Composition targeting epidermal growth factor receptor, method for producing the same, and method for using the same

Abstract

This specification provides, in particular, an antibody-binding domain for the differentiation antigen group 3 T cell receptor (CD3), an antibody-binding domain for the epidermal growth factor receptor (EGFR), a cleavable linker sequence, and a protease-activatable bispecific fusion protein, such as a protease-activatable T cell engager, as well as therapeutic uses and methods.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 459,828, filed on 17 April 2023, and U.S. Provisional Patent Application No. 63 / 463,273, filed on 1 May 2023, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Epidermal growth factor receptor (EGFR), also known as ErbB1 and HER1, is a receptor tyrosine kinase involved in cell proliferation. EGFR overexpression or abnormal activity is associated with numerous cancers and is therefore an attractive target for therapeutic intervention. While approved therapies exist, their effectiveness may be hindered by toxicity and / or poor stability.

[0003] There has been a long-standing and unmet need for therapeutic interventions for EGFR-expressing tumors, including stable antibody-based therapies with improved therapeutic indices. [Overview of the project]

[0004] This disclosure provides, in particular, antigen-binding molecules with binding specificity to EGFR, antigen-binding molecules with binding specificity to CD3, and bispecific antigen-binding molecules that bind to both EGFR and CD3, for use in therapeutic settings where specific targeting of EGFR-expressing cells and T cell-mediated killing are desired. The embodiments disclosed herein address a long-standing unmet need for EGFR-targeted cancer therapies, including T cell engagers (TCEs) with increased therapeutic index. The embodiments of this disclosure also address a long-standing, yet unmet need for therapeutic intervention for immunologically cold tumors expressing EGFR, such as solid tumors.

[0005] In one embodiment, the present disclosure is a chimeric polypeptide comprising a bispecific antibody domain, the bispecific antibody domain comprising a first antigen-binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to the differentiated antigen group 3 T cell receptor (CD3), wherein the first antigen-binding domain comprises 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). Acid sequence, and the proline (P) residue at position 40 of FR2 (alternating with amino acid residue 42 for SEQ ID NO: 450); the valine (V) residue at position 67 of FR3 (alternating with amino acid residue 69 for SEQ ID NO: 450); the valine (V) residue at position 71 of FR3 (alternating with amino acid residue 73 for SEQ ID NO: 450); the asparagine (N) residue at position 76 of FR3 (alternating with amino acid residue 78 for SEQ ID NO: 450); the valine (V) residue at position 89 of FR3 (alternating with amino acid residue 94 for SEQ ID NO: 450). The present invention provides a chimeric polypeptide comprising a VH domain containing at least one alanine (A) residue at position 93 of FR3 (alternatingly referred to as amino acid residue 98 for SEQ ID NO: 450) and / or a leucine (L) residue at position 108 of FR4 (alternatingly referred to as amino acid residue 114 for SEQ ID NO: 450) (FR numbering follows Kabat), and a VL domain containing 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), wherein the chimeric polypeptide further comprises a mask polypeptide which is conjugated to the bispecific antibody domain via a linker containing a protease-cleavable release segment located between the mask polypeptide and the bispecific antibody domain, thereby enabling the mask polypeptide to reduce the binding of the bispecific antibody domain to CD3 or EGFR, and the protease-cleavable release segment to be cleavable by at least one protease present in the tumor.

[0006] In some embodiments, the VH domain includes an asparagine (N) residue at position 76 of FR3. In some embodiments, the VH domain includes an alanine (A) residue at position 93 of FR3. In some embodiments, the VH domain includes 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 includes 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.

[0007] In some embodiments, the VL domain comprises at least one of the following: a tyrosine (Y) residue at position 87 of FR3 (alternatingly referred to as amino acid residue 87 for SEQ ID NO: 451) and / or a glutamine (Q) residue at position 100 of FR4 (alternatingly referred to as amino acid residue 100 for SEQ ID NO: 451), with FR numbering following 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.

[0008] 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). The VL domain comprises X1IX2X3TQSPX4X5LSX6SX7GX8RX9TX 10 X 11 CQASQDISNYLNWYQQKPGX 12 APX 13LLIYDASNLETGX 14 PX 15 RFSGSGSGTDFTX 16 TISX 17 LX 18 PEDX 19 AX 20 It contains 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 corresponds to I or L, X 11 corresponds to T or S, X 12 corresponds to K or Q, X 13 corresponds to K or R, X 14 corresponds to V or I, X 15 corresponds to S, D, or A, X 16 corresponds to F or L, X 17 corresponds to S or R, X 18 corresponds to Q or E, X 19 corresponds to I or F, X 20 corresponds to T or V (SEQ ID NO: 577).

[0009] In one embodiment, the present 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 epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to differentiation antigen group 3 T cell receptor (CD3), and the chimeric polypeptide further comprises a mask polypeptide linked to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain so that the mask polypeptide can reduce the binding of the bispecific antibody domain to CD3 or EGFR, wherein the protease-cleavable release segment cannot be cleaved by legmine in human plasma, or legmine cleaves the protease-cleavable release segment in human plasma at a rate less than 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmine.

[0010] In one embodiment, the disclosure relates to a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain that specifically binds to epidermal growth factor receptor (EGFR) and a second antigen-binding domain that binds to differentiation antigen group 3 T cell receptor (CD3), wherein 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, and the chimeric polypeptide further comprises a mask polypeptide conjugated to the bispecific antibody domain via a linker comprising a protease-cleavable release segment located between the mask polypeptide and the bispecific antibody domain, wherein the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or EGFR, and the protease-cleavable release segment is cleavable by at least one protease present in the tumor.

[0011] In some embodiments, Tm is determined by differential scanning fluorescence (DSF).

[0012] In some embodiments, the thermal stability ratio is determined by i) incubating the input amount of chimeric polypeptide at 62°C for 30 minutes to denature a portion of the input amount of chimeric polypeptide; ii) measuring the amount of monomeric chimeric polypeptide remaining after step i; and iii) dividing the amount of monomeric chimeric polypeptide by the input amount of chimeric polypeptide to generate the thermal stability ratio.

[0013] In some embodiments, the amount of monomeric chimeric polypeptide is measured by mass spectrometry.

[0014] In one embodiment, the present disclosure is a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen-binding domain that specifically binds to a cancer cell antigen and a second antigen-binding domain that binds to the differentiated antigen group 3 T cell receptor (CD3), the second antigen-binding domain comprising a VH domain containing 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 the CDR1 amino acid sequence of RSSNGAVTSSNYAN (SEQ ID NO: 1), GTNKRAP (SEQ ID NO: 4) The present invention provides a chimeric polypeptide comprising a VL domain containing the CDR2 amino acid sequence of and the CDR3 amino acid sequence of ALWYPNLWV (SEQ ID NO: 6), wherein the chimeric polypeptide is a mask polypeptide, and is conjugated to the bispecific antibody domain via a linker containing a protease-cleavable release segment located between the mask polypeptide and the bispecific antibody domain, thereby enabling the mask polypeptide to reduce the binding of the bispecific antibody domain to CD3 or cancer cell antigens, and the protease-cleavable release segment to be cleavable by at least one protease present in the tumor.

[0015] In some embodiments, the second antigen-binding domain includes (i) a VL domain comprising the amino acid sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127), and (ii) a VH domain comprising the amino acid sequence EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126).

[0016] In some embodiments, cancer cell antigens include human alpha-4 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, MUC16βhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3; 9-O-acetyl-GD3, GM2, Globo H, Fucosyl GM1, GD2, Carbonic anhydrase IX, CD44v6, Sonic Hedgehog (Shh), Wue-1, Plasma cell antigen 1, Melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, Prostatic 6-transmembrane epithelial antigen (STEAP), Mesothelin, A33 antigen, Prostatic stem cell antigen (PSCA), Ly-6, Desmoglein 4, Fetal acetylcholine receptor (fnAChR), CD25, Cancer antigen 19-9 (CA19-9), Cancer antigen 125 (CA-125), Müllerian inhibitor receptor type II (MISIIR), Siallylated Tn These include antigens (sTN), fibroblast-activating antigen (FAP), endothialin (CD248), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Kola antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMPs, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, or EphA2.

[0017] In some embodiments, the cancer cell antigen is EGFR.

[0018] In some embodiments, the chimeric polypeptide includes a structural arrangement defined from the N-terminus to the C-terminus as (first antigen-binding domain)-(second antigen-binding domain)-(linker)-(mask polypeptide), (second antigen-binding domain)-(first antigen-binding domain)-(linker)-(mask polypeptide), (mask polypeptide)-(linker)-(first antigen-binding domain)-(second antigen-binding domain), or (mask polypeptide)-(linker)-(second antigen-binding domain)-(first antigen-binding domain), where each hyphen is a covalent linkage or polypeptide linker.

[0019] In some embodiments, the mask polypeptide is an extended-length non-natural polypeptide (ELNN).

[0020] In some embodiments, the linker further includes a spacer.

[0021] In some embodiments, the protease-cleavable release segment is fused to the bispecific antibody domain via a spacer.

[0022] In some embodiments, the spacer is characterized by (i) at least 90% of its amino acids being glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof, and (ii) containing at least three amino acids selected from the group consisting of G, A, S, T, E, and P.

[0023] In some embodiments, the spacer is 9 to 14 amino acids long.

[0024] In some embodiments, the spacer comprises at least four amino acids selected from the group consisting of G, A, S, T, E, and P.

[0025] In some embodiments, the amino acids of the spacer consist of A, E, G, S, P, and / or T.

[0026] In some embodiments, the spacer can be cleaved by a non-mammalian protease.

[0027] In some embodiments, the non-mammalian protease is Glu-C.

[0028] In some embodiments, the spacer includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the sequences listed in Table C.

[0029] In some embodiments, the spacer includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to GTSESATPES or GTATPESGPG.

[0030] 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.

[0031] In some embodiments, the chimeric polypeptide comprises a first mask polypeptide linked to a first antigen-binding domain via a first linker, the first linker comprising a first protease-cleavable releasing segment (RS1) that can be cleaved by at least one protease present in the tumor, and the second mask polypeptide is linked to a second antigen-binding domain via a second linker, the second linker comprising a second protease-cleavable releasing segment (RS2) that can be cleaved by at least one protease present in the tumor.

[0032] In some embodiments, the chimeric polypeptide includes a structural configuration defined as (mask1)-(linker1)-(first antigen-binding domain)-(second antigen-binding domain)-(linker2)-(mask2), (mask1)-(linker1)-(second antigen-binding domain)-(first antigen-binding domain)-(linker2)-(mask2), (mask2)-(linker2)-(first antigen-binding domain)-(second antigen-binding domain)-(linker1)-(mask1), or (mask2)-(linker2)-(second antigen-binding domain)-(first antigen-binding domain)-(linker1)-(mask1), where each is individually a covalent or polypeptide linker.

[0033] In some embodiments, the first mask polypeptide is a first ELNN (ELNN1), and the second mask polypeptide is a second ELNN (ELNN2).

[0034] In some embodiments, the chimeric polypeptide includes a structural arrangement 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), where each is individually a covalent bond or polypeptide linker.

[0035] In some embodiments, the linker 1 further includes a first spacer (spacer 1).

[0036] In some embodiments, linker 2 further includes a second spacer (spacer 2).

[0037] In some embodiments, RS1 is fused to the bispecific antibody domain via spacer 1, and / or RS2 is fused to the bispecific antibody domain via spacer 2.

[0038] In some embodiments, the chimeric polypeptide includes a structural arrangement 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), where each is individually a covalent bond or polypeptide linker.

[0039] 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) contain at least three amino acids selected from the group consisting of G, A, S, T, E, and P.

[0040] In some embodiments, spacer 1 and / or spacer 2 are 9 to 14 amino acid long.

[0041] In some embodiments, spacer 1 and / or spacer 2 contain at least four amino acids selected from the group consisting of G, A, S, T, E, and P.

[0042] In some embodiments, the amino acids of spacer 1 and / or spacer 2 consist of A, E, G, S, P, and / or T.

[0043] In some embodiments, spacer 1 and / or spacer 2 include amino acid sequences that have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the sequences listed in Table C.

[0044] In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to GTSESATPES or GTATPESGPG.

[0045] In some embodiments, the amino acid sequence of the first ELNN is 250 to 350 amino acids long, and the amino acid sequence of the second ELNN is 500 to 600 amino acids long.

[0046] In some embodiments, 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.

[0047] In some embodiments, RS1 and / or RS2 include an amino acid sequence comprising the sequence:EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is any amino acid other than N. In some embodiments, X is S.

