pI) of less than or equal to 6.6.In some embodiments, the antigen-binding fragment (AF) may exhibit an isoelectric point (pI) of 6.0 to 6.6 (inclusive). In some embodiments, the antigen-binding fragment (AF) may exhibit a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the isoelectric point (pI) of the reference antigen-binding fragment composed of the sequence shown in SEQ ID NO: 206 (see Table 5e). In some embodiments, the antigen-binding fragment (AF) may have a binding affinity (K) between about 10 nM and about 400 nM. D The antigen-binding fragment (AF) has a constant specific binding affinity to human CD3 (such as that determined in an in vitro antigen-binding assay containing human CD3 antigen). In some embodiments, the antigen-binding fragment (AF) may have a binding affinity (K) of less than about 10 nM, or less than about 50 nM, or less than about 100 nM, or less than about 150 nM, or less than about 200 nM, or less than about 250 nM, or less than about 300 nM, or less than about 350 nM, or less than about 400 nM. D ) specifically binds to human CD3 (as determined in in vitro antigen binding assays). In some embodiments, the antigen-binding fragment (AF) may exhibit a binding affinity to CD3 that is at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times weaker than that of the antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 206 (see Table 5e) (as determined by the corresponding binding affinity (K) in in vitro antigen binding assays). D (as determined by)

[0495] In some embodiments, the VL and VH of the antigen-binding fragment are fused via relatively long linkers consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids, which, when joined together, possess a flexible characteristic. In some embodiments, the VL and VH of any of the scFv embodiments described herein are linked via relatively long linkers having the sequence SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, VL and VH of any of the scFv embodiments described herein are linked by a relatively long linker having the sequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 82), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 83), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 84), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 85). In some embodiments, AF1 and AF2 are linked together by a short linker having 3, 4, 5, 6, or 7 amino acids of hydrophilic amino acids. In some embodiments, the short linker sequences are identified herein as SGGGGS (SEQ ID NO: 86), GGGGS (SEQ ID NO: 87), GGSGGS (SEQ ID NO: 88), GGS, or GSP. In some embodiments, this disclosure provides compositions comprising single-chain biantibodies, wherein, after 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 linkers, and the second and third variable domains are fused via one of the aforementioned relatively long linkers. In some embodiments, the short linkers and relatively long linkers are chosen to prevent incorrect pairing of adjacent variable domains, thereby facilitating the formation of single-chain configurations of VL and VH containing the first and second antigen-binding fragments.

[0496] Table 5b. Exemplary CD3 CDR Sequences

[0497]

[0498] Table 5c. Exemplary CD3 FR sequences

[0499]

[0500]

[0501] Table 5d: Exemplary CD3 VL and VH sequences

[0502]

[0503]

[0504]

[0505] Table 5e: Exemplary CD3 scFv sequences

[0506]

[0507]

[0508]

[0509]

[0510] Anti-EGFR binding domain

[0511] Anti-EGFR antibodies, their fragments, and fusion proteins containing such antibodies and / or fragments are also provided.

[0512] In some embodiments, this disclosure provides paTCE compositions comprising a first binding domain having binding affinity for the tumor-specific marker EGFR and a second binding domain binding effector cell antigens (such as CD3 antigen).

[0513] In some embodiments, the first binding domain is an scFv domain comprising a VH domain and a VL domain. Non-limiting examples of VH and VL domain sequences are provided in Table 5f. In some embodiments, the binding domain having binding affinity for the tumor-specific biomarker EGFR is an scFv domain comprising the VH and VL domains listed in Table 5f. In some embodiments, the binding domain having binding affinity for EGFR is an scFv domain comprising three CDRs from the VH domain listed in Table 5f and three CDRs from the VL domain listed in Table 5f.

[0514] In some embodiments, this disclosure provides a paTCE composition comprising a first moiety binding domain having binding affinity for the tumor-specific marker EGFR, the first moiety binding domain comprising the anti-EGFR VH and VL sequences shown in Table 5f. In some embodiments, the binding domain comprises approximately 10 -10M to 10 -7 M of K D Values, such as those determined in in vitro binding assays. In some embodiments, the binding has a K+ value of about 1 nM to 10 nM. D Values, such as those determined in in vitro binding assays. In some embodiments, the binding has a K+ value of approximately 2 nM. D Values, as determined in an in vitro binding assay. It is clearly anticipated that the paTCE composition may contain any of the binding domains disclosed herein or sequence variants thereof, provided that the variants exhibit binding specificity to the antigen.

[0515] Table 5f. Anti-EGFR VH and VL sequences

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539] In some embodiments, the anti-EGFR VH domain comprises the amino acid sequence QVQLQX1X2GX3GLX4KPSETLSLTCX5VX6GGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS, where X1 corresponds to E or Q; X2 corresponds to S or W; X3 corresponds to P or A; X4 corresponds to V or L; X5 corresponds to T or A; and X6 corresponds to S or Y (SEQ ID NO: 576); and the anti-EGFR VL domain comprises X1IX2X3TQSPX4X5LSX6SX7GX s RX9TX 10 X 11 CQASQDISNYLNWYQQKPGX 12 APX 13 LLIYDASNLETGX 14 PX 15 RFSGSGSGTDFTX 16 TISX 17 LX 18 PEDX 19 AX 20 The amino acid sequence of YYCQHFDHLPLAFGQGTKVEIK, where X1 corresponds to D or E; X2 corresponds to Q or V; X3 corresponds to M or L; X4 corresponds to S, G, or A; X5 corresponds to S or T; X6 corresponds to L or A; X7 corresponds to P or V; X8 corresponds to D or E; X9 corresponds to V or A; X 10 Corresponding to I or L; X 11 Corresponding to T or S; X 12 Corresponding to K or Q; X 13 Corresponding to K or R; X 14 Corresponding to V or I; X 15 Corresponding to S, D, or A; X 16 Corresponding to F or L; X 17 Corresponding to S or R; X 18 Corresponding to Q or E; X 19 Corresponding to I or F; and X20 Corresponding to T or V (SEQ ID NO: 577);

[0540] Each EGFR antibody described in Table 5f contains the following CDR sequence: HCDR1 – GGSVSSGDYYWT (SEQ ID NO: 562) HCDR2 – HIYYSGNTNYNPSLKS (SEQ ID NO: 563) HCDR3 – DRVTGAFDI (SEQ ID NO: 564) LCDR1 – QASQDISNYLN (SEQ ID NO: 565) LCDR2 – DASNLET (SEQ ID NO: 566) LCDR3 – QHFDHLPLA (SEQ ID NO: 567)

[0541] In some embodiments, this disclosure provides an anti-EGFR antibody VH region comprising the following CDRs: VH region CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with GGSVSSGDYYWT; VH region CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with HIYYSGNTNYNPSLKS; and VH region CDR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with DRVTGAFDI.

[0542] In some embodiments, this disclosure provides an anti-EGFR antibody VL region comprising the following CDRs: VL region CDR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with QASQDISNYLN; VL region CDR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with DASNLET; and VL region CDR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with QHFDHLPLA.

[0543] In some embodiments, the VH region of the anti-EGFR antibody includes the following framework regions (FR): VH region FR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with QVQLQESGPGLVKPSETLSLTCTVS; VH region FR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with WIRQPPGKGLEWIG. % or 100% identity; VH region FR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR; and VH region FR4, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with WGQGTLVTVSS.

[0544] In some embodiments, the VL region of the anti-EGFR antibody includes the following frame regions (FR): VL region FR1, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with DIQMTQSPSSLSASVGDRVTITC; VL region FR2, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with WYQQKPGKAPKLLIY. % or 100% identity; VL region FR3, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC; and VL region FR4, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or 100% identity with FGQGTKVEIK.