[0048] In one embodiment, the present disclosure relates to 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, and the chimeric polypeptide further comprises a first ELNN, which is conjugated to the first antigen-binding domain via a first linker comprising a first protease-cleavable release segment (RS1) located between the first ELNN and the first antigen-binding domain, thereby enabling the first ELNN to reduce the binding of the first antigen-binding domain to the cancer cell antigen, and RS The invention provides a chimeric polypeptide wherein 1 is cleavable by at least one protease present in the tumor, and the chimeric polypeptide further comprises a second ELNN, which is a second ELNN conjugated to the second antigen-binding domain via a second linker containing a second protease-cleavable release segment (RS2) located between the second ELNN and the second antigen-binding domain, thereby enabling the second ELNN to reduce the binding of the first antigen-binding domain to the effector cell antigen, the RS2 is cleavable by at least one protease present in the tumor, the first ELNN has a shorter amino acid sequence than the second ELNN, and the cancer cell antigen is not EGFR.

[0049] In some embodiments, the chimeric polypeptide includes a structural arrangement 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), where each is individually a covalent bond or polypeptide linker.

[0050] In some embodiments, the linker 1 further includes a first spacer (spacer 1).

[0051] In some embodiments, linker 2 further includes a second spacer (spacer 2).

[0052] In some embodiments, RS1 is fused to the bispecific antibody domain via spacer 1, and / or RS2 is fused to the bispecific antibody domain via spacer 2.

[0053] In some embodiments, the chimeric polypeptide includes a structural arrangement 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), where each is individually a covalent bond or polypeptide linker.

[0054] In one embodiment, the present disclosure relates to a chimeric polypeptide comprising a bispecific antibody domain, comprising a formula from the N-terminus to the C-terminus including: 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), or 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 has binding specificity to cancer cell antigens, and the second antigen-binding domain is expressed on effector cells. The present invention provides a chimeric polypeptide having binding specificity to a cell antigen, wherein each of the two components individually comprises a covalent linkage or a polypeptide linker, and the mask 1 is a polypeptide capable of reducing the binding of the first antigen-binding domain to its target, and the mask 2 is a polypeptide capable of reducing the binding of the second antigen-binding domain to its target, wherein if the chimeric polypeptide comprises formula 1, spacer 1 consists of A, E, G, S, P, and / or T residues, if the chimeric polypeptide comprises formula 2, spacer 2 consists of A, E, G, S, P, and / or T residues, and if the chimeric polypeptide comprises formula 3, spacer 1 and / or spacer 2 consist of A, E, G, S, P, and / or T residues, and the cancer cell antigen is not EGFR.

[0055] In some embodiments, each link is individually a covalent linkage. In some embodiments, each link is individually a covalent bond. In some embodiments, each link is a peptide bond. In some embodiments, each link is individually a polypeptide linker of 5 amino acids or less.

[0056] In some embodiments, the second antigen-binding domain has binding specificity to human CD3 and cynomolgus monkey CD3. In some embodiments, the second antigen-binding domain has binding specificity to human CD3. In some embodiments, the effector cell antigen is the differentiated antigen group 3 T cell receptor (CD3). In some embodiments, CD3 is CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta. In some embodiments, CD3 is CD3 epsilon.

[0057] In some embodiments, mask 1 is a first ELNN, and mask 2 is a second ELNN.

[0058] 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) contain at least three amino acids selected from the group consisting of G, A, S, T, E, and P.

[0059] In some embodiments, spacer 1 and / or spacer 2 are 9 to 14 amino acid long.

[0060] In some embodiments, spacer 1 and / or spacer 2 contain at least four amino acids selected from the group consisting of G, A, S, T, E, and P.

[0061] In some embodiments, the amino acids of spacer 1 and / or spacer 2 consist of A, E, G, S, P, and / or T.

[0062] In some embodiments, spacer 1 and / or spacer 2 are cleavable by non-mammalian proteases.

[0063] The chimeric polypeptide according to claim 71, wherein the non-mammalian protease is Glu-C.

[0064] In some embodiments, spacer 1 and / or spacer 2 include amino acid sequences that have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the sequences listed in Table C.

[0065] In some embodiments, spacer 1 and / or spacer 2 include an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to GTSESATPES or GTATPESGPG.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] In some embodiments, the amino acid sequence of the first ELNN is 250 to 350 amino acids long, and the amino acid sequence of the second ELNN is 500 to 600 amino acids long.

[0070] In some embodiments, 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.

[0071] In some embodiments, the first antigen-binding domain comprises a first antibody or its antigen-binding fragment, and the second antigen-binding domain comprises a second antibody or its antigen-binding fragment.

[0072] In some embodiments, the first antigen-binding domain is Fab, scFv, or ISVD.

[0073] In some embodiments, the second antigen-binding domain is Fab, scFV, or ISVD.

[0074] In some embodiments, ISVD is a VHH domain.

[0075] In some embodiments, the first antigen-binding domain is scFV.

[0076] In some embodiments, the second antigen-binding domain is scFV.

[0077] In some embodiments, an antibody domain linker is present between the first antigen-binding domain and the second antigen-binding domain.

[0078] In some embodiments, the antibody domain linker includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the sequences listed in Table A or B.

[0079] In some embodiments, the antibody domain linker consists of G and S amino residues.

[0080] In some embodiments, the antibody domain linker is approximately 6 to 12 residues long.

[0081] In some embodiments, the antibody domain linker includes the amino acid sequence GGGGS or GGGGSGGGS.

[0082] In some embodiments, the first antigen-binding domain and / or the second antigen-binding domain comprises an scFv including a VL domain, a VH domain, and a linker between the VL domain and the VH domain, wherein the linker consists of A, E, G, S, P, and / or T residues.

[0083] In some embodiments, the linker is characterized by (i) at least 90% of its amino acids being glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), proline (P), or any combination thereof, and (ii) containing at least three amino acids selected from the group consisting of G, A, S, T, E, and P.

[0084] In some embodiments, the linker between the VL domain and the VH domain is 25-35 amino acids long.

[0085] In some embodiments, the linker between the VL domain and the VH domain contains at least four amino acids selected from the group consisting of G, A, S, T, E, and P.

[0086] In some embodiments, the amino acids of the linker between the VL domain and the VH domain consist of A, E, G, S, P, and / or T.

[0087] In some embodiments, the linker between the VL domain and the VH domain can be cleaved by a non-mammalian protease.

[0088] In some embodiments, the non-mammalian protease is Glu-C.

[0089] In some embodiments, the linker between the VL domain and the VH domain contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0090] In some embodiments, the second antigen-binding domain has an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with the VL domain CDR1;GTNKRAP which contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the following CDR:RSSX1GAVTX2SNYAN (wherein X1 corresponds to T or N, and X2 corresponds to T or S). VL domain CDR2;ALWYX4NLWV (wherein X4 corresponds to S or P) containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VL domain CDR3;GFTFX8TYAMN (wherein X8 corresponds to S or N) containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VH domain CDR1;-RIRX 10 KX 11 NX 12 YATYYADSVKX 13 (In the formula, X 10 This corresponds to T or S, and X 11 This corresponds to R or Y, and X 12 This corresponds to D or N, and X 13 VH domain CDR2;HX containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to (corresponding to G or D). 14 NFGNSYVSWFAX 15 (In the formula, X 14 This corresponds to E or G, and X 15The VH domain CDR3 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to H or Y.

[0091] In some embodiments, the second antigen-binding domain includes 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).

[0092] In some embodiments, the second antigen-binding domain includes a VH domain comprising the amino acid sequence EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126), and a VL domain comprising the amino acid sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127).

[0093] In some embodiments, the first antigen-binding domain is a VL domain CDR1 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDRs: QASQDISNYLN; VL domain CDR2 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to DASNLET; VL domain CDR2 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QHFDHLPLA. The VH domain CDR2 includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to domain CDR3;GGSVSSGDYYWT; and VH domain CDR3 includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to domain CDR1;DRVTGAFDI; and VH domain CDR3 includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to domain CDR3;GGSVSSGDYYWT.

[0094] In some embodiments, the VH domain includes 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, with FR numbering following Kabat. In some embodiments, the VH domain includes an asparagine (N) residue at position 76 of FR3. In some embodiments, the VH domain includes an alanine (A) residue at position 93 of FR3. In some embodiments, the VH domain includes 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 includes 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.

[0095] In some embodiments, the VL domain comprises at least one of the tyrosine (Y) residue at position 87 of FR3 and / or the glutamine (Q) residue at position 100 of FR4, with FR numbering following 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.

[0096] In some embodiments, the first antigen-binding domain includes 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.

[0097] In some embodiments, the first antigen-binding domain includes i) a VH domain containing the amino acid sequence of SEQ ID NO: 468 and a VL domain containing the amino acid sequence of SEQ ID NO: 469; ii) a VH domain containing the amino acid sequence of SEQ ID NO: 466 and a VL domain containing the amino acid sequence of SEQ ID NO: 467; iii) a VH domain containing the amino acid sequence of SEQ ID NO: 490 and a VL domain containing the amino acid sequence of SEQ ID NO: 491; iv) a VH domain containing the amino acid sequence of SEQ ID NO: 492 and a VL domain containing the amino acid sequence of SEQ ID NO: 493; v) a VH domain containing the amino acid sequence of SEQ ID NO: 514 and a VL domain containing the amino acid sequence of SEQ ID NO: 515; vi) a VH domain containing the amino acid sequence of SEQ ID NO: 516 and a VL domain containing the amino acid sequence of SEQ ID NO: 517; vii) a VH domain containing the amino acid sequence of SEQ ID NO: 538 and a VL domain containing the amino acid sequence of SEQ ID NO: 539; or viii) a VH domain containing the amino acid sequence of SEQ ID NO: 540 and a VL domain containing the amino acid sequence of SEQ ID NO: 541.

[0098] In some embodiments, the VL domain is the N-terminus of the VH domain. In some embodiments, the VL domain is the C-terminus of the VH domain.

[0099] In some embodiments, the second antigen-binding domain includes an scFV comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or 100% identity with respect to:

[0100] [Table 1]

[0101] In some embodiments, the first antigen-binding domain includes an scFV comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or 100% identity with respect to:

[0102] [Table 2]

[0103] In some embodiments, the RS includes a protease cleavage site that can be cleaved by at least one protease listed in Table 6.

[0104] In some embodiments, RS includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the sequences listed in Table 7a.

[0105] In some embodiments, RS can be disconnected by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.

[0106] In some embodiments, RS is not cleavable by Regmine.

[0107] In some embodiments, RS is not cleavable by regmine in human blood, plasma, or serum.

[0108] In some embodiments, RS is not cleavable during incubation with regmine of about 1 nM or less for about 20 hours.

[0109] In some embodiments, RS is not cleavable during incubation with legmine at a concentration of approximately 1 nM or less in human blood, plasma, or serum for approximately 20 hours.

[0110] In some embodiments, legmaine cleaves RS in human plasma at a rate less than 50% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmaine.

[0111] In some embodiments, legmaine cleaves RS in human plasma at a rate less than 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmaine.

[0112] In some embodiments, legmaine cleaves RS in human plasma at a rate less than 10% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmaine.

[0113] In some embodiments, legmaine cleaves RS in human plasma at a rate less than 5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmaine.

[0114] In some embodiments, legmaine cleaves RS in human plasma at a rate less than 2.5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmaine.

[0115] In some embodiments, RS1 and / or RS2 include protease cleavage that can be cleaved by at least one protease listed in Table 6.

[0116] In some embodiments, RS1 and / or RS2 include amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the sequences listed in Table 7a.

[0117] In some embodiments, RS1 and / or RS2 can be disconnected by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.

[0118] In some embodiments, RS1 and / or RS2 are not cleavable by Regmine.

[0119] In some embodiments, RS1 and / or RS2 are not cleavable by legmine in human blood, plasma, or serum.

[0120] In some embodiments, RS1 and / or RS2 are not cleavable during incubation with regmine of about 1 nM or less for about 20 hours.

[0121] In some embodiments, RS1 and / or RS2 are not cleavable during incubation with legmine at a concentration of about 1 nM or less in human blood, plasma, or serum for about 20 hours.

[0122] In some embodiments, legmaine cleaves RS1 and / or RS2 in human plasma at a rate less than 50% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmaine.

[0123] In some embodiments, legmaine cleaves RS1 and / or RS2 in human plasma at a rate less than 25% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmaine.

[0124] In some embodiments, legmaine cleaves RS1 and / or RS2 in human plasma at a rate less than 10% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmaine.

[0125] In some embodiments, legmaine cleaves RS1 and / or RS2 in human plasma at a rate less than 5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmaine.

[0126] In some embodiments, legmaine cleaves RS1 and / or RS2 in human plasma at a rate less than 2.5% of the rate at which RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by legmaine.

[0127] 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.

[0128] 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.

[0129] In some embodiments, RS1 and / or RS2 include a protease-cleavable amino acid sequence comprising the sequence:EAGRSASHTPAGLTGP (SEQ ID NO: 7628).

[0130] In some embodiments, RS1 and RS2 are the same.

[0131] In some embodiments, RS1 and RS2 are different.

[0132] In some embodiments, the first ELNN and the second ELNN are each characterized in that (i) at least 90% of the amino acids of 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 amino acids selected from the group consisting of G, A, S, T, E, and P.

[0133] In some embodiments, the first ELNN and the second ELNN are further characterized by (i) each containing at least 100 amino acid residues; and (ii) each containing a plurality of non-repeating sequence motifs having a length of 9 to 14 amino acids, wherein the plurality of non-repeating sequence motifs constitute a set of non-repeating sequence motifs, and each non-repeating sequence motif in the set of non-repeating sequence motifs is repeated at least twice in the ELNN.

[0134] In some embodiments, the multiple non-overlapping sequence motifs include at least one non-overlapping sequence motif that occurs only once within the ELNN.

[0135] In some embodiments, the non-overlapping sequence motif includes one or any combination of the sequence motifs listed in Table 1.

[0136] In some embodiments, the non-overlapping sequence motif includes at least two, three, or four of the sequence motifs listed in Table 1.

[0137] In some embodiments, the non-overlapping sequence motif includes one or any combination of GTSTEPSEGSAP, GTSESATPESGP, GSGPGTSESATP, GSIEPATSGSETP, GSPAGSTSTEE, and GTSPSATPESGP.

[0138] In some embodiments, each of the first ELNN and the second ELNN contains at least four amino acids selected from the group consisting of G, A, S, T, E, and P.

[0139] In some embodiments, the amino acids of the first ELNN and the second ELNN each consist of A, E, G, S, P, and / or T.

[0140] 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 amino acid sequence of the first ELNN is 250 to 350 amino acids long, and the amino acid sequence of the second ELNN is 500 to 600 amino acids long. In some embodiments, 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.

[0141] In some embodiments, the first ELNN and / or the second ELNN include an amino acid sequence that is at least 85% identical to the amino acid sequences listed in Table 3a or 3b.

[0142] In some embodiments, the first ELNN is

[0143] [Table 3] It contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity.

[0144] In some embodiments, the second ELNN is

[0145] [Table 4] It contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity.

[0146] In some embodiments, the chimeric polypeptide comprises one or more barcode fragments.

[0147] In some embodiments, the chimeric polypeptide comprises two or more barcode fragments.

[0148] In some embodiments, each barcode segment is different from all other barcode segments.

[0149] In some embodiments, each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that can be released from the chimeric polypeptide upon complete digestion of the chimeric polypeptide by a non-mammalian protease.

[0150] In some embodiments, the non-mammalian protease is Glu-C.

[0151] In some embodiments, the chimeric polypeptide includes a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG, SGSETPGT, and GTSESATP.

[0152] In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences:

[0153] [Table 5] (In the formula, each "." represents a Glu-C cleavage site, and n is any integer between 0 and 50).

[0154] In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences:

[0155] [Table 6] (In the formula, each "." represents a Glu-C cleavage site, and n is any integer between 0 and 30).

[0156] In some embodiments, n is any integer between 1 and 20. In some embodiments, n is any integer between 5 and 15. In some embodiments, n is any integer between 3 and 7. In some embodiments, n is any integer between 5 and 10. In some embodiments, n is 9. In some embodiments, n is 4.

[0157] In some embodiments, X nThese are PGTGTSAT, PGSGPGT, PGTTPGTT, PGTPPTST, PGTSPSAT, PGTGSAGT, PGTGGAGT, PGTSPGAT, PGTSGSGT, PGTSSAST, PGTGAGTT, PGTGSTST, GSEPATSG, APGTSTEP, PGTAGSGT, PGTSSGGT, PGTGPAT, PGTPGTGT, PGTGGPTT, or PGTGSGST.

[0158] In some embodiments, X n These are TGTS, SGP, TTPG, TPPT, TSPS, TGSA, TGGA, TSPG, TSGS, TSSA, TGAG, TGST, EPAT, GTST, TAGS, TSSG, TAGP, TPGT, TGGP, or TGSG.

[0159] In some embodiments, neither the N-terminal nor the C-terminal amino acids of the chimeric polypeptide are included in the barcode fragment.

[0160] In some embodiments, the chimeric polypeptide comprises an ELNN containing a non-overlapping sequence motif occurring only once within the ELNN, and the ELNN further comprises a barcode fragment containing at least a portion of the non-overlapping sequence motif occurring only once within the ELNN.

[0161] In some embodiments, the chimeric polypeptide comprises a first ELNN containing a first barcode fragment and a second ELNN containing a second barcode fragment, wherein neither the first nor the second barcode fragment contains glutamic acid directly adjacent to another glutamic acid (if present) in the ELNN containing the barcode fragment.

[0162] In some embodiments, at least one of the barcode fragments contains glutamic acid at its C-terminus.

[0163] In some embodiments, at least one of the barcode fragments has an N-terminal amino acid immediately preceding glutamic acid in a chimeric polypeptide.

[0164] In some embodiments, the glutamic acid preceding the N-terminal amino acid of the barcode fragment is not directly adjacent to another glutamic acid.

[0165] In some embodiments, at least one of the barcode segments does not contain the second glutamate at any position other than the C-terminus of the barcode segment, unless proline follows immediately after the second glutamate.

[0166] In some embodiments, the chimeric polypeptide comprises a single polypeptide chain, and the chimeric polypeptide comprises a barcode fragment located within the polypeptide chain, 10 to 200 amino acids or 10 to 125 amino acids from the N-terminus or C-terminus of the chimeric polypeptide.

[0167] In some embodiments, the first ELNN is located at the N-terminus of the bispecific antibody domain, and the first barcode fragment is located within 200, 150, 100, or 50 amino acids of the N-terminus of the chimeric polypeptide.

[0168] In some embodiments, the second ELNN is located at the C-terminus of the bispecific antibody domain, and the second barcode fragment is located within 200, 150, 100, or 50 amino acids of the C-terminus of the chimeric polypeptide.

[0169] In some embodiments, at least one of the barcode segments is at least 4 amino acids long. In some embodiments, at least one of the barcode segments is 4-20, 5-15, 6-12, or 7-10 amino acids long.

[0170] In some embodiments, each mask polypeptide includes one barcode fragment listed in Table 2 or disclosed in Table 3a.

[0171] In some embodiments, the chimeric polypeptide comprises a barcode fragment having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to SGPGSGPGTSE or SGPGTSPSATPE.

[0172] In some embodiments, the chimeric polypeptide comprises one barcode fragment having an amino acid sequence that is at least 95% identical to SGPGSGPGTSE, and another barcode fragment having an amino acid sequence that is at least 95% identical to SGPGTSPSATPE.

[0173] In some embodiments, the barcode fragment consists of A, E, G, S, P, and / or T residues.

[0174] In some embodiments, the barcode fragment is part of the mask peptide.

[0175] In some embodiments, the mask peptide is a first ELNN or a second ELNN.

[0176] In one embodiment, the 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% identity, or 100% identity, to:

[0177] [Table 7]

[0178] In some embodiments, the chimeric polypeptide has the following amino acid sequence:

[0179] [Table 8] Includes.

[0180] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a chimeric polypeptide described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in liquid form or frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake that is reconstituted before administration.

[0181] In one embodiment, the present disclosure provides an injection device comprising a pharmaceutical composition described herein. In some embodiments, the injection device includes a syringe.

[0182] In one embodiment, the present disclosure provides polynucleotide sequences encoding the chimeric polypeptides described herein.

[0183] In one embodiment, the present disclosure provides an expression vector comprising a polynucleotide sequence encoding a chimeric polypeptide described herein.

[0184] In one embodiment, the present disclosure provides a host cell comprising an expression vector containing a polynucleotide sequence encoding a chimeric polypeptide described herein.

[0185] In one embodiment, the present disclosure provides a method for producing the chimeric polypeptide described herein. In some embodiments, the method further includes isolating the chimeric polypeptide from a host cell.

[0186] In one embodiment, the present disclosure provides a method for treating cancer in a subject requiring treatment for cancer, comprising the step of administering an effective amount of a chimeric polypeptide described herein to the subject.

[0187] In some embodiments, cancer includes solid tumors.

[0188] In some embodiments, cancer is a carcinoma, sarcoma, or melanoma.

[0189] In some embodiments, cancer expresses EGFR.

[0190] In some embodiments, cancer overexpresses EGFR.

[0191] In some embodiments, the cancer comprises cells expressing, on average, 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.

[0192] In some embodiments, the cancer comprises cells having one or more oncogenic mutations in the EGFR gene.

[0193] In some embodiments, the cancer comprises cells having EGFR gene amplification.

[0194] In some embodiments, the cells include an increase of 2–5 times, 2–10 times, 2–15 times, 2–30 times, 2–50 times, 3–5 times, 3–10 times, 3–15 times, 3–30 times, 3–50 times, 5–10 times, 5–15 times, 5–30 times, or 5–50 times compared to non-cancerous cells of the same tissue type.

[0195] In some embodiments, 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.

[0196] In some embodiments, the cancer is lung cancer.

[0197] In some embodiments, the lung cancer is non-small cell lung cancer.

[0198] In some embodiments, the cancer is colorectal cancer.

[0199] In some embodiments, the cancer is squamous cell carcinoma of the head and neck.

[0200] In some embodiments, the cancer is breast cancer.

[0201] In some embodiments, the cancer is triple-negative breast cancer.

[0202] In some embodiments, the cancer is brain cancer.

[0203] In some embodiments, the brain cancer is glioblastoma.

[0204] In some embodiments, the method further comprises administering a checkpoint inhibitor.

[0205] In some embodiments, the checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0206] In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0207] In some embodiments, the checkpoint inhibitor is pembrolizumab or cemiplimab.

[0208] In one embodiment, the present disclosure relates to an antibody or antigen-binding fragment thereof that specifically binds to EGFR, 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), wherein the proline (P) residue is at position 40 of FR2, the valine (V) residue is at position 67 of FR3, the valine (V) residue is at position 71 of FR3, the asparagine (N) residue is at position 76 of FR3, and the 89 The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to EGFR, comprising a VH domain containing at least one of a valine (V) residue at position 1, an alanine (A) residue at position 93 of FR3, and / or a leucine (L) residue at position 108 of FR4, and whose FR numbering follows Kabat, and a VL domain containing 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).

[0209] In some embodiments, the VH domain includes an asparagine (N) residue at position 76 of FR3. In some embodiments, the VH domain includes an alanine (A) residue at position 93 of FR3. In some embodiments, the VH domain includes 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 includes 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.

[0210] In some embodiments, the VL domain comprises at least one of the tyrosine (Y) residue at position 87 of FR3 and / or the glutamine (Q) residue at position 100 of FR4, with FR numbering following 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.

[0211] In some embodiments, the antibody or antigen-binding fragment includes a VH domain containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, 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% identity, or 100% identity, with SEQ ID NO: 577.

[0212] In some embodiments, the antibody comprises i) a VH domain containing the amino acid sequence of SEQ ID NO: 468 and a VL domain containing the amino acid sequence of SEQ ID NO: 469; ii) a VH domain containing the amino acid sequence of SEQ ID NO: 466 and a VL domain containing the amino acid sequence of SEQ ID NO: 467; hi) a VH domain containing the amino acid sequence of SEQ ID NO: 490 and a VL domain containing the amino acid sequence of SEQ ID NO: 491; iv) a VH domain containing the amino acid sequence of SEQ ID NO: 492 and a VL domain containing the amino acid sequence of SEQ ID NO: 493; v) a VH domain containing the amino acid sequence of SEQ ID NO: 514 and a VL domain containing the amino acid sequence of SEQ ID NO: 515; vi) a VH domain containing the amino acid sequence of SEQ ID NO: 516 and a VL domain containing the amino acid sequence of SEQ ID NO: 517; vii) a VH domain containing the amino acid sequence of SEQ ID NO: 538 and a VL domain containing the amino acid sequence of SEQ ID NO: 539; or viii) a VH domain containing the amino acid sequence of SEQ ID NO: 540 and a VL domain containing the amino acid sequence of SEQ ID NO: 541.