[0545] In some embodiments, this disclosure provides for including sequences The anti-EGFR antibody VH region, or its CDR, containing the sequence QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS (SEQ ID NO: 468); and the anti-EGFR antibody VL region, or its CDR, containing the sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQGTKVEIK (SEQ ID NO: 469).

[0546] In some embodiments, this disclosure provides an EGFR-resistant binding domain (e.g., scFv) comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with the following sequences: DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYKCHFDHLPLAFGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS (SEQ ID NO: 449).

[0547] In some embodiments (e.g., Table 5f), the VL and VH of the antigen-binding fragment are fused by relatively long linkers consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids, which, when joined together, possess a flexible characteristic. In some embodiments, the VL and VH of any of the scFv embodiments described herein (e.g., Table 5f) are linked by relatively long linkers having the sequence SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, VL and VH of any of the scFv embodiments described herein are linked by a relatively long linker having the sequence GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 82), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 83), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 84), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 85). In some embodiments, AF1 and AF2 are linked together by a short linker having 3, 4, 5, 6, or 7 amino acids of hydrophilic amino acids. In some embodiments, the short linker sequences are identified herein as SGGGGS (SEQ ID NO: 86), GGGGS (SEQ ID NO: 87), GGSGGS (SEQ ID NO: 88), GGS, or GSP. In some embodiments, this disclosure provides compositions comprising single-chain biantibodies, wherein, after 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 linkers, and the second and third variable domains are fused via one of the aforementioned relatively long linkers. In some embodiments, the short linkers and relatively long linkers are chosen to prevent incorrect pairing of adjacent variable domains, thereby facilitating the formation of single-chain configurations of VL and VH containing the first and second antigen-binding fragments.

[0548] In some embodiments, this disclosure provides an EGFR-binding antigen-binding fragment (e.g., AF1 or AF2) that exhibits enhanced stability compared to EGFR-binding antibodies or antigen-binding fragments known in the art. In some embodiments, the EGFR antigen-binding fragments of this disclosure are designed to confer a higher degree of stability to chimeric bispecific antigen-binding fragment compositions into which they are integrated, resulting in improved expression and recovery of the fusion protein, increased shelf life, and enhanced stability upon administration to a subject. In some embodiments, the anti-EGFR AF of this disclosure exhibits a higher degree of thermostability compared to certain EGFR-binding antibodies and antigen-binding fragments known in the art. In some embodiments, the anti-EGFR AF of this disclosure exhibits a higher degree of thermostability compared to antigen-binding fragments comprising panitumumab in VH and VL. In some embodiments, the anti-EGFR AF of this disclosure exhibits a higher degree of thermostability compared to EGFR.2 as disclosed in PCT International Patent Application Publication No. WO / 2020 / 264208. In some embodiments, the anti-EGFR AF of this disclosure exhibits lower immunogenicity in humans compared to certain EGFR-binding antibodies and antigen-binding fragments known in the art. In some embodiments, the immunogenicity of the anti-EGFR AF of this disclosure is lower than that of the VH and VL antigen-binding fragments comprising panitumumab. In some embodiments, the immunogenicity of the anti-EGFR AF of this disclosure is lower than that of EGFR.2 as disclosed in PCT International Patent Application Publication No. WO / 2020 / 264208. In some implementations, the degree of immunogenicity of AF is determined by immunogenicity prediction methods such as TELEPITOPAN (described in Zhang et al. PLoS One. 2012; 7(2):e30483. doi: 10.1371 / journal.pone.0030483, PMID: 22383964, the entire contents of which are incorporated herein by reference) or NetMHCpan-4.1 and NetMHCiPlan-4.0 (each described in Reynisson et al., Nucleic Acids Res 2020; 48(W1):W449-W454. doi: 10.1093 / nar / gkaa379., PMID:32406916, the entire contents of which are incorporated herein by reference). In some embodiments, anti-EGFR AFs used as components of chimeric bispecific antigen-binding fragment compositions into which they are integrated exhibit favorable pharmaceutical properties, including high thermal stability and low aggregation tendency, resulting in improved expression and recovery during manufacturing and storage, as well as promoting a long serum half-life.Biophysical properties, such as thermal stability, are often limited by antibody variable domains, which vary considerably in their inherent properties. 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). In some embodiments, thermal stability is measured by the “melting temperature” (T). m The melting temperature is determined by [method / method], defined as the temperature at which half of the molecules are denatured. The melting temperature of each heterodimer indicates its thermal stability. [The determination of T] m The in vitro determination of the heterodimer is known in the art, including 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), or as described in the following examples.

[0549] In some embodiments of the peptides disclosed herein, the antigen-binding fragment (e.g., AF1 or AF2) may exhibit higher thermal stability than the anti-EGFR-binding fragments comprising VH of SEQ ID NO: 450 and VL of SEQ ID NO: 451 (see Table 5f), as demonstrated in in vitro assays by the higher melting temperature (T0) of the first antigen-binding fragment relative to the anti-EGFR-binding fragment. m As demonstrated by ); or after incorporating the first antigen-binding fragment into the test bispecific antigen-binding domain, the T of the test bispecific antigen-binding domain is... m The test bispecific antigen-binding domain is higher than the control bispecific antigen-binding domain, wherein the test bispecific antigen-binding domain includes a first antigen-binding fragment and a reference antigen-binding fragment binding antigens other than EGFR; and wherein the control bispecific antigen-binding domain consists of an anti-EGFR binding fragment comprising VH of SEQ ID NO: 450 and VL of SEQ ID NO: 451 (see Table 5f) and a reference antigen-binding fragment. In some embodiments, the melting temperature (T) of the first antigen-binding fragment is... mThis can be compared to the T-cell anti-EGFR binding fragment containing VH of SEQ ID NO: 450 and VL of SEQ ID NO: 451. 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 (see Table 5f). In some embodiments, the melting temperature (T) of the first antigen-binding fragment... m This can be compared to the T-cell anti-EGFR binding fragment containing VH of SEQ ID NO: 450 and VL of SEQ ID NO: 451. m 2°C to 15°C higher, or 3°C to 15°C higher, or 4°C to 15°C higher, or 5°C to 15°C higher (see Table 5f).

[0550] In some embodiments, the peptide of any of the subject composition embodiments described herein comprises an antigen-binding fragment (AF) that specifically binds to human EGFR. The antigen-binding fragment (AF) can specifically bind to human EGFR. In some embodiments, the antigen-binding fragment (AF) can have a binding affinity (K0) 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 The constant specifically binds to human EGFR, as determined in an in vitro antigen-binding assay containing human EGFR antigen. In some embodiments, the peptide of any of the subject composition embodiments described herein comprises an antigen-binding fragment (AF) with a binding affinity (K) weaker than about 10 nM, or about 50 nM, or about 100 nM, or about 150 nM, or about 200 nM, or about 250 nM, or about 300 nM, or about 350 nM, or weaker than about 400 nM. D It specifically binds to human EGFR, as measured in in vitro antigen binding assays. For clarity, K... D The antigen-binding fragment (AF) of 400 is compared to K D The 10 nM antigen-binding fragment binds its ligand more weakly. In some embodiments, the peptide of any of the subject composition embodiments described herein comprises an antigen-binding fragment (AF) that specifically binds to human EGFR with a binding affinity at least 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times weaker than the antigen-binding fragment consisting of the amino acid sequences in Table 5f, as indicated by the corresponding binding affinity (K) in an in vitro antigen binding assay. D The measurements were taken by [the relevant authority].

[0551] In some embodiments, this disclosure provides a bispecific polypeptide comprising an antigen-binding fragment (AF) that exhibits a binding affinity for EGFR that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or at least 1000 times weaker than that of the anti-EGFR AF embodiments described herein incorporated into the subject polypeptide (anti-EGFR AF), as indicated by the corresponding binding affinity (K0) in an in vitro antigen binding assay. D The measurements were taken by [the relevant authority].

[0552] The binding affinity of the subject composition to the target ligand can be determined, for example, using binding or competitive binding assays, such as the Biacore assay or ELISA assay using a chip to bind receptors or binding proteins 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.

[0553] In some embodiments, this disclosure provides an antigen-binding fragment (AF) (anti-EGFR AF) incorporated into a chimeric bispecific polypeptide composition that binds EGFR and is engineered to have an isoelectric point (pI) that imparts enhanced stability to the composition compared to corresponding compositions comprising EGFR-binding antibodies or antigen-binding fragments known in the art. In some embodiments, the polypeptide of any of the subject composition embodiments described herein comprises an EGFR-binding AF (anti-EGFR AF) wherein the anti-EGFR AF exhibits a pI between 6.0 and 6.6 (inclusive). In some embodiments, the polypeptide of any of the subject composition embodiments described herein comprises an EGFR-binding AF (anti-EGFR AF) wherein the anti-EGFR AF exhibits a pI at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the pI of a reference antigen-binding fragment. In some embodiments, the peptide of any of the subject composition embodiments described herein comprises an EGFR-binding AF (anti-EGFR AF) fused to another CD3 antigen-binding AF (anti-CD3 AF), wherein the anti-EGFR AF exhibits a pI 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 AF binding the CD3 antigen or its epitope. In some embodiments, the peptide of any of the subject composition embodiments described herein comprises an EGFR-binding AF (anti-EGFR AF) fused to a CD3 antigen-binding AF (anti-CD3 AF), wherein the AF exhibits a pI within at least about 0.1 to about 1.5, or at least about 0.3 to about 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 to about 0.9 pH units of the pI of the anti-EGFR AF. Specifically, it is anticipated that such designs, with the pI of two of the antigen-binding fragments within such a range, will result in fusion antigen-binding fragments that will confer a higher degree of stability to the chimeric bispecific antigen-binding fragment composition into which they are integrated, leading to improved expression and enhanced recovery of the fusion protein in a soluble, non-aggregated form, increased shelf life of the formulated chimeric bispecific peptide composition, and enhanced stability when the composition is administered to a subject. In some embodiments, having two AFs (anti-EGFR AF and anti-CD3 AF) within a relatively narrow pI range allows selection of buffers or other solutions in which both AFs (anti-EGFR AF and anti-CD3 AF) are stable, thereby promoting overall stability of the composition. In some embodiments, the antigen-binding fragments (AFs) may exhibit an isoelectric point (pI) of less than or equal to 6.6.In some implementations, the antigen-binding fragment (AF) may exhibit an isoelectric point (pI) of 6.0 to 6.6 (inclusive of the terminal value).

[0554] Unless otherwise stated, the amino acid residues in the antibody domains, antigen-binding domains, or variable domains of fragments thereof described herein are numbered according to the Kabat numbering scheme. The Kabat numbers for EGFR.2 VH (SEQ ID NO: 450) and VL (SEQ ID NO: 451) are provided below.

[0555] Table 5g: Kabat numbers for EGFR.2 VH (SEQ ID NO: 450) and VL (SEQ ID NO: 451)

[0556]

[0557]

[0558]

[0559]

[0560] Linkers and spacers between antibody regions in bispecific antibodies

[0561] In some embodiments of the peptides disclosed herein, a pair of light chain variable regions (VL) and heavy chain variable regions (VH) of the antigen-binding fragment can be linked by a linker or a long linker (e.g., a linker having hydrophilic amino acids). In some embodiments, a first antigen-binding fragment (AF1) (e.g., an scFv domain, such as an anti-EGFR scFv domain) and a second antigen-binding fragment (AF2) (e.g., scFv, such as an anti-CD3 scFv) are linked by a linker or a long linker (e.g., a linker having hydrophilic amino acids). In some embodiments, the linker connecting the light chain variable regions (VL) and heavy chain variable regions (VH) of the antigen-binding fragments (e.g., the first antigen-binding fragment (AF1) and / or the second antigen-binding fragment (AF2)) can (each independently) contain an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences shown in Table A. In some embodiments, the linkers connecting the light chain variable region (VL) and heavy chain variable region (VH) of the antigen-binding fragments (e.g., the first antigen-binding fragment (AF1) and / or the second antigen-binding fragment (AF2)) may (each independently) contain the same amino acid sequence as the sequence shown in Table A. In some embodiments of the peptides disclosed herein, two antigen-binding fragments (e.g., the first antigen-binding fragment and the second antigen-binding fragment) may be fused together by a peptide linker or a short linker. In some embodiments, the peptide linker connecting the two antigen-binding fragments (e.g., the first antigen-binding fragment and the second antigen-binding fragment) may contain an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in Table B. In some embodiments, the peptide linker connecting the two antigen-binding fragments (e.g., the first antigen-binding fragment and the second antigen-binding fragment) may contain the same amino acid sequence as the sequence shown in Table B. In some cases, the first antigen-binding fragment is a single-chain variable fragment (scFv). In some cases, the second antigen-binding fragment is a single-chain variable fragment (scFv). The two single-chain variable fragments of the first and second antigen-binding fragments can be linked together by peptide linkers. In some embodiments of the peptides disclosed herein, the linker for linking the scFv of the first antigen-binding fragment (e.g., anti-EGFR scFv) and the linkers for linking the VL and VH of the second antigen-binding fragment (e.g., anti-CD3 scFv) can be GGGGSGGGS (SEQ ID NO: 125) of Table A.In other embodiments, the linker for connecting the VL and VH of the antigen-binding fragment (e.g., anti-CD3 scFv) may be SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, this disclosure provides a polypeptide comprising a single-chain biantibody, wherein, after folding, a first domain (VL or VH) pairs with a last domain (VH or VL) to form one 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...

[0562] Short linkers of hydrophilic amino acids identified in this paper by the sequences shown in Table B are fused together, and the second and third variable domains are fused together by long linkers identified in Table A. In some embodiments, the short and long linkers are selected to prevent incorrect pairing of adjacent variable domains, thereby facilitating the formation of single-chain configurations of VL and VH containing the first and second binding portions.

[0563] Table A: Intramolecular Long Joints

[0564]

[0565] Table B: Intermolecular short junctions

[0566]

[0567] Spacer and TCE release segment

[0568] This article includes fusion proteins containing TCE components that become biologically active or have increased biological activity upon release from ELNN by cleaving optional cleavage sequences incorporated into optional spacer sequences within the fusion protein, as described herein, for example.

[0569] In some embodiments, spacers may be provided to enhance the expression of the fusion protein from the host cell and / or reduce steric hindrance, allowing the TCE component to present its desired tertiary structure and / or to interact appropriately with its target molecules. For spacers and methods for identifying desired spacers, see, for example, George, et al. (2003) Protein Engineering 15:871-879, which is specifically incorporated herein by reference. In some embodiments, the spacer comprises one or more peptide sequences of 1 to 50 amino acid residues, or about 1 to 25 residues, or about 1 to 10 residues in length. The spacer sequence, excluding cleavage sites, may comprise any of 20 natural L amino acids and will preferably comprise sterically unhindered hydrophilic amino acids, which may include, but are not limited to, glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P). In some embodiments, the spacer may be polyglycine or polyalanine, or a mixture primarily of glycine and alanine residues. In some embodiments, the spacer polypeptide, excluding the cleavage sequence, is substantially free of secondary structure. In some embodiments, one or both spacer sequences in the paTCE fusion protein composition may each further contain a cleavage sequence, which may be the same or different, wherein the cleavage sequence may be acted upon by a protease to release TCE from the fusion protein.