[0213] In one embodiment, the present disclosure provides an anti-CD3 antibody or its antigen-binding fragment comprising a VH domain including the CDR1 amino acid sequence of CDR: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 including 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).

[0214] In some embodiments, the VL domain comprises the amino acid sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127), and the VH domain comprises the amino acid sequence EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126).

[0215] Various features of this disclosure are specifically described in the appended claims. A better understanding of the features and advantages of this disclosure can be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings. [Brief explanation of the drawing]

[0216] [Figure 1] A schematic diagram of an example EGFR-targeted paTCE is shown. [Figure 2] This is a schematic diagram of the proposed mechanism of action of the exemplary paTCE described herein. [Figure 3]Figure 1 shows schematic diagrams of fully unmasked paTCE (uTCE) from an exemplary paTCE, as well as the individually masked metabolites paTCE(1x-N) and paTCE(1x-C). [Figure 4] A schematic diagram of the antibody framework screening is shown. To identify anti-EGFR antigen-binding fragments with improved properties, CDRs of the donor anti-EGFR antibody, panitumumab, were transplanted in a combined manner into framework regions from approved monoclonal antibody therapies. paTCE libraries containing anti-EGFR antigen-binding fragments were screened for stability, expression, and binding. [Figure 5-1] Figures 5A to 5D show the results from the screening of EGFR-targeted paTCE for thermal stability (Figure 5A), binding affinity (Figure 5B), and thermal stability ratio, which represents the amount of thermally stable monomer remaining at 62°C compared to the protein input (Figure 5C). Figure 5D shows a simulated ribbon structure of an anti-EGFR antibody fragment related to EGFR. [Figure 5-2] Figures 5A to 5D show the results from the screening of EGFR-targeted paTCE for thermal stability (Figure 5A), binding affinity (Figure 5B), and thermal stability ratio, which represents the amount of thermally stable monomer remaining at 62°C compared to the protein input (Figure 5C). Figure 5D shows a simulated ribbon structure of an anti-EGFR antibody fragment related to EGFR. [Figure 5-3] Figures 5A to 5D show the results from the screening of EGFR-targeted paTCE for thermal stability (Figure 5A), binding affinity (Figure 5B), and thermal stability ratio, which represents the amount of thermally stable monomer remaining at 62°C compared to the protein input (Figure 5C). Figure 5D shows a simulated ribbon structure of an anti-EGFR antibody fragment related to EGFR. [Figure 6] This shows the PTE score evaluation for anti-CD3 antibody fragments using the internal PTE algorithm v22. [Figure 7A] The amino acid sequences of RSR-2295 and RSR-3213, along with the alignment of proteases capable of cleaving them, are shown. [Figure 7B]This shows in vitro protease digestion of paTCE using RSR-2295 or RSR-3213. The RSR-3213 sequence is modified to substantially reduce cleavage by legmine. [Figure 8A] Figures 8A and 8B show the relative plasma stability of paTCE using RSR-2295 or RSR-3213, measured on day 0 and day 7. In Figure 8A, RSR-2295 was measured using the SCy5.5 fluorophore, and RSR-3213 was measured using the Scy7.5 fluorophore. In Figure 8B, RSR-2295 was measured using the Scy7.5 fluorophore, and RSR-3213 was measured using the Scy5.5 fluorophore. Figure 8C shows the in vivo cleavage observed from tumor homogenates from three different mouse tumor models. For each set of bars in the bar graph (i.e., 1x-C%, 1x-N%, uTCE%), each bar represents, from left to right, B1, B2, B3, B4, A1, A2, A3, A4, 43-1, 43-2, 43-3, and 43-4. B1-B4 represent four different mice from the first tumor model (NCI-N87). A1-A4 represent four different mice from the second tumor model (HT-29). 43-1-43-4 represent four different mice from the third tumor model (HT-55). Figure 8D shows the percentage of the total of the three metabolites + paTCE (paTCE, 1x-N, 1x-C, and uTCE) when using RSR-2295 or RSR-3213. [Figure 8B]Figures 8A and 8B show the relative plasma stability of paTCE using RSR-2295 or RSR-3213, measured on day 0 and day 7. In Figure 8A, RSR-2295 was measured using the SCy5.5 fluorophore, and RSR-3213 was measured using the Scy7.5 fluorophore. In Figure 8B, RSR-2295 was measured using the Scy7.5 fluorophore, and RSR-3213 was measured using the Scy5.5 fluorophore. Figure 8C shows the in vivo cleavage observed from tumor homogenates from three different mouse tumor models. For each set of bars in the bar graph (i.e., 1x-C%, 1x-N%, uTCE%), each bar represents, from left to right, B1, B2, B3, B4, A1, A2, A3, A4, 43-1, 43-2, 43-3, and 43-4. B1-B4 represent four different mice from the first tumor model (NCI-N87). A1-A4 represent four different mice from the second tumor model (HT-29). 43-1-43-4 represent four different mice from the third tumor model (HT-55). Figure 8D shows the percentage of the total of the three metabolites + paTCE (paTCE, 1x-N, 1x-C, and uTCE) when using RSR-2295 or RSR-3213. [Figure 8C]Figures 8A and 8B show the relative plasma stability of paTCE using RSR-2295 or RSR-3213, measured on day 0 and day 7. In Figure 8A, RSR-2295 was measured using the SCy5.5 fluorophore, and RSR-3213 was measured using the Scy7.5 fluorophore. In Figure 8B, RSR-2295 was measured using the Scy7.5 fluorophore, and RSR-3213 was measured using the Scy5.5 fluorophore. Figure 8C shows the in vivo cleavage observed from tumor homogenates from three different mouse tumor models. For each set of bars in the bar graph (i.e., 1x-C%, 1x-N%, uTCE%), each bar represents, from left to right, B1, B2, B3, B4, A1, A2, A3, A4, 43-1, 43-2, 43-3, and 43-4. B1-B4 represent four different mice from the first tumor model (NCI-N87). A1-A4 represent four different mice from the second tumor model (HT-29). 43-1-43-4 represent four different mice from the third tumor model (HT-55). Figure 8D shows the percentage of the total of the three metabolites + paTCE (paTCE, 1x-N, 1x-C, and uTCE) when using RSR-2295 or RSR-3213. [Figure 8D]Figures 8A and 8B show the relative plasma stability of paTCE using RSR-2295 or RSR-3213, measured on day 0 and day 7. In Figure 8A, RSR-2295 was measured using the SCy5.5 fluorophore, and RSR-3213 was measured using the Scy7.5 fluorophore. In Figure 8B, RSR-2295 was measured using the Scy7.5 fluorophore, and RSR-3213 was measured using the Scy5.5 fluorophore. Figure 8C shows the in vivo cleavage observed from tumor homogenates from three different mouse tumor models. For each set of bars in the bar graph (i.e., 1x-C%, 1x-N%, uTCE%), each bar represents, from left to right, B1, B2, B3, B4, A1, A2, A3, A4, 43-1, 43-2, 43-3, and 43-4. B1-B4 represent four different mice from the first tumor model (NCI-N87). A1-A4 represent four different mice from the second tumor model (HT-29). 43-1-43-4 represent four different mice from the third tumor model (HT-55). Figure 8D shows the percentage of the total of the three metabolites + paTCE (paTCE, 1x-N, 1x-C, and uTCE) when using RSR-2295 or RSR-3213. [Figure 9] This shows the relative tumor uptake of paTCE using RSR-2295 or RSR-3213. The plasma:tumor ratio was calculated in three different mouse tumor models (four mice per tumor model). Each of the three different tumor models contains "Mouse 1", each of the three different tumor models contains "Mouse 2", each of the three different tumor models contains "Mouse 3", and each of the three different tumor models contains "Mouse 4". [Figure 10] Figures 10A to 10C show cytotoxicity curves for exemplary donor cells HT-29 (Figure 10A), MDA-MB-231 (Figure 10B), and A-431 (Figure 10C). [Figure 11-1] Figures 11A and 11B show in vitro cytokine induction assays from representative HT-29 donors. Induction of IFNγ (Figure 11A), TNFα (Figure 11B), IL-6 (Figure 11C), and IL-10 (Figure 11D) is shown. [Figure 11-2]Figures 11A and 11B show an in vitro cytokine induction assay from a representative HT-29 donor. Induction of IFNγ (Figure 11A), TNFα (Figure 11B), IL-6 (Figure 11C) and IL-10 (Figure 11D) is shown. [Figure 12] Shows the expression of CD69, CD25 and PD-1 in CD4+ and CD8+ T cells. [Figure 13] It is a figure showing the in vitro plasma stability of AMX-525 from samples of healthy human donors, human cancer donors (8 pancreas, 2 head and neck, 4 ovary), healthy cynomolgus monkeys, healthy mice, and tumor-bearing mice (HT-29 transplanted CDX). [Figure 14] Shows the tumor growth curve in mice bearing HT-29 tumors. [Figure 15] Shows the tumor growth curve in mice bearing LoVo tumors. [Figure 16] Shows immunohistochemistry (IHC) images and corresponding quantification of CD8+ T cells in tumor tissues from a LoVo xenograft mouse model. [Figure 17] Shows IHC images and corresponding quantification of CD4+ T cells in tumor tissues from a LoVo xenograft mouse model. [Figure 18] Shows IHC images and corresponding quantification of PD-L1 expression in tumor tissues from a LoVo xenograft mouse model. [Figure 19] Shows the tumor growth curve in mice bearing MDA-MB-231 tumors. [Figure 20] Shows the efficacy of AMX-525 as shown by the tumor growth curve in mice bearing SK-OV-3 ovarian tumors. NSG-MHCI / II DKO mice were subcutaneously inoculated with SK-OV-3 tumor cells (day 0), transplanted with PBMC (day 18), and treated with the test article shown on the days indicated by the arrows. [Figure 21]The efficacy of the combination of AMX-525 and the anti-PD-1 antibody pembrolizumab is demonstrated by tumor growth curves in mice carrying SK-OV-3 ovarian tumors. NSG-MHCI / IIDKO mice were subcutaneously inoculated with SK-OV-3 tumor cells (day 0), transplanted with PBMCs (day 18), and treated with the test product indicated by the arrows. [Modes for carrying out the invention]

[0217] There is a significant unmet need in cancer therapy for EGFR-targeted bispecific therapeutic modalities that are effective against solid tumors, particularly those residing in an immunologically cold microenvironment. While TCEs have been shown to be effective in inducing remission in certain cancers, their extreme potency and on-target, off-tumor toxicity in healthy tissue have hindered the development of widespread therapeutics.

[0218] While not bound by any scientific theory, TCEs form crosslinks between T cells and tumor cells, activating T cell-mediated tumor cell killing and initiating a cytokine amplification cascade. This cytokine amplification cascade promotes further tumor cell killing and can potentially provide long-term immunity. T cells activated by TCEs release cell-soluble perforin / granzymes in a manner independent of antigen-MHC recognition. This creates a twofold response: direct tumor cell death and amplification of tumor killing through the initiation of a potent cytokine response from tumor cells. Direct tumor cell death results in the release of tumor antigens. Cytokine responses include, among others, an increase in interferon-γ, which stimulates CD8 T cell activity and antigen presentation by APCs; an increase in IL-2, which leads to increased proliferation of activated T cells; and an increase in CXCL9 and 10 responses, which increases T cell recruitment. The release of tumor antigens and the initiation of the cytokine response together result in the activation of the endogenous T cell response, which potentially triggers epitope diffusion and induces long-term immunity.

[0219] One toxic challenge with TCEs stems from the fact that many tumor targets are expressed to some extent in healthy tissues, and normal cells can also produce cytokine responses that lead to cytokine release syndrome (CRS). These two potent responses in healthy tissues to TCE-induced T cell activation often result in an overall lack of acceptable therapeutic indices for these drugs.

[0220] This disclosure addresses an unmet need and provides a protease-activatable TCE (paTCE) that is superior in one or more embodiments to conventional antibody therapies or bispecific antibody therapies that are active at injection, including improved terminal phase half-life, improved stability, targeted delivery, and / or improved therapeutic ratio with reduced damage to healthy tissue.

[0221] This specification includes compounds, compositions, and methods that overcome the shortcomings of existing TCEs by providing EGFR-targeted paTCEs (referred to herein as EGFR-paTCEs and exemplified as AMX-525).