[0570] Table C: Exemplary spacers between release segments and bispecific antibody domains

[0571]

[0572] In some embodiments of the peptides disclosed herein, release regions (RS) (e.g., a first release region (RS1), a second release region (RS2), etc.) may be fused to a bispecific antibody domain (BsAb) via spacers. In some embodiments, the spacers may (independently) contain at least four types of amino acids: glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P). In some embodiments, the peptides disclosed herein may comprise a first release region fused to the bispecific antibody domain via a first spacer and a second release region fused to the bispecific antibody domain via a second spacer. In some embodiments, the spacers (e.g., a first spacer, a second spacer, etc.) may (independently) contain an amino acid sequence having at least (about) 80%, at least (about) 90%, or 100% sequence identity with the sequences shown in Table C. In some embodiments, the spacers (e.g., a first spacer, a second spacer, etc.) may (independently) contain the same amino acid sequence as the sequences shown in Table C.

[0573] In some embodiments, the incorporation of the cleavage sequence into the fusion protein is designed to allow the release of the TCE, which becomes active or more active upon release from one or more ELNNs. In some embodiments, the cleavage sequence is located sufficiently close to the TCE sequence, typically within 18, 12, 6, or 2 amino acids from the end of the TCE sequence, such that any remaining residues attached to the TCE after cleavage do not significantly interfere with the activity of the TCE (e.g., such as receptor binding), but still provide sufficient proximity to the protease to enable cleavage of the cleavage sequence. In some embodiments, the cleavage site is a sequence that can be cleaved by endogenous proteases in mammalian subjects, such that paTCE can be cleaved after administration to a subject. In such cases, paTCE can act as a circulating reservoir of TCE. Examples of cleavage sites contemplated herein include, but are not limited to, polypeptide sequences that can be cleaved by endogenous proteases in mammals listed in Table 6.

[0574] In some embodiments, the paTCE fusion protein includes spacer sequences containing one or more cleavage sequences configured to release TCE from the fusion protein upon action by a protease. In some embodiments, the spacer sequences do not contain cleavage sequences. In some embodiments, the one or more cleavage sequences may be sequences having at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%) sequence identity with sequences from Table 7a or Table 7b.

[0575] In some embodiments, this disclosure provides a TCE-releasing peptide (or releasing segment (RS)) that is a substrate of one or more mammalian proteases associated with or produced by cells found in or near diseased tissue. Such proteases may include, but are not limited to, protease classes such as metalloproteinases, cysteine ​​proteases, aspartic proteases, and serine proteases, including but not limited to the proteases listed in Table 6. RS can be particularly used for incorporation into the subject recombinant peptide to confer an inactive form that can be activated by cleavage of the RS by a mammalian protease. As described herein, incorporating RS into the subject recombinant peptide composition and linking the incorporated binding moiety to an ELNN (exemplary configurations of which are described herein) such that upon cleavage of RS by one or more proteases with RS as a substrate, the binding moiety and the ELNN are released from the composition, and the binding moiety, no longer shielded by the ELNN, regains its full potential to bind its ligand.

[0576] Table 6: Proteases in target tissues

[0577]

[0578]

[0579] In some embodiments, this disclosure provides an activatable recombinant polypeptide comprising a first release segment (RS1) sequence having at least 88%, or at least 94%, or 100% sequence identity with the sequences identified in Table 7a upon optimal alignment, wherein RS1 is a substrate of one or more mammalian proteases. In some embodiments, RS is further engineered to remove the pod protein cleavage site. In some embodiments, this disclosure provides an activatable recombinant polypeptide comprising an RS1 and a second release segment (RS2) sequence, wherein, upon optimal alignment, each of the RS1 and RS2 sequences has at least 88%, or at least 94%, or 100% sequence identity with the sequences identified herein by the sequences shown in Table 7a, wherein each of RS1 and RS2 is a substrate of one or more mammalian proteases. In some embodiments, RS1 and RS2 are not used as substrates of pod proteins.

[0580] In some embodiments, this disclosure provides an activatable recombinant polypeptide comprising a first RS (RS1) sequence that, upon optimal alignment, has at least 90%, at least 93%, at least 97%, or 100% sequence identity with the sequences identified in Table 7b, wherein the RS1 is a substrate of one or more mammalian proteases. In some embodiments, this disclosure provides an activatable recombinant polypeptide comprising an RS1 and a second release segment (RS2) sequence, each of which, upon optimal alignment, has at least 88%, or at least 94%, or 100% sequence identity with the sequences identified herein by the sequences shown in Table 7b, wherein RS1 and RS2 are each a substrate of one or more mammalian proteases (e.g., at one, two, or three cleavage sites within each release segment sequence). In some embodiments of the activatable recombinant polypeptide comprising RS1 and RS2, the two release segments may be identical. In some embodiments of the activatable recombinant polypeptide comprising RS1 and RS2, the two release segments may be different.

[0581] This disclosure considers release segments that are substrates for one, two, or three different classes of proteases, including metalloproteinases, cysteine ​​proteases, aspartic proteases, or serine proteases, as listed in Table 6. In some embodiments, the paTCE comprises RS (e.g., RS1 and RS2) that act as substrates for one or more proteases that are tightly associated with or co-located with tumors or cancer cells, and upon cleavage of the RS, the originally…

[0582] The binding moieties shielded by the ELNN of the paTCE (and therefore having lower binding affinity for their respective ligands) are released from the ELNN and regain their full potential to bind target cell ligands and effector cell ligands. In some embodiments, the paTCE comprises RSs (e.g., RS1 and RS2), each containing an amino acid sequence that serves as a substrate for one or more cellular proteases (including, but not limited to, the proteases in Table 6) located within the target cell. In some embodiments, the RSs are substrates for two or three classes of proteases that cleave different moieties of each RS. In some embodiments, each RS serving as a substrate for two, three, or more classes of proteases has two, three, or more distinct cleavage sites, but cleavage by a single protease still results in the release of the binding moieties from the ELNN.

[0583] In some embodiments, the RS of this disclosure used for incorporation into fusion proteins (such as paTCE) is a substrate of one or more proteases, including but not limited to transmembrane peptidase, enkephalinase (CD10), PSMA, BMP-1, deintegrin and metalloproteinase (ADAM), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM with platelet motif (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloproteinase 1, etc. Matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3 (MMP-3, lysozyme 1), matrix metalloproteinase-7 (MMP-7, matrix lysozyme 1), matrix metalloproteinase-8 (MMP-8, collagenase 2), matrix metalloproteinase-9 (MMP-9, gelatinase B), matrix metalloproteinase-10 (MMP-10, lysozyme 2), matrix metalloproteinase-11 (MMP-11, lysozyme 3), matrix metalloproteinase-12 (MMP-12, macrophage elastase), matrix metalloproteinase-13 (MMP-13, collagenase 3), matrix metalloproteinase-1 4 (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 lysin 2), matrix metalloproteinase-27 (MMP-27, CMMP), podocyte oleoresin, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X, cathepsin D, cathepsin E, secretase, urokinase ( The following proteins are listed: uPA, 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), proteolytic enzyme, proteolytic enzyme-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast activating protein (FAP), kallikrein-related peptidases (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14. In some embodiments, RS is a substrate of ADAM17. In some embodiments, RS is a substrate of BMP-1.In some embodiments, RS is a substrate of cathepsin. In some embodiments, RS is a substrate of HtrA1. In some embodiments, RS is a substrate of podin. In some embodiments, RS is a substrate of MMP-1. In some embodiments, RS is a substrate of MMP-2. In some embodiments, RS is a substrate of MMP-7. In some embodiments, RS is a substrate of MMP-9. In some embodiments, RS is a substrate of MMP-11. In some embodiments, RS is a substrate of MMP-14. In some embodiments, RS is a substrate of uPA. In some embodiments, RS is a substrate of proteolytic enzyme. In some embodiments, RS is a substrate of MT-SP1. In some embodiments, RS is a substrate of neutrophil elastase. In some embodiments, RS is a substrate of thrombin. In some embodiments, RS is a substrate of TMPRSS3. In some embodiments, RS is a substrate of TMPRSS4. In some embodiments, the RS of the recombinant polypeptide composition is a substrate of at least two proteases, including but not limited to podin, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and proteolytic enzymes. In some embodiments, the RS of the recombinant polypeptide composition is a substrate of podin, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and proteolytic enzymes. In certain embodiments, the RS of the recombinant polypeptide composition is not a substrate of podin. In some embodiments, the RS of the recombinant polypeptide composition is a substrate of uPA, proteolytic enzymes (also referred to as MT-SP1 and ST14), MMP2, MMP7, MMP9, and MMP14. In some embodiments, the RS of the recombinant polypeptide composition is a substrate of uPA, proteolytic enzymes, MMP2, MMP7, MMP9, and MMP14, but not podin.