[0222] AMX-525 contains the amino acid sequence described as Sequence ID No. 1000. While not bound by any scientific theory, it is understood that the paTCE described herein leverages the dysregulated protease activity present in tumors against healthy tissue, enabling an expansion of the therapeutic index. The paTCE core comprises antigen-binding domains, one of which targets CD3 and the other targets EGFR. The two antigen-binding domains may, in exemplary embodiments, be two different antibody formats (e.g., single-chain antibody fragment (scFv) and VHH, etc.) or the same antibody format (e.g., scFv, etc.). Many different antibody fragments or formats may be used.

[0223] In some embodiments, the EGFR-targeted paTCE comprises a first portion which is an scFv that binds to EGFR, and a second portion which is an scFv that binds to CD3. One or more (e.g., two) unstructured polypeptide masks are bound to the core. In some embodiments, these unstructured polypeptide masks sterically reduce target engagement to either the tumor target and / or CD3, and also extend the protein half-life. In some embodiments, the unstructured polypeptide masks are extended-length non-natural polypeptides (ELNNs).

[0224] In some embodiments, the properties of ELNNs also minimize immunogenicity potential because the lack of a stable tertiary structure is unfavorable for antibody binding, and the absence of a hydrophobic aromatic positively charged residue that functions as an anchor residue for peptide MHC II binding reduces the potential of the T cell epitope.

[0225] In some embodiments, the base of the ELNN or the protease cleavage site of the ELNN allows for proteolytic activation of paTCE in the tumor microenvironment, unleashing smaller, highly potent TCEs that can redirect cytotoxic T cells to kill target-expressing tumor cells. In some embodiments, in healthy tissue where protease activity is tightly regulated, paTCEs remain largely inactive, thus expanding the therapeutic index compared to unmasked TCEs.

[0226] In some embodiments, in addition to localized activation, the short half-life of the unmasked TCE form further broadens the therapeutic index while providing T-cell immune efficacy that improves the eradication of solid tumors. In some embodiments, the release site used in paTCE can be cleaved across a wide range of tumors by proteases collectively involved in all cancer hallmarks (growth; survival and mortality; angiogenesis; invasion and metastasis; inflammation; and immune evasion). Thus, by utilizing enhanced protease activity that is upregulated at all stages of cancer and tumor development but tightly regulated in healthy tissues, the TCE activity of paTCE is localized to the tumor.

[0227] term As used herein, the following terms have their meanings unless otherwise specified.

[0228] As used herein and in the claims, the singular forms "a," "an," and "the" include multiple referents unless otherwise clearly indicated. For example, the term "cell" includes multiple cells, including mixtures thereof, unless otherwise clearly indicated by the context.

[0229] Furthermore, as used herein, “and / or” is to be interpreted as a specific disclosure of each of two particular features or components, whether or not they are accompanied by the other. Accordingly, as used herein in phrases such as “A and / or B,” the term “and / or” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, as used in phrases such as “A, B, and / or C,” the term “and / or” is intended to include each of the following embodiments: “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).

[0230] Whenever an aspect is described herein using the phrase "including," it should be understood that other aspects similar in respect to "consisting of" and / or "essentially consisting of" are also provided.

[0231] Numerical ranges include both ends of the number defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino-carboxyl direction. The headings provided herein are not limitations of the various aspects of this disclosure. Therefore, the terms defined immediately thereunder are more fully defined by referring to the entirety of this specification.

[0232] The term “about” is used herein to mean approximately, roughly, around, or within that range. When the term “about” is used with a numerical range, it modifies the range by extending the boundary above and below the stated numerical value. Generally, the term “about” can modify a numerical value by a variation above or below the stated value, for example, 10 percent above or below (higher or lower). In some embodiments, the term indicates a deviation 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% from the stated numerical value. In some embodiments, “about” indicates a deviation of ±10% from the stated numerical value. In some embodiments, “about” indicates a deviation of ±5% from the stated numerical value. In some embodiments, "approximately" indicates a deviation of ±4% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±3% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±2% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±1% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.9% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.8% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.7% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.6% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.5% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.4% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.3% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.1% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.05% from the indicated value. In some embodiments, "approximately" indicates a deviation of ±0.01% from the given value.

[0233] With respect to naturally occurring compounds, the term “isolated” refers to a compound (i.e., a polypeptide or polynucleotide) that does not exist in its natural state (e.g., does not contain naturally associated components in nature, but contains them to varying degrees). No specific level of purification is required. For example, isolated polypeptides can be readily extracted from their natural or natural environment. Recombinant-produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of this disclosure, as are natural or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any preferred technique. “Isolate” and “isolated” may also, depending on the context, indicate the degree of separation from the original source or environment.

[0234] 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 all included within the definition of “polypeptide.” The term “polypeptide” also encompasses amino acid polymers modified by any other operation (e.g., post-translational modification), such as disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or conjugation with labeling components. Depending on the context, the term “polypeptide” may also be used to refer to a protein containing two or more polymers of two or more amino acids.

[0235] "Host cells" include individual cells (e.g., in culture) containing exogenous polynucleotides. Host cells may include offspring of a single host cell. Offspring may not necessarily be completely identical to the original parent cell (in morphology or in the genome of the whole DNA complement) due to naturally occurring or genetically engineered mutations.

[0236] A "fusion" or "chimeric" polypeptide or protein comprises a first polypeptide moiety linked to a second polypeptide moiety that is not naturally linked in nature. In some embodiments, these moieties may typically exist in separate proteins and be combined in the fusion polypeptide, or they may typically exist in the same protein but be arranged in a new configuration in the fusion polypeptide, or these moieties may be combined from different sources. In some embodiments, a fusion or chimeric protein comprises two or more moieties that do not exist in nature (e.g., human-made, designed, or generated moieties such as binding domains, masks, linkers, barcodes, and other polypeptides provided herein). Chimeric proteins may be produced, for example, by chemosynthesis or by recombinant expression (e.g., producing and translating polynucleotides in which peptide regions are encoded in a desired relationship).

[0237] The terms “conjugated,” “linked,” “fused,” and “fused” may be used interchangeably herein, depending on the context. These terms may refer to the covalent joining of two or more chemical (e.g., polypeptide) elements or components by any means including chemical conjugation or recombinant means.

[0238] 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 “identical%”, “identity%”, or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids (where applicable) that are identical in the optimal alignment between the sequences being compared. This percentage may be purely statistical, and the differences between the two sequences may, but may not, be randomly distributed over the entire length of the sequences being compared. The comparison of two sequences is usually performed by comparing the sequences with respect to a segment or “window of comparison” after the optimal alignment in order to identify local regions of the corresponding sequences. For example, optimal alignment for comparison can be performed manually, or with the help of local homology algorithms by Smith and Waterman, 1981, Ads App.Math.2, 482, by Neddleman and Wunsch, 1970, J.Mol.Biol.48, 443, by identity search algorithms by Pearson and Lipman, 1988, Proc.Natl.Acad.Sci.USA 88, 2444, or with the help of computer programs using algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).In some embodiments, the percentage of identity between two sequences is determined using the BLASTN or BLASTP algorithm available on the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word size set to 28; (hi) maximum match within the query range set to 0; (iv) a match / mismatch score set to 1, -2; (v) a gap cost set linearly; and (vi) a filter for low complexity regions used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expected threshold set to 10; (ii) a word size set to 3; (iii) maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) a gap cost set to exist: 11, extended: 1; and (vi) a conditional composition score matrix adjustment. Where 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 the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).BESTFIT uses the local homology algorithm described in Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the best segment of homology 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 in this disclosure, the percentage of identity is naturally calculated over the entire length of the reference polypeptide sequence, and parameters are set so that a homology gap of up to 5% of the total number of amino acids in the reference sequence is acceptable.

[0239] As used herein, the terms “mask polypeptide,” “mask,” and “masking portion” refer to polypeptides capable of reducing the binding of an antigen-binding domain (e.g., an antibody) to a target antigen, in the context of fusion proteins (such as chimeric polypeptides) provided herein. Exemplary mask polypeptides include, but are not limited to, the ELNN polypeptides described herein. Additional mask polypeptides include polypeptides comprising albumin, proline, serine, and alanine; coiled-coil domains; albumin-binding domains; Fc domains; and binding domains specific to the conserved region of the antibody-variable domain. Mask polypeptides are described in further detail in Lucchi et al. (ACS Cent Sci. 2021 May 26;7(5):724-738).

[0240] As used herein, the terms “ELNN polypeptide” and “ELNN” are synonymous and refer to an elongated polypeptide comprising a substantially non-repeating sequence (e.g., polypeptide motif) that is not naturally occurring, primarily composed of small hydrophilic amino acids, which has little to no secondary or tertiary structure under physiological conditions. ELNN polypeptides include unstructured hydrophilic polypeptides comprising a repeating motif of six natural amino acids (G, A, P, E, S, and / or T). In some embodiments, ELNN polypeptides comprise multiple motifs of the six natural amino acids (G, A, P, E, S, T), where the motifs are identical or combinations of different motifs. In some embodiments, when ELNN polypeptides are ligated to proteins, including T cell engagers disclosed herein, they can confer certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties. Such desirable properties include, but are not limited to, improved pharmacokinetic parameters and solubility characteristics, as well as improved therapeutic index. ELNN polypeptides are known in the art, and a non-exclusive description and examples of ELNN polypeptides known as XTEN® polypeptides are available in Schellenberger et al., (2009) Nat Biotechnol 27(12):1186-90, Brandl et al., (2020) Journal of Controlled Release 327:186-197, and Radon et al., (2021) Advanced Functional Materials 31, 2101633 (pages 1-33), the entire contents of each of these are incorporated herein by reference.

[0241] In some embodiments, the repeatability of an ELNN sequence refers to trimer repeatability and can be measured by a computer program or algorithm, or by other means known in the art. In some embodiments, the trimer repeatability of an ELNN can be evaluated by determining the number of occurrences of overlapping trimer sequences within the polypeptide. For example, a polypeptide of 200 amino acid residues has 198 overlapping 3-amino acid sequences (trimers), but the number of unique trimer sequences depends on the amount of repeatability in the sequence. In some embodiments, a score (hereinafter, "subsequence score") can be generated that reflects the degree of trimer repeatability in the entire polypeptide sequence. In this context, the "subsequence score" means the sum of occurrences of each unique trimer frame across 200 consecutive amino acid sequences of the polypeptide divided by the absolute number of unique trimer subsequences within the 200 amino acid sequences. Examples of such subsequence scores derived from the first 200 amino acids of repeating and non-repeat polypeptides are presented in Example 73 of International Patent Application Publication No. 2010 / 091122(A1), which is incorporated by reference in whole.

[0242] In some embodiments, in the context of ELNNs, “substantially non-repeating sequences” means ELNN sequences in which (1) there are few or no examples of four identical amino acids in the sequence of the ELNN sequence, and (2) the ELNN has a subsequence score of 12 or 10 or less (as defined in the preceding paragraphs herein), or there is no pattern in the order of the sequence motifs constituting the polypeptide sequence from N-terminus to C-terminus.

[0243] The term “single-stranded variable fragment” (scFV) corresponds to an antigen-binding domain consisting of at least one heavy-strand variable domain (VH) linked to at least one light-strand variable domain (VL). The VH and VL may be linked to any linker recognized in the art, including, but not limited to, SESATPESGPGTSPGATPESGPGTSESATP. In some embodiments, the scFV includes a VH domain and a VL domain from the N-terminus to the C-terminus. In other embodiments, the scFv includes a VL domain and a VH domain from the N-terminus to the C-terminus. Tandem scFvs, such as divalent scFvs (di-scFvs), are scFvs that include multiple scFvs linked in tandem. A di-scFv includes two VH domains and two VL domains, and each scFv has the same or different (e.g., bispecific) target specificity. In some embodiments, the scFvs described herein are monovalent or divalent scFvs.

[0244] The term "Immunoglobulin Single Variable Domain" (ISVD) defines an immunoglobulin molecule formed by the presence of an antigen-binding site on a single immunoglobulin domain. This distinguishes an ISVD from "conventional" immunoglobulins (e.g., monoclonal antibodies) or their fragments (Fab, Fab', F(ab')2, scFv, di-scFv, etc.) which have two immunoglobulin domains, particularly two variable domains, that interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen-binding site. In this case, complementarity-determining regions (CDRs) from both the VH and VL contribute to the antigen-binding site, meaning a total of six CDRs are involved in antigen-binding site formation. In contrast, in ISVDs, only three CDRs from a single domain contribute to antigen-binding site formation.