[0584] Table 7a: TCE Release Segment Sequence .

[0585]

[0586]

[0587]

[0588] Table 7b: Release Segment Sequence

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598] In some embodiments, paTCE comprises RS1 and RS2 with different cleavage rates and efficiencies for various proteases (RS1 and RS2 being substrates for the proteases). Since a given protease can be present in tumors at varying concentrations, this disclosure provides RSs with higher or lower cleavage efficiencies for a given protease compared to healthy tissue or circulation. This ensures that when paTCE approaches cancer cells or tissue and their co-localized proteases, paTCE preferentially converts from an inactive form to an active form (i.e., by separating and releasing the binding moiety and ELNN from paTCE after RS ​​cleavage) compared to the cleavage rate of RSs in healthy tissue or circulation, such that the released binding moiety of TCE is more capable of binding to ligands within the tumor compared to the inactive form retained in circulation. This selective design improves the therapeutic index of the resulting composition, thereby producing fewer side effects compared to conventional therapeutic agents that do not incorporate such site-specific activation.

[0599] In some implementations, the cleavage efficiency is the log2 value of the ratio of the percentage of test substrate containing cleaved RS to the percentage of cleaved control substrate AC1611 when subjected to the protease in these biochemical assays. The reactions are performed with an initial substrate concentration of 6 μM, incubated at 37°C for 2 hours, and then terminated by the addition of EDTA. The ratio of cleavage percentage is established by analyzing the amount of digested products and uncleaved substrate using non-reducing SDS-PAGE. The cleavage efficiency can be calculated as follows: Log2 Therefore, a cleavage efficiency of -1 means that the test substrate is cleaved by 50% compared to the control substrate, while a cleavage efficiency of +1 means that the test substrate is cleaved by 200% compared to the control substrate. A higher cleavage rate of the test protease relative to the control will result in higher cleavage efficiency, and a slower cleavage rate of the test protease relative to the control will result in lower cleavage efficiency. When testing the rate of cleavage by a single protease in an in vitro biochemical assay, the control RS sequence AC1611 (RSR-1517) with the amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001) was established as having an appropriate baseline cleavage efficiency for the proteases podin, MMP-2, MMP-7, MMP-9, MMP-14, uPA, and proteolytic enzymes. RS libraries were generated by selectively substituting amino acids at individual positions in the RS peptides, and the groups were evaluated against seven proteases, providing an overview of guidelines for establishing appropriate amino acid substitutions to achieve RSs with the desired cleavage efficiency. In some embodiments, when preparing RS with the desired cleavage efficiency, it is preferred to use the hydrophilic amino acids A, E, G, P, S, and T for substitution. However, other L-amino acids can be substituted at a given position to modulate the cleavage efficiency, as long as the RS retains at least some sensitivity to cleavage by a given protease. Conservative substitution of amino acids in a peptide to preserve or affect activity is entirely within the knowledge and ability of those skilled in the art. In some embodiments, this disclosure provides an RS that is cleaved by a protease, including but not limited to MMP-2, MMP-7, MMP-9, MMP-14, uPA, or a proteolytic enzyme (also known as MT-SP1), exhibiting at least 0.2 log2, 0.4 log2, 0.8 log2, or 1.0 log2 higher cleavage efficiency in an in vitro biochemical competitive assay compared to the control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO. 7001) being cleaved by the same protease. In some embodiments, this disclosure provides an RS that is cleaved by a protease, including but not limited to MMP-2, MMP-7, MMP-9, MMP-11, uPA, or a proteolytic enzyme, having a cleavage efficiency of at least 0.2 log2, 0.4 log2, 0.8 log2, or 1.0 log2 lower in an in vitro biochemical competitive assay compared to the cleavage of a control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO. 7001) by the same protease.In some embodiments, this disclosure provides RS, wherein a protease (including, but not limited to, MMP-2, MMP-7, MMP-9, MMP-14, uPA, or a protein lyase) cleaves RS at a rate at least 2-fold, at least 4-fold, at least 8-fold, or at least 16-fold faster than a control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO. 7001). In some embodiments, this disclosure provides RS, wherein a protease (including, but not limited to, MMP-2, MMP-7, MMP-9, MMP-14, uPA, or a protein lyase) cleaves RS at a rate at least 2-fold, at least 4-fold, at least 8-fold, or at least 16-fold slower than a control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO. 7001).

[0600] In some embodiments, RS comprises the amino acid sequence EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is any amino acid except N. In some embodiments, X is S. In some embodiments, X is T. In some embodiments, X is Y. In some embodiments, X is Q. In some embodiments, X is G. In some embodiments, X is A. In some embodiments, X is V. In some embodiments, X is C. In some embodiments, X is P. In some embodiments, X is L. In some embodiments, X is I. In some embodiments, X is M. In some embodiments, X is F. In some embodiments, X is K. In some embodiments, X is R. In some embodiments, X is H. In some embodiments, X is D. In some embodiments, X is E. In some embodiments, RS is not cleaved by pod proteins. In some embodiments, RS cannot be cleaved by pod proteins in human blood, plasma, or serum.

[0601] In some embodiments, RS becomes uncut after incubation with about 1 nM or less of pod protein for about 20 hours. In some embodiments, RS is cleaved by pod protein more slowly or less efficiently than RSR-2295 (EAGRSANHTPAGLTGP). In some embodiments, the rate at which RS is cleaved by pod protein is less than about 50% of the rate at which pod protein cleaves RSR-2295 (EAGRSANHTPAGLTGP). In some embodiments, the rate at which RS is cleaved by pod protein is less than about 25% of the rate at which pod protein cleaves RSR-2295. In some embodiments, the rate at which RS is cleaved by pod protein is less than about 10% of the rate at which pod protein cleaves RSR-2295. In some embodiments, the rate at which RS is cleaved by pod protein is less than about 5% of the rate at which pod protein cleaves RSR-2295. In some embodiments, the rate at which RS is cleaved by pod protein is less than about 2.5% of the rate at which pod protein cleaves RSR-2295.

[0602] In some embodiments, the rate at which strychnoids cleave RSR is less than about 50% of the rate at which strychnoids cleave RSR-2295 (EAGRSANHTPAGLTGP) in human plasma. In some embodiments, the rate at which strychnoids cleave RSR is less than about 25% of the rate at which strychnoids cleave RSR-2295 in human plasma. In some embodiments, the rate at which strychnoids cleave RSR is less than about 10% of the rate at which strychnoids cleave RSR-2295 in human plasma. In some embodiments, the rate at which strychnoids cleave RSR is less than about 5% of the rate at which strychnoids cleave RSR-2295 in human plasma. In some embodiments, the rate at which strychnoids cleave RSR is less than about 2.5% of the rate at which strychnoids cleave RSR-2295 in human plasma.

[0603] In some embodiments, this disclosure provides a paTCE comprising multiple RSs, wherein each RS sequence is identified herein by the sequence groups shown in Table 7a, and the RSs are linked to each other by 1 to 6 amino acids, which are glycine, serine, alanine, and threonine. In some embodiments, the paTCE comprises a first RS and a second RS different from the first RS, wherein each RS sequence is identified herein by the sequences shown in Table 7a, and the RSs are linked to each other by 1 to 6 amino acids, which are glycine, serine, alanine, and threonine. In some embodiments, the paTCE comprises a first RS, a second RS different from the first RS, and a third RS different from the first and second RS, wherein each sequence is identified herein by the sequences shown in Table 7a, and the first RS, second RS, and third RS are linked to each other by 1 to 6 amino acids, which are glycine, serine, alanine, and threonine. In some embodiments, the multiple RSs of the paTCE may be linked to form a sequence that can be cleaved by various proteases at different cleavage rates or cleavage efficiencies. In some embodiments, this disclosure provides a paTCE comprising RS1 and RS2, each having a sequence shown in Table 7a or Table 7b, and ELNNs (e.g., ELNN1 and ELNN2), such as those described herein, wherein RS1 is fused between ELNN1 and the binding moiety, and RS2 is fused between ELNN2 and the binding moiety. In some embodiments, paTCE is more readily cleaved in target tissues expressing multiple proteases (e.g., cancerous tissue) compared to healthy tissue or when in normal circulation, resulting in fragments with the binding moiety that will more readily penetrate the target tissue; for example, a tumor, and have an enhanced ability to bind and connect cancer cells and effector cells.

[0604] In some embodiments, paTCE comprises a first release segment (RS1) located between a first ELNN and a bispecific antibody. In some embodiments, the polypeptide further comprises a second release segment (RS2) located between the bispecific antibody and a second ELNN. In some embodiments, RS1 and RS2 are sequence-identical. In some embodiments, RS1 and RS2 are sequence-dissimilar. In some embodiments, RS1 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences or subsets thereof identified in Tables 7a or 7b herein. In some embodiments, RS2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences or subsets thereof identified in Tables 7a or 7b herein. In some implementations, RS1 and RS2 are each substrates used for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence.

[0605] In some embodiments, paTCE further includes one or more reference fragments (e.g., barcode fragments) that can be released from the paTCE upon digestion by the protease. In some embodiments, the one or more reference fragments are single reference fragments whose sequence and molecular weight differ from all other peptide fragments that can be released from the polypeptide after digestion by the protease.

[0606] Example pacCE

[0607] In some embodiments, paTCE comprises an amino acid sequence having at least (about) 80% sequence identity with the sequences shown in Table D (SEQ ID NO: 1000 to SEQ ID NO: 1007) or a subset thereof. In some embodiments, paTCE comprises an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity with the sequences shown in SEQ ID NO: 1000 to SEQ ID NO: 1007 or a subset thereof. In some embodiments, paTCE comprises an amino acid sequence having at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity with the sequences shown in SEQ ID NO: 1000 to SEQ ID NO: 1007. It is clearly contemplated that the compositions of this disclosure may comprise sequence variants of the amino acid sequences shown in Table D, such as adapter sequences with substitutions or insertions or purified tag sequences attached thereto, provided that the variants exhibit substantially similar or identical one or more biological activities and / or activation mechanisms.

[0608] Table D: Exemplary amino acid sequences of polypeptides

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617] Reorganization

[0618] Also provided are the polynucleotides encoding any of the polypeptides disclosed herein and / or the reverse complementary sequences of such polynucleotides.

[0619] The disclosures herein include expression vectors containing a polynucleotide sequence, such as any polynucleotide sequence described in the preceding paragraphs, and a regulatory sequence operatively linked to the polynucleotide sequence.

[0620] The disclosure herein includes host cells containing expression vectors, such as any expression vectors described in the preceding paragraphs. In some embodiments, the host cell is a prokaryote. In some embodiments, the host cell is *Escherichia coli* (E. coli). In some embodiments, the host cell is a mammalian cell.

[0621] In some embodiments, this disclosure provides methods for manufacturing the subject composition. In some embodiments, such methods include culturing host cells containing a nucleic acid construct encoding a polypeptide described herein (such as paTCE) under conditions that promote polypeptide expression, followed by recovery of the polypeptide using standard purification methods (e.g., column chromatography, HPLC, etc.), wherein the composition is recovered in which at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the binding fragments of the expressed polypeptide or paTCE fusion polypeptide are correctly folded. In some embodiments of the preparation method, the expressed polypeptide is recovered in which at least 90%, or at least 95%, or at least 97%, or at least 99% of the polypeptide is recovered in a monomeric, soluble form.

[0622] In some embodiments, this disclosure relates to the preparation of peptides (such as paTCE fusion peptides) at high fermentation expression levels of functional proteins using *E. coli* or mammalian host cells, and to methods for providing expression vectors encoding peptides suitable for producing cytotoxic active peptide compositions at high expression levels. In some embodiments, the method includes the steps of: 1) preparing a polynucleotide encoding a peptide 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 protein expression in a biological system; 3) transforming a suitable host cell with the expression vector; and 4) culturing the host cell in a conventional nutrient medium under conditions suitable for expressing the peptide composition. When desired, the host cell is *E. coli*. 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 (e.g., after activation). In some embodiments, this disclosure provides a method for producing a peptide comprising culturing a host cell containing a vector encoding a peptide-containing peptide in a fermentation reaction under conditions conducive to the efficient expression of the peptide product.

[0623] Pharmaceutical Composition

[0624] The pharmaceutical compositions disclosed herein include polypeptides (such as paTCE) (such as any polypeptide described herein) and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions are formulated for intradermal, subcutaneous, intravenous, intraarterial, intraperitoneal, intravitreal, intrathecal, or intramuscular administration. In some embodiments, the pharmaceutical compositions are formulated for intravenous injection. In some embodiments, the pharmaceutical compositions are in liquid or frozen form. In some embodiments, the pharmaceutical compositions are formulated as lyophilized powders reconstituted prior to administration.

[0625] The pharmaceutical composition may be administered for treatment via any suitable route. In some embodiments, the dose is administered intradermally, subcutaneously, intravenously, intraarterially, intraperitoneally, intrathecally, or intramuscularly. In some embodiments, the subject is a mouse, rat, monkey, or human. In a preferred embodiment, the subject is a human.

[0626] In some embodiments, the pharmaceutical composition may be administered subcutaneously, intramuscularly, or intravenously. In some embodiments, the pharmaceutical composition is administered in a therapeutically effective amount. In some embodiments, a therapeutically effective amount results in an increased time spent by the fusion protein within the therapeutic window compared to the corresponding TCE of the fusion protein not linked to the ELNN and administered to the subject in an equivalent amount.

[0627] In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the composition may be supplied as a lyophilized powder or cake for reconstitution prior to administration. In some embodiments, the composition may also be supplied in liquid or frozen form, which can be administered directly to the subject.

[0628] Drug kit

[0629] In some embodiments, this disclosure provides a kit for facilitating the use of paTCE. In some embodiments, the kit includes (a) a first container containing a pharmaceutically effective amount of paTCE in a lyophilized composition; and (b) a second container containing a diluent for reconstituted lyophilized formulation. In some embodiments, the kit further includes instructions for storing the kit, information on cancers treatable with paTCE, instructions for reconstituted lyophilized formulation, and / or instructions for administration.