[0245] Considering the above definition, the antigen-binding domains of conventional four-chain antibodies (such as IgG, IgM, IgA, IgD, or IgE molecules known in the art), or Fab fragments, F(ab')2 fragments, disulfide-linked Fv fragments, or scFV fragments, or diabodies derived from such conventional four-chain antibodies (all known in the art), are not usually considered single immunoglobulin variable domains. In these cases, binding to each epitope of the antigen does not usually occur by a single immunoglobulin domain, but by a pair of (associated) immunoglobulin domains, such as light-chain and heavy-chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains that bind together to the epitopes of each antigen.

[0246] In contrast, a single immunoglobulin variable domain can specifically bind to an antigen epitope without pairing with an additional immunoglobulin variable domain. The binding site of a single immunoglobulin variable domain is formed by a single VH, single VHH, or single VL domain.

[0247] Therefore, a single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof, insofar as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit that is essentially derived from 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), insofar as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit that is primarily derived from 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).

[0248] The immunoglobulin single variable domain (ISVD) may be a heavy chain ISVD such as VH, VHH, or for example, camelized VH or humanized VHH. In some embodiments, it is VHH containing camelized VH or humanized VHH. The heavy chain ISVD may be derived from a conventional four-chain antibody or a heavy chain antibody.

[0249] For example, an immunoglobulin monovariate domain may be a monodomain antibody (or an amino acid sequence suitable for use as a monodomain antibody), a "dAb", or a dAb (or an amino acid sequence suitable for use as a dAb); another monovariate domain, or any suitable fragment of any of these.

[0250] In some embodiments, the immunoglobulin single variable domain may be a nanobody® molecule or a suitable antigen-binding fragment thereof. Nanobody® is a registered trademark of Ablynx N.V.

[0251] The "VHH domain," also known as VHH, VHH region, VHH antibody fragment, and VHH antibody, was originally described as the antigen-binding immunoglobulin variable domain of "heavy chain antibodies" (i.e., "antibodies lacking a light chain," 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 four-chain antibodies (referred to herein as "VH domain," "VH region," and "VH") and the light chain variable domains present in conventional four-chain antibodies (referred to herein as "VL domain," "VL region," and "VL"). For further description of VHH, see the review by Muyldermans (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0252] A “vector” is a nucleic acid molecule that transfers an inserted nucleic acid molecule into and / or between host cells. In some embodiments, the vector self-replicates in a suitable host. The term includes vectors that primarily function for the insertion of DNA or RNA into cells, vector replicaters that primarily function for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Also included are vectors that provide two or more of the above functions. An “expression vector” is a polynucleotide that, when introduced into a suitable host cell, can be used for the transcription of mRNA, which is translated into polypeptides. In some embodiments, an “expression system” is a suitable host cell containing an expression vector that can function to produce a desired expression product.

[0253] The terms “treatment” or “treating” and “improving” may be used interchangeably herein. These terms refer to approaches to obtain beneficial or desirable outcomes, including but not limited to therapeutic benefits. “Therapeutic benefit” means the eradication or improvement of the underlying disorder being treated. In some embodiments, the therapeutic benefit is achieved by the eradication or improvement of one or more physiological symptoms associated with the underlying disease condition, such that improvement is observed in the subject, even though the subject may still be suffering from the underlying disorder. In some embodiments, the therapeutic benefit includes slowing or halting the growth of one or more tumors. In some embodiments, the therapeutic benefit includes reducing the size of one or more tumors. In some embodiments, the therapeutic benefit includes eradicating one or more tumors from the subject. In some embodiments, the therapeutic benefit includes causing the death of cancer cells.

[0254] As used herein, the term “therapeutic dose” means the amount of a biologically active agent (e.g., a fusion protein provided herein as part of a pharmaceutical composition) that, when administered to a subject in a single or repeated dose, is capable of having any detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease condition or state. Such effect does not need to be absolute to be beneficial. A disease condition may refer to a disorder or disease, such as cancer or the symptoms of cancer.

[0255] Antigen-binding domain, cleavage sequence, barcode fragment, and fusion polypeptide 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. Included herein are fusion polypeptides comprising (i) one or more mask polypeptides (e.g., ELNN), (ii) a bispecific antibody (BsAb, e.g., TCE) linked to the mask polypeptide, and (iii) one or more protease-cleavable release segments (RS), wherein the RS is located between the mask polypeptide and the BsAb.

[0256] In some embodiments, the anti-EGFR antibody provided herein has the following sequence: It includes a VH domain containing QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS (Sequence ID 468), and a VL domain containing the following sequence:DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQGTKVEIK (Sequence ID 469).

[0257] In some embodiments, the anti-CD3 antibody provided herein has the sequence: VH domain containing the CDR area of ​​the VH domain containing EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (Sequence ID 126), and / or array: Includes a VL domain containing a CDR of a VL domain including ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (Sequence ID 127).

[0258] Also provided is a BsAb comprising, for example, an anti-EGFR antibody and / or an anti-CD3 antibody as disclosed herein. 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 comprising the VH and VL pairs disclosed herein and an anti-CD3 scFV region comprising the VH and VL pairs disclosed herein. In some embodiments, the BsAb is a TCE.

[0259] In some embodiments, the fusion polypeptide comprises a first ELNN (such as the ELNN described herein). In some embodiments, the polypeptide further comprises a second ELNN (such as the ELNN described herein). In some embodiments, the polypeptide comprises an ELNN ("N-terminal ELNN") at or near its N-terminus. In some embodiments, the polypeptide comprises an ELNN ("C-terminal ELNN") at or near its C-terminus. In some embodiments, the polypeptide comprises both an N-terminal ELNN and a C-terminal ELNN.

[0260] In some embodiments, the fusion polypeptide comprises a BsAb, where a first ELNN is bound to the N-terminus of the BsAb by a first RS, and a second ELNN is bound to the C-terminus of the BsAb by a second RS. In some embodiments, each RS is cleavable by a protease as described herein. In some embodiments, each RS comprises an RS sequence as disclosed herein. In some embodiments, the fusion polypeptide is paTCE.

[0261] This specification includes polypeptide sequences that can be used, for example, to link one polypeptide moiety to another in a fusion protein. For example, useful linkers are provided that are cleaved by multiple proteases but not by legmine. In some embodiments, such linkers may be used outside the context of antibodies, such as those described herein.

[0262] In some embodiments, the fusion polypeptide (e.g., one or more ELNNs of paTCE, and / or other parts of the fusion polypeptide such as a linker or spacer sequence) may include one or more barcode fragments (e.g., as described herein) that are capable of releasing (e.g., configured to release) the fusion polypeptide upon cleavage or digestion of the fusion polypeptide (e.g., paTCE) by a protease. In some embodiments, the protease is a non-mammalian protease. In some embodiments, each barcode fragment has a different sequence and molecular weight from all other peptide fragments (including all other barcode fragments, if present) that are capable of releasing from the polypeptide upon complete digestion of the polypeptide by the protease, thereby making it unique and allowing its presence to be detected by techniques such as mass spectrometry.

[0263] Elongated recombinant polypeptide (ELNN) Chain length and amino acid composition In some embodiments, ELNN contains at least 100 or at least 150 amino acids. In some embodiments, ELNN has an amino acid length of 100 to 3,000 or 150 to 3,000. In some embodiments, ELNN has an amino acid length of 100 to 1,000 or 150 to 1,000. In some embodiments, ELNN has 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, and a few The amino acid length is at least (approximately) 850, at least (approximately) 900, at least (approximately) 950, at least (approximately) 1,000, at least (approximately) 1,100, at least (approximately) 1,200, at least (approximately) 1,300, at least (approximately) 1,400, at least (approximately) 1,500, at least (approximately) 1,600, at least (approximately) 1,700, at least (approximately) 1,800, at least (approximately) 1,900, or at least (approximately) 2,000. In some embodiments, 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, many The amino acid length is at most (approximately) 850, at most (approximately) 900, at most (approximately) 950, at most (approximately) 1,000, at most (approximately) 1,100, at most (approximately) 1,200, at most (approximately) 1,300, at most (approximately) 1,400, at most (approximately) 1,500, at most (approximately) 1,600, at most (approximately) 1,700, at most (approximately) 1,800, at most (approximately) 1,900, or at most (approximately) 2,000.In some embodiments, ELNN is approximately 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, and 800. The amino acid lengths are approximately 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000, or within a range between any two of the above. In some embodiments, at least 90% of the amino acid residues of 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 ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P). In some embodiments, ELNN comprises at least three different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, ELNN comprises at least four different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, ELNN comprises at least five different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, ELNN consists of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, ELNN comprises the amino acids G, A, S, T, E, and P. In some embodiments, ELNN (e.g., ELNN1, ELNN2, etc.) is characterized by (i) containing at least 100 or at least 150 amino acids, (ii) at least 90% of the amino acid residues of ELNN being glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P), and (hi) containing 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 a glutamic acid residue, whether or not the side-chain carboxyl is deprotonated. In some embodiments, the ELNN-containing fusion polypeptide comprises a first ELNN and a second ELNN. In some embodiments, the sum of 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.

[0264] Non-repeating array motifs In some embodiments, the ELNN comprises or is formed from a plurality of non-repeating sequence motifs. In some embodiments, at least one of the non-repeating sequence motifs is repeating (or repeats at least twice in the ELNN). In some embodiments, the ELNN comprises at least one other non-repeating sequence motif that is non-repeating (or found only once in the ELNN). In some embodiments, the plurality of non-repeating sequence motifs comprises (a) a set of (repeating) non-repeating sequence motifs in which each non-repeating sequence motif in the set is repeated at least twice in the ELNN, and (b) a non-repeating (non-repeating) sequence motif that occurs (or is found) only once in the ELNN. In some embodiments, each non-repeating sequence motif is 9–14 (or 10–14, or 11–13) amino acid length. In some embodiments, each non-repeating sequence motif is 12 amino acid length. In some embodiments, the multiple non-repeating sequence motifs include a set of non-repeating (repeating) sequence motifs, where each non-repeating sequence motif in the set of non-repeating sequence motifs is (1) repeated at least twice in the ELNN and (2) 9 to 14 amino acid lengths. In some embodiments, the set of (repeating) non-repeating sequence motifs includes dodecamer sequence motifs identified herein by sequence numbers 179-200 and 1715-1722 in Table 1. In some embodiments, the set of (repeating) non-repeating sequence motifs includes dodecamer sequence motifs identified herein by sequence numbers 186-189 in Table 1. In some embodiments, the set of (repeating) non-repeating sequence motifs includes at least two, at least three, or all four of the dodecamer sequence motifs of sequence numbers 186-189 in Table 1. In some embodiments, the ELNN further includes sequences other than the dodecamer sequence motifs shown in Table 1. In some embodiments, the ELNN includes sequences not in Table 1, such as ASSATPESGP, GSGPGTSESATP, or GTSESATP. In some embodiments, ELNN includes sequences not listed in Table 1, such as ATPESGP, GTSPSATPESGP, or GTSESAGEPEA. In some embodiments, ELNN includes barcode sequences.

[0265] [Table 9] * This indicates individual motif sequences that, when used together in various permutations, create a "family sequence".

[0266] Confirmation of unstructured polypeptides In various embodiments, the ELNN components (or multiple ELNN components) of a fusion protein have an unstructured three-dimensional structure under physiological conditions, regardless of the polymer length (e.g., elongated length). For example, the ELNN is characterized by a large degree of structural freedom of the peptide backbone. In some embodiments, the ELNN is characterized by the absence of long-range interactions as determined by NMR. In some embodiments, the disclosure provides an ELNN that, under physiological conditions, resembles the structure of a denatured sequence with little secondary structure. In some embodiments, the ELNN may substantially lack secondary structure under physiological conditions. As used in this context, “little secondary structure” means that, as measured or determined by the means described herein, less than 50% of the ELNN amino acid residues of the ELNN contribute to the secondary structure. As used in this context, “substantially secondary” means that, as measured or determined by the means described herein, 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 of the ELNN sequence do not contribute to the secondary structure.

[0267] 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 secondary structure of an ELNN can be measured spectrophotometrically, for example, by circular dichroism spectroscopy in the "far ultraviolet" spectral region (190–250 nm). Secondary structural elements such as alpha-helices and beta-sheets each give rise to characteristic shapes and sizes in the CD spectrum. Secondary structures can also be predicted for polypeptide sequences using certain computer programs or well-known algorithms such as the Chou-Fasman algorithm (Chou, PY, et al. (1974) Biochemistry, 13:222-45) and the Garnier-Osguthorpe-Robson ("GOR") algorithm (Gamier J, Gibrat JF, Robson B. (1996), U.S. Patent Application Publication No. 20030228309(A1) (the entire contents of which are incorporated herein by reference), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553). For a given sequence, the algorithm can predict whether some or no secondary structure is present, expressed, for example, as the total and / or percentage of residues in sequences that form an alpha-helix or beta-sheet, or as the percentage of residues in sequences that are predicted to result in random coil formation (lack of secondary structure).

[0268] In some embodiments, the ELNN used in the fusion protein composition may have an alpha-helix percentage in the range of 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 beta-sheet percentage in the range of 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 an alpha-helix percentage in the range of 0% to less than about 5% and a beta-sheet percentage in the range of 0% to less than about 5%, as determined by the Chou-Fasman algorithm. In some embodiments, the ELNN of the fusion protein composition has an alpha-helix percentage of less than about 2% and a beta-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 embodiments, 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%, as determined by the GOR algorithm.

[0269] Net charge In some embodiments, the ELNN polypeptide may have unstructured characteristics conferred by the incorporation of net-charged amino acid residues and / or a reduction in the proportion of hydrophobic amino acids in the ELNN sequence. The overall net charge and net charge density can be controlled, for example, by modifying 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 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.

[0270] Since most tissues and surfaces in humans or animals have a net negative charge, ELNNs can be optionally designed to have a net negative charge to minimize nonspecific interactions between ELNN-containing compositions and various surfaces such as blood vessels, healthy tissues, or various receptors. While not bound by any particular theory, ELNNs can adopt an open conformation due to the electrostatic repulsive forces between individual amino acids of the ELNN polypeptide, which carry a high net negative charge individually and are distributed across the sequence of the ELNN polypeptide. Such a distribution of net negative charge in the extended sequence length of ELNN can result in an unstructured conformation, which in turn can lead to an effective increase in the hydrodynamic radius. Therefore, in some embodiments, ELNNs contain glutamine such that glutamine accounts for about 8, 10, 15, 20, 25, or even about 30% of the amino acids in the sequence. The ELNNs in the compositions of this disclosure generally have no positively charged amino acids or have a low content of positively charged amino acids. In some embodiments, the ELNN may have less than 10% positively charged amino acid residues, or less than 7%, 5%, or 2% positively charged amino acid residues. However, the disclosure envisions a polypeptide in which a limited number of positively charged amino acids, such as lysine, can be incorporated into the ELNN to enable conjugation between the epsilonamine of lysine and a reactive group on a peptide, a linker crosslink, or a reactive group on a drug or small molecule that is conjugated to the ELNN skeleton.

[0271] In some embodiments, ELNN may contain charged residues separated by other residues such as serine or glycine, which may result in better expression or purification behavior. Based on net charge, ELNN of the composition in question may have isoelectric points (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, ELNN has isoelectric points between 1.5 and 4.5. In some embodiments, ELNN incorporated into a paTCE fusion protein carries a net negative charge under physiological conditions, contributing to an unstructured three-dimensional structure and reduced binding of ELNN components to mammalian proteins and tissues.

[0272] Since hydrophobic amino acids can confer structure to polypeptides, in some embodiments, the hydrophobic amino acid content in ELNN is less than 5%, less than 2%, or less than 1%. In some embodiments, ELNN does not contain hydrophobic amino acids. In some embodiments, the amino acid content of methionine and tryptophan in the ELNN components of the paTCE fusion protein is less than 5%, less than 2%, most preferably less than 1%. In some embodiments, ELNN has a sequence having less than 10% positively charged amino acid residues, or about less than 7%, about less than 5%, or about less than 2% positively charged amino acid residues, with the sum of methionine and tryptophan residues being less than 2% of the total ELNN sequence, and the sum of asparagine and glutamine residues being less than 10% of the total ELNN sequence. In some embodiments, ELNN does not contain methionine or tryptophan residues.

[0273] Increased hydrodynamic radius In some embodiments, ELNNs may have a high hydrodynamic radius, conferring a correspondingly increased apparent molecular weight to the paTCE fusion protein incorporating the ELNN. Conjugation of ELNNs to a BsAb (e.g., TCE) sequence may result in a paTCE composition having an increased hydrodynamic radius, increased apparent molecular weight, and increased apparent molecular weight factor compared to a BsAb (e.g., TCE) not conjugated to an ELNN. For example, in some therapeutic applications where an extended half-life is desirable, one or more ELNNs with a high hydrodynamic radius may be incorporated into a fusion protein containing a BsAb (e.g., TCE) to effectively expand the hydrodynamic radius of the fusion protein beyond a glomerular pore size of approximately 3–5 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), resulting in reduced renal clearance of the circulating protein. In some embodiments, the hydrodynamic radius of a protein is determined by its molecular weight and its structure, including its shape and compactness. While not bound 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 certain amino acids in a sequence lacking the potential to confer secondary structure. In some embodiments, the open, elongated, and unstructured conformation of an ELNN polypeptide has a larger proportional hydrodynamic radius compared to polypeptides of comparable sequence length and / or molecular weight that have a secondary structure and / or tertiary structure, such as a typical globular protein. Methods for determining the hydrodynamic radius, such as the use of size exclusion chromatography (SEC) as described in U.S. Patents 6,406,632 and 7,294,513, are well known in the art.In some embodiments, the addition of increasing the ELNN length results in a proportional increase in the parameters of hydrodynamic radius, apparent molecular weight, and apparent molecular weight factor, allowing for the adjustment of paTCE to a desired characteristic cutoff apparent molecular weight or hydrodynamic radius. Thus, in some embodiments, the paTCE fusion protein can be configured with an ELNN such that the fusion protein may 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 conferred by the ELNN in the paTCE fusion protein may result in a reduction in the renal clearance of the resulting fusion protein, leading to a corresponding increase in terminal phase half-life, an increase in mean residence time, and / or a decrease in renal clearance rate.

[0274] In some embodiments, ELNNs of selected length and sequence (or multiple ELNNs, such as two ELNNs) can be selectively incorporated into paTCE to produce a fusion protein having 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 more under physiological conditions. In some embodiments, selective ligation of ELNNs (or multiple ELNNs, such as two ELNNs) of selected length and sequence to BsAb (e.g., TCE) can yield a paTCE fusion protein having, under physiological conditions, 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 apparent molecular weight factors, or at least 20 or more apparent molecular weight factors. In some embodiments, the paTCE fusion protein has, under physiological conditions, 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. In some embodiments, the fusion polypeptide exhibits an apparent molecular weight factor of greater than about 6 under physiological conditions.

[0275] Increased terminal phase half-life In some embodiments, the fusion polypeptide containing an ELNN (such as paTCE) has a terminal phase half-life that is at least twice as long, at least three times longer, at least four times longer, or at least five times longer compared to the corresponding biologically active polypeptide that is not linked to an ELNN.

[0276] In some embodiments, administering a therapeutically effective dose of paTCE fusion protein to a subject in need results in at least a twofold, at least threefold, at least fourfold, or at least fivefold increase in the time the fusion protein remains within the therapeutic window compared to the corresponding BsAb (e.g., TCE) not linked to ELNN when administered to the subject at an equivalent dose.

[0277] In some embodiments, the TCE released from paTCE upon protease cleavage contains one or more short polypeptides (e.g., amino acids with an amino acid length of about 30, 25, 20, 15, 14, 13, 12, 11, 10, or less) that do not contain amino acids other than G, A, P, E, S, and / or T. For example, the 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 into one or more spacer or linker sequences that remain as part 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 the 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid position of the N-terminal amino acid, or the sequence includes the N-terminus). In some embodiments, the TCE released from paTCE is GTATPESGPG on the C-terminal side of the TCE (for example, the nearest amino acid in the sequence is at the 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid position of the N-terminal amino acid, or the sequence includes the N-terminus). In some embodiments, the TCE includes an internal linker (for example, between the VL and VH regions of scFV) containing a polypeptide sequence that does not contain amino acids other than G, A, P, E, S, and / or T, such as SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

[0278] Low immunogenicity In some embodiments, the present disclosure provides compositions in which ELNNs have low immunogenicity or are substantially non-immunogenic. Several factors, such as substantially non-repeating sequences, unstructured three-dimensional structures, high solubility, low degree of autoaggregation or absence of autoaggregation, low degree of proteolytic sites or absence of proteolytic sites in the sequence, and low degree of epitopes or absence of epitopes in ELNNs, may contribute to the low immunogenicity of ELNNs.

[0279] Those skilled in the art will generally understand that polypeptides having highly repeating short amino acid sequences (e.g., sequences of 200 amino acids in length containing a limited set of trimers or tetramers with an average of 20 or more repeats) and / or polypeptides having consecutive repeating amino acid residues (e.g., sequences of pentamers or hexamers having identical amino acid residues) tend to aggregate, form higher-order structures, or form contacts to produce crystalline or pseudocrystalline structures.

[0280] In some embodiments, the ELNN sequence is substantially non-repeating, (1) the ELNN sequence does not have three consecutive amino acids of the same type unless the amino acid is serine, in which case three or fewer consecutive amino acids may be serine residues, and (2) the ELNN does not contain three amino acid sequences (trimers) that occur more than 16, 14, 12, or 10 times within the sequence of at least 200 amino acids in length of the ELNN (e.g., the entire span of the ELNN which is at least the length of the amino acids). While not bound by any scientific theory, such substantially non-repeating sequences allow for the design of long sequences of ELNNs with a relatively low frequency of charged amino acids, which are less prone to aggregation and therefore more likely to aggregate if the sequence or amino acid residues are more repeating.

[0281] Structural epitopes can be formed by regions on the protein surface composed of multiple discontinuous amino acid sequences of a protein antigen. While not bound by any scientific theory, precise protein folding can transform these sequences into clearly defined, stable spatial configurations or epitopes that can be recognized as "foreign" by the host humoral immune system, leading to antibody production against the protein and / or triggering a cell-mediated immune response. In the latter case, the immune response to the protein in an individual is largely influenced by T cell epitope recognition, which correlates with the peptide bond specificity of the individual's HLA-DR allotype. Engagement of MHC class II peptide complexes by congeneral T cell receptors on the T cell surface, along with cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T cell. This activation can lead to cytokine release, further activating other lymphocytes such as B cells to produce antibodies, or activating T killer cells as a complete cellular immune response.

[0282] While not bound by any scientific theory, the ability of a peptide to bind to a given MHC class II molecule for presentation on the surface of an antigen-presenting cell (APC) can depend on several factors, most notably its primary sequence. In some embodiments, lower immunogenicity can be achieved by designing an ELNN that resists antibody processing in antigen-presenting cells and / or by selecting a sequence that does not bind sufficiently to the MHC receptor. In some embodiments, an ELNN-containing fusion protein has a substantially non-repeating ELNN polypeptide designed to reduce binding to the MHC II receptor and to avoid the formation of epitopes for T cell receptor or antibody binding, resulting in lower immunogenicity. While not bound by any scientific theory, avoidance of immunogenicity is, in part, a direct result of the flexibility of the ELNN's conformation, i.e., the lack of secondary structure due to the selection and order of amino acid residues. Of particular interest, for example, are sequences that tend to adopt a compactly folded conformation in aqueous solution or under physiological conditions that may produce conformational epitopes. Administration of ELNN-containing fusion proteins using conventional therapeutic practices and dosages generally does not result in the formation of neutralizing antibodies against ELNN and can reduce the immunogenicity of BsAb (e.g., TCE) fusion partners in paTCE compositions.

[0283] In some embodiments, the ELNN used in the target fusion protein may substantially omit epitopes recognized by human T cells. Excluding such epitopes for the purpose of generating less immunogenic proteins has been previously disclosed, see, for example, International Publications 98 / 52976, 02 / 079232, and 00 / 3317, which are incorporated herein by reference. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) J Immunol Methods, 281:95-108). Of particular interest are peptide sequences that can be oligomerized without generating T cell epitopes or non-human sequences. This can be achieved by testing direct repeats of these sequences for the presence of T cell epitopes and for the generation of non-human 6-15 mers, particularly 9-mer sequences, and then modifying the design of the ELNN sequence to exclude or disrupt the epitope sequences. In some embodiments, ELNNs become substantially non-immunogenic by limiting the number of ELNN epitopes predicted to bind to MHC receptors. Along with the reduction in the number of epitopes capable of binding to MHC receptors, there is a simultaneous reduction in the potential for T cell activation and T cell helper function, a reduction in B cell activation or upregulation, and a reduction in antibody production. The low degree of predicted T cell epitopes can be determined by epitope prediction algorithms such as TEPITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17:555-61), as shown in Example 74 of International Patent Application Publication No. 2010 / 144502(A2), which is incorporated entirely by reference. A form of TEPITOPE score for a given peptide frame in a protein is disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555. The score should be at least above 20log, approximately 10 to approximately -10 (10e 10 K D ~10e -10 K DThis range corresponds to the binding constraints of ) and can be reduced by avoiding hydrophobic amino acids such as M, I, L, V, or F that can function as anchor residues during peptide display on MHC. In some embodiments, the ELNN components incorporated into paTCE do not have T cell epitopes predicted by a TEPITOPE score of about -5 or greater, or -6 or greater, or -7 or greater, or -8 or greater, or a TEPITOPE score of -9 or greater. As used herein, the score of "-9 or greater" includes TEPITOPE scores from 10 to -9 (inclusive), but does not include the score of -10, as -10 is less than -9.