[0630] Treatment

[0631] This document discloses the use of polypeptides (such as any polypeptides described herein) in the preparation of medicaments for treating a disease in a subject. In some embodiments, the specific disease to be treated will depend on the selection of the bioactive protein. In some embodiments, the disease is cancer. This document includes the patCE polypeptide for treating cancer. In some cases, the cancer or tumor expresses EGFR. In some embodiments, the cancer or tumor is a solid tumor. In some embodiments, the cancer is carcinoma, sarcoma, or melanoma. In some embodiments, the cancer is carcinoma. In some embodiments, the cancer is sarcoma. In some embodiments, the cancer is melanoma.

[0632] EGFR is one of the most frequently altered oncogenes in solid tumors. EGFR activation promotes processes responsible for tumor growth and progression, including proliferation and maturation, angiogenesis, invasion, metastasis, and inhibition of apoptosis. Pathological alterations of EGFR in cancer include kinase-activating mutations in EGFR and / or overexpression of the EGFR protein. Kinase-activating mutations lead to increased tyrosine kinase activity in EGFR. EGFR protein overexpression may or may not be associated with EGFR gene amplification. Furthermore, wild-type EGFR protein is commonly overexpressed in many types of solid cancers and is often associated with a negative prognosis. EGFR alterations in solid cancers known in this art, for example, are described in Thomas R. and Weihua Z. Front. Oncol. 9:800 (2019) and Singal et al. Cancer Control 14(3):295-304 (2007), each of which is incorporated herein by reference in its entirety. Current EGFR inhibitors, including tyrosine kinase inhibitors and monoclonal antibody inhibitors, have shown limited efficacy and have been challenged by innate and acquired resistance in clinical practice.

[0633] In some embodiments, cancer is associated with EGFR overexpression (e.g., relative to non-cancerous cells of the same tissue type). In some embodiments, cancer comprises cells expressing at least 3,000; 5,000; 10,000; 20,000; 30,000; 40,000; 50,000; 60,000; 70,000; 80,000; 90,000; 100,000; or 200,000 EGFR proteins per cell on average. In some embodiments, cancer comprises cells having one or more oncogenic mutations in the EGFR gene. In some embodiments, cancer comprises cells with EGFR gene amplification. In some implementations, the cells contain 2 to 5 times, 2 to 10 times, 2 to 15 times, 2 to 30 times, 2 to 50 times, 3 to 5 times, 3 to 10 times, 3 to 15 times, 3 to 30 times, 3 to 50 times, 5 to 10 times, 5 to 15 times, 5 to 30 times, or 5 to 50 times more EGFR gene copies than non-cancerous cells of the same tissue type.

[0634] In some implementations, the cancer is lung cancer, colorectal cancer, head and neck cancer, breast cancer, pancreatic cancer, brain cancer, liver cancer, kidney cancer, ovarian cancer, prostate cancer, esophageal cancer, cervical cancer, or bladder cancer. In some implementations, the cancer is lung cancer. In some implementations, the lung cancer is non-small cell lung cancer. In some implementations, the cancer is colorectal cancer. In some implementations, the cancer is squamous cell carcinoma of the head and neck. In some implementations, the cancer is breast cancer. In some implementations, the cancer is triple-negative breast cancer. In some implementations, the cancer is brain cancer. In some implementations, the brain cancer is glioblastoma.

[0635] In some implementation schemes, cancer includes anaplastic and medullary thyroid carcinoma, appendix cancer, arrhenoblastoma, biliary tract cancer, bladder cancer, breast cancer, cholangiocarcinoma, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, intraepithelial malignant tumors with malignant ascites, esophageal cancer, Ewing sarcoma, fallopian tube cancer, follicular carcinoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor (GIST), gastroesophageal junction cancer, urogenital tract cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, liver cancer, HR+ and HER2+ breast cancer, Hurthle cell cancer, inflammatory breast cancer, and Kaposi's sarcoma. Sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroendocrine carcinoma, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary carcinoma, parathyroid carcinoma, peritoneal cancer, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, gastric cancer, testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial carcinoma, uterine cancer, serous uterine carcinoma, vaginal cancer, vulvar cancer, or Wilms tumor.

[0636] This disclosure includes a method of treating a subject for a disease, the method comprising administering to the subject in need a therapeutically effective amount of a pharmaceutical composition, such as any pharmaceutical composition described herein. In some embodiments, the disease is cancer. In some embodiments, the subject is a mouse, rat, monkey, or human. In some embodiments, the subject is a human.

[0637] In some embodiments, the bispecific EGFR-targeting compositions of this disclosure (such as paTCE) may be combined with one or more checkpoint inhibitors. In some embodiments of such combination therapy, paTCE may be combined with an antagonist of the cell surface receptor programmed cell death protein 1 (also known as PD-1) and / or an antagonist of PD-L1. As used herein, the term “combination” or “combination therapy” corresponds to the administration of two or more different compounds (e.g., EGFR paTCE and a checkpoint inhibitor) as part of a treatment regimen. The two or more compounds may be administered simultaneously or sequentially. Prior to administration, the two or more compounds may be combined into a single composition. Each compound in the combination may be administered alone as part of a defined dosing regimen.

[0638] PD-1 plays a crucial role in downregulating the immune system and promoting self-tolerance by inhibiting T-cell inflammatory activity. The binding of PD-1 ligands PD-L1 and PD-L2 to the PD-1 receptor found on T cells inhibits T-cell proliferation and cytokine production. Upregulation of PD-1 ligands occurs in some tumors, and signaling through this pathway can contribute to suppressing tumor-active T-cell immune surveillance. Anti-PD-1 antibodies bind to the PD-1 receptor and block its interaction with PD-L1 and PD-L3, releasing the inhibition of PD-1 pathway-mediated immune responses, including anti-tumor immune responses.

[0639] Those skilled in the art are aware of the various anti-PD-1 antibodies that can be used. In some embodiments, an exemplary anti-PD-1 antibody used in combination with the compounds of the present invention is pembrolizumab (Keytruda). ® In some embodiments, the anti-PD-1 antibody used in combination with the above compounds is nivolumab (Opdivo). ® In some embodiments, the anti-PD-1 antibody used in combination with the above-described compounds is pitilizumab (Medivation).

[0640] Additive PD-1 antibodies known to those skilled in the art include AAGEN-2034 (Agenus), AMP-224 (Medimmune), BCD-100 (Biocad), BGBA-317 (Beigene), BI-754091 (Boehringer Ingelheim), CBT-501 (Genor Biopharma), CC-90006 (Celgene), cimipril (Regeneron Pharmaceuticals), durvalumab + MEDI-0680 (Medimmune), GLS-010 (Harbin Gloria Pharmaceuticals), IBI-308 (Eli Lilly), JNJ-3283 (Johnson & Johnson), and JS-001 (Shanghai Junshi Bioscience). Co.), MEDI-0680 (Medimmune), MGA-012 (MacroGenics), MGD-013 (Marcogenics), pazopanib hydrochloride + pembrolizumab (Novartis), PDR-001 (Novartis), PF-06801591 (Pfizer), SHR-1210 (Jiangsu Hengrui Medicine Co.), TSR-042 (Tesaro Inc.), LZM-009 (Livzon Pharmaceutical Group Inc.) and ABBV-181 (AbbVie Inc.).

[0641] In some embodiments of the combination therapy disclosed herein, the anti-PD-1 antibody is pembrolizumab (Keytruda). ® ).

[0642] In some embodiments, the compositions of the present invention are combined with anti-PD-L1 antibodies. Exemplary anti-PD-L1 antibodies used in the combinations of this invention can be selected from the group consisting of: MedImmune LLC (dvalumab), Atezolizumab (Hoffmann-La Roche Ltd, Chugai Pharmaceutical Co Ltd), Avelumab (Merck KGaA), CX-072 (CytomX Therapeutics Inc), BMS-936559 (ViiVHealthcare Ltd), SHR-1316 (Jiangsu Hengrui Medicine Co Ltd), M-7824 (Merck KGaA), LY-3300054 (Eli Lilly and Co), FAZ-053 (Novartis AG), KN-035 (AlphaMab Co Ltd), CA-170 (Curis Inc), CK-301 (TG Therapeutics Inc), CS-1001 (CStone Pharmaceuticals Co Ltd), HLX-10 (Shanghai Henlius Biotech Co Ltd). Ltd), MCLA-145 (Merus NV), MSB-2311 (MabSpace Biosciences (MabSpace Biosciences (Suzhou) Co Ltd) and MEDI-...

Claims

1. A chimeric polypeptide comprising a bispecific antibody domain, The bispecific antibody domain comprises a first antigen-binding domain that specifically binds to the epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to the cluster 3 T cell receptor (CD3). The first antigen-binding domain includes: The VH structural domain contains The CDR1 amino acid sequence of GGSVSSGDYYWT (SEQ ID NO: 562), the CDR2 amino acid sequence of HIYYSGNTNYNPSLKS (SEQ ID NO: 563), and the CDR3 amino acid sequence of DRVTGAFDI (SEQ ID NO: 564); and At least one of the following: a proline (P) residue at position 40 of FR2, a valine (V) residue at position 67 of FR3, a valine (V) residue at position 71 of FR3, an asparagine (N) residue at position 76 of FR3, a valine (V) residue at position 89 of FR3, an alanine (A) residue at position 93 of FR3, and / or a leucine (L) residue at position 108 of FR4, wherein the FR numbering is based on Kabat; and The VL structural domain, the VH structural domain includes The CDR1 amino acid sequence of QASQDISNYLN (SEQ ID NO: 565), the CDR2 amino acid sequence of DASNLET (SEQ ID NO: 566), and the CDR3 amino acid sequence of QHFDHLPLA (SEQ ID NO: 567); and The chimeric polypeptide further comprises a masking polypeptide that binds to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the masking polypeptide and the bispecific antibody domain, such that the masking polypeptide can reduce the binding of the bispecific antibody domain to CD3 or EGFR, and wherein the protease-cleavable release segment can be cleaved by at least one protease present in the tumor.

2. The chimeric polypeptide according to claim 1, wherein the VH domain comprises an asparagine (N) residue at position 76 of FR3.

3. The chimeric polypeptide according to claim 1 or 2, wherein the VL domain comprises at least one of the following: a tyrosine (Y) residue at position 87 in FR3 and / or a glutamine (Q) residue at position 100 in FR4, wherein the FR numbering is based on Kabat.

4. The chimeric polypeptide according to any one of claims 1 to 3, wherein: The VH domain includes The amino acid sequence of QVQLQX1X2GX3GLX4KPSETLSLTCX5VX6GGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS, where X1 corresponds to E or Q; X2 corresponds to S or W; X3 corresponds to P or A; X4 corresponds to V or L; X5 corresponds to T or A; and X6 corresponds to S or Y (SEQ ID NO: 576); and The VL domain includes X1IX2X3TQSPX4X5LSX6SX7GX8RX9TX 10 X 11 CQASQDISNYLNWYQQKPGX 12 APX 13 LLIYDASNLETGX 14 PX 15 RFSGSGSGTDFTX 16 TISX 17 LX 18 PEDX 19 AX 20 The amino acid sequence of YYCQHFDHLPLAFGQGTKVEIK, where X1 corresponds to D or E; X2 corresponds to Q or V; X3 corresponds to M or L; X4 corresponds to S, G, or A; X5 corresponds to S or T; X6 corresponds to L or A; X7 corresponds to P or V; X8 corresponds to D or E; X9 corresponds to V or A; X 10 Corresponding to I or L; X 11 Corresponding to T or S; X 12 Corresponding to K or Q; X 13 Corresponding to K or R; X 14 Corresponding to V or I; X 15 Corresponding to S, D, or A; X 16 Corresponding to F or L; X 17 Corresponding to S or R; X 18 Corresponding to Q or E; X 19 Corresponding to I or F; and X 20 Corresponding to T or V (SEQ ID NO: 577).

5. A chimeric polypeptide comprising a bispecific antibody domain. The bispecific antibody domain comprises a first antigen-binding domain that specifically binds to the epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to the cluster 3 T cell receptor (CD3). The chimeric polypeptide further comprises a masking polypeptide that binds to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the masking polypeptide and the bispecific antibody domain, such that the masking polypeptide reduces the binding of the bispecific antibody domain to CD3 or EGFR, wherein the protease-cleavable release segment is not cleaved by podin in human plasma, or wherein the rate at which podin cleaves the protease-cleavable release segment in human plasma is less than approximately 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by podin.

6. A chimeric polypeptide comprising a bispecific antibody domain, The bispecific antibody domain comprises a first antigen-binding domain that specifically binds to the epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to the cluster 3 T cell receptor (CD3). The chimeric polypeptide has a melting temperature (Tm) greater than 62°C and / or a thermal stability ratio greater than 0.5 at 62°C; The chimeric polypeptide further comprises a masking polypeptide that binds to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the masking polypeptide and the bispecific antibody domain, such that the masking polypeptide can reduce the binding of the bispecific antibody domain to CD3 or EGFR, and wherein the protease-cleavable release segment can be cleaved by at least one protease present in the tumor.

7. A chimeric polypeptide comprising a bispecific antibody domain. The bispecific antibody domain comprises a first antigen-binding domain that specifically binds to cancer cell antigens and a second antigen-binding domain that binds to the cluster 3 T cell receptor (CD3). The second antigen-binding domain includes: The VH domain comprises the CDR1 amino acid sequence of GFTFSTYAMN (SEQ ID NO: 12), the CDR2 amino acid sequence of RIRTKRNDYATYYADSVKG (SEQ ID NO: 14), and the CDR3 amino acid sequence of HENFGNSYVSWFAH (SEQ ID NO: 10); and VL domain, which contains the CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO: 1), the CDR2 amino acid sequence of GTNKRAP (SEQ ID NO: 4), and the CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 6). The chimeric polypeptide further comprises a masking polypeptide that binds to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the masking polypeptide and the bispecific antibody domain, such that the masking polypeptide can reduce the binding of the bispecific antibody domain to CD3 or the cancer cell antigen, and wherein the protease-cleavable release segment can be cleaved by at least one protease present in the tumor.

8. The chimeric polypeptide of claim 7, wherein the second antigen-binding domain comprises: (i) the VL domain, the VL domain comprising the amino acid sequence (SEQ ID NO: 127) of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL; and (ii) The VH domain, which contains the amino acid sequence of EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRlRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126).

9. The chimeric polypeptide according to any one of claims 1 to 8, wherein the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side, the structural arrangement being defined as: (first antigen-binding domain)-(second antigen-binding domain)-(linker)-(masking polypeptide), (second antigen-binding domain)-(first antigen-binding domain)-(linker)-(masking polypeptide), (masking polypeptide)-(linker)-(first antigen-binding domain)-(second antigen-binding domain) or (masking polypeptide)-(linker)-(second antigen-binding domain)-(first antigen-binding domain), wherein each - is a covalently linked or polypeptide linker.

10. The chimeric polypeptide according to any one of claims 1 to 9, wherein the masking polypeptide is an elongated non-natural polypeptide (ELNN).

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