[0284] In some embodiments, ELNNs, including those incorporated into a target paTCE fusion protein, can be made substantially non-immunogenic by limiting known proteolytic sites from the ELNN sequence, reducing the processing of the ELNN into small peptides that can bind to MHC II receptors. In some embodiments, the ELNN sequence can be made substantially non-immunogenic by using a sequence that substantially lacks secondary structure, conferring resistance to many proteases due to the high entropy of the structure. Thus, the reduction of the TEPITOPE score and the elimination of known proteolytic sites from the ELNN can make ELNN compositions containing the ELNN of a paTCE fusion protein composition substantially unbound by mammalian receptors, including receptors of the immune system. In some embodiments, the ELNN of a paTCE fusion protein has a Kb greater than 100 nM for mammalian receptors. D Binding, or a K+500 nM K to mammalian cell surface or circulating polypeptide receptors. D Alternatively, K greater than 1 μM D It may have.

[0285] Additionally, the substantially non-repeating sequence and lack of corresponding epitopes in such embodiments of ELNN may limit the ability of B cells to bind to or be activated by ELNN. In some embodiments, ELNN may come into contact with many different B cells across its extended sequence, while each individual B cell may come into contact with only one or a few individual ELNNs. As a result, ELNN may typically have a much lower tendency to stimulate B cell proliferation, and therefore an immune response. In some embodiments, paTCE may have reduced immunogenicity compared to the corresponding BsAb (e.g., TCE) that is not fused to a mask polypeptide such as ELNN. In some embodiments, up to three parenteral doses of paTCE administered to a mammal may produce detectable anti-paTCE IgG at a serum dilution of 1:100, but not at a dilution of 1:1000. In some embodiments, up to three parenteral doses of paTCE administered to a mammal may produce detectable anti-BsAb (e.g., TCE) IgG at a serum dilution of 1:100, but not at a dilution of 1:1000. In some embodiments, up to three parenteral doses of paTCE administered to a mammal may produce detectable anti-ELNN IgG at a serum dilution of 1:100, but not at a dilution of 1:1000. In some embodiments, the mammal may be, for example, a mouse, rat, rabbit, cynomolgus monkey, or human. In some embodiments, the mammal is human.

[0286] Compared to these less repeating sequences (such as those with three consecutive identical amino acids), the additional features of certain ELNNs having substantially non-repeating sequences are that non-repeating ELNNs may form weaker contact (e.g., monovalent interactions) with antibodies, thereby resulting in the possibility of lower immunoclearance, allowing paTCE compositions to persist for an increased period in circulation.

[0287] In some embodiments, biologically active polypeptides containing ELNNs (BsAbs, e.g., TCE) exhibit lower immunogenicity compared to fusion polypeptides not linked to any ELNN, and this immunogenicity is confirmed by measuring the production of IgG antibodies that selectively bind to the biologically active polypeptide after administration of equivalent doses to the subject.

[0288] Barcode fragment 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, second, or 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 terms “barcode fragment” (or “barcode” or “barcode sequence”) may refer to either a portion of a polypeptide cleavably fused within the polypeptide, or a peptide fragment resulting from its release from the polypeptide.

[0289] In some embodiments, the barcode fragment may be (1) a part of the ELNN containing at least a portion of a (non-repeating, non-overlapping) sequence motif that occurs (or is found) only once within the ELNN, and (2) differs in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide upon cleavage or complete digestion of the polypeptide by a protease.

[0290] In some embodiments, the barcode fragment does not contain the N-terminal or C-terminal amino acids of the fusion polypeptide. As described herein, in some embodiments, the barcode fragment is releaseable (e.g., configured to be releaseable) during Glu-C digestion of the fusion polypeptide. In some embodiments, the barcode fragment is located in ELNN and does not contain a glutamate directly adjacent to another glutamate (if present) in ELNN. In some embodiments, the barcode fragment has glutamate at its C-terminus. Those skilled in the art will understand that if the C-terminus of the barcode fragment is cleavably fused within a polypeptide (such as ELNN), it can refer to the "last" (or most C-terminal) amino acid residue in the barcode fragment, even if other non-barcode amino acid residues are located C-terminal to the barcode fragment within the polypeptide (e.g., ELNN). In some embodiments, the barcode fragment has an N-terminal amino acid immediately preceded by a glutamate residue. In some embodiments, the glutamate residue preceding the N-terminal amino acid is not directly adjacent to another glutamate residue. In some embodiments, the barcode fragment does not contain a (second) glutamate residue at any position other than the C-terminus of the barcode fragment, unless proline immediately follows glutamate. In some embodiments, the barcode fragment is located at a certain distance from either the N-terminus or the C-terminus of the polypeptide, where this distance is 10 to 150 or 10 to 125 amino acids. 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 within a range of any of the aforementioned. In some embodiments, the barcode fragment is located within 200 amino acids, 150 amino acids, 100 amino acids, or 50 amino acids from the N-terminus of the polypeptide.In some embodiments, the barcode fragment is located at a position 10-200, 30-200, 40-150, or 50-100 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 within a range between any of the above. In some embodiments, the barcode fragment is located within 200, 150, 100, or 50 amino acids from the C-terminus of the polypeptide. In some embodiments, the barcode fragment is located at a position 10-200, 30-200, 40-150, or 50-100 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 directly adjacent to another glutamic acid, if present in ELNN, (ii) having a glutamic acid at its C-terminus, (iii) having an N-terminal amino acid immediately preceding a glutamic acid residue, and (iv) being located at a certain distance from either the N-terminus or the C-terminus of the polypeptide, with a distance of 10-150 amino acids or 10-125 amino acids in length. In some embodiments, the barcode fragment is located in the ELNN and (i) does not contain the N-terminal or C-terminal amino acid of the polypeptide, (ii) does not contain a glutamic acid directly adjacent to another glutamic acid in the ELNN, (iii) has glutamic acid at its C-terminus, (iv) has an N-terminal amino acid immediately preceding a glutamic acid residue, and (v) is located at a certain distance from either the N-terminus or C-terminus of the polypeptide, the distance being 10 to 150 or 10 to 125 amino acid lengths. In some embodiments, the glutamic acid residue preceding the N-terminal amino acid is not directly adjacent to another glutamic acid residue. In some embodiments, the barcode fragment does not contain glutamic acid residues at any position other than the C-terminus of the barcode fragment unless a proline immediately follows the glutamic acid.Depending on the context herein, when referring to a location within a polypeptide sequence, the term “distance” may refer to the number of amino acid residues from the N-terminus of the polypeptide to the most N-terminal amino acid residue of the barcode fragment, or from the C-terminus of the polypeptide to the most C-terminal amino acid residue of the barcode fragment. In some embodiments, for a barcoded ELNN fused to a biologically active polypeptide, at least one barcode fragment (or at least two, or three) contained in the barcoded ELNN is located at at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 amino acids from the biologically active polypeptide. In some embodiments, the barcode fragment is at least 4, at least 5, at least 6, at least 7, or at least 8 amino acid lengths. In some embodiments, the barcode fragment is at least 4 amino acid lengths. In some embodiments, the barcode fragment is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid lengths, or within a range of any of the aforementioned values. In some embodiments, the barcode fragment is 4–20, 5–15, 6–12, or 7–10 amino acid lengths. In some embodiments, as used herein, the barcode fragment comprises amino acid sequences identified by SEQ ID NOs. 68–79 and SEQ ID NOs. 1010–1027 in Table 2.

[0291] [Table 10]

[0292] In some embodiments, each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that can be released from the chimeric polypeptide described herein upon complete digestion of the chimeric polypeptide by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

[0293] In some embodiments, the chimeric polypeptides disclosed herein include a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG, SGSETPGT, and GTSESATP.

[0294] In some embodiments, the chimeric polypeptide disclosed herein has the following amino acid sequence: 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 The formula comprises at least one of 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 has the following amino acid sequence:PE.SGX n PE.SG, PE.GSX n SE.GG, PE.TPX n SE.TG, PE.SGX n Includes at least one of SE.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 nThese are SGPGTGTSATPE, SGPGSGPGTSE, SGPGTTPGTTPE, SGPGTPPTSTPE, SGPGTSPSATPE, SGPGTGSAGTPE, SGPGTGGAGTPE, SGPGTSPGATPE, SGPGTSGSGTPE, SGPGTSSASTPE, SGPGTGAGTTPE, SGPGTGSTSTPE, TPGSEPATSGSE, GSAPGTSTEPSE, SGPGTAGSGTPE, SGPGTSSGGTPE, SGPGTAGPATPE, SGPGTPGTGTPE, SGPGTGGPTTPE, or SGPGTGSGSTPE.

[0295] In some embodiments, the chimeric polypeptide has the following amino acid sequence:

[0296] [Table 11] The formula includes at least one of the following (wherein each "." is a Glu-C cleavage site and n is any integer from 0 to 50). In some embodiments, the chimeric polypeptide has the following amino acid sequence:

[0297] [Table 12] The formula includes at least one of the following: (wherein 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, X nThis is PGTGTSAT, PGSGPGT, PGTTPGTT, PGTPPTST, PGTSPSAT, PGTGSAGT, PGTGGAGT, PGTSPGAT, PGTSGSGT, PGTSSAST, PGTGAGTT, PGTGSTST, GSEPATSG, APGTSTEP, PGTAGSGT, PGTSSGGT, PGTGPAT, PGTPGTGT, PGTGGPTT, or PGTGSGST. In some embodiments, X n These are TGTS, SGP, TTPG, TPPT, TSPS, TGSA, TGGA, TSPG, TSGS, TSSA, TGAG, TGST, EPAT, GTST, TAGS, TSSG, TAGP, TPGT, TGGP, or TGSG.

[0298] In some embodiments, the barcode is designed to have improved analytical properties. In some embodiments, such barcodes may be released by a non-mammalian protease such as Glu-C at a relatively low concentration. This facilitates better detection, for example, through LC / MS, and also enables the measurement of peptides produced from the cleavable linker, thereby enabling the measurement of the cleavage product, for example, using LC / MS.

[0299] In some embodiments of fusion proteins containing ELNNs, the fusion protein has a single polypeptide chain, and the polypeptide chain contains 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 (e.g., paTCE) contains a first ELNN and a second ELNN, the first ELNN being 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 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.

[0300] In some embodiments, the ELNN further comprises one or more additional barcode fragments, each of which has a different sequence and molecular weight from all other peptide fragments that can be released from the polypeptide upon complete digestion of the polypeptide by a protease. In some embodiments, the barcoded ELNN comprises only one barcode fragment. In some embodiments, the barcoded ELNN comprises a set of barcode fragments, including a first barcode fragment such as those described herein. In some embodiments, the set of barcode fragments comprises a second barcode fragment (or further barcode fragments), such as those described herein. In some embodiments, the set of barcode fragments comprises a third barcode fragment, such as those described herein.

[0301] A set of fused barcode fragments within an N-terminal ELNN may be referred to as the N-terminal set of the barcode ("N-terminal set"). A set of fused barcode fragments within a C-terminal ELNN may be referred to as the C-terminal set of the barcode ("C-terminal set"). In some embodiments, the N-terminal set includes a first barcode fragment and a second barcode fragment. In some embodiments, the N-terminal set further includes a third barcode fragment. In some embodiments, the C-terminal set includes a first barcode fragment and a second barcode fragment. In some embodiments, the C-terminal set further includes a third barcode fragment. In some embodiments, the polypeptide includes 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) differs in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide upon complete digestion of the polypeptide by a protease.

[0302] This specification includes mixtures comprising multiple polypeptides of varying lengths, wherein the mixture comprises a set of first polypeptides and a set of second polypeptides. In some embodiments, each polypeptide in the set of first polypeptides includes a barcode fragment having a sequence and molecular weight different from all other fragments that can be released from the set of first polypeptides (a) by protease digestion and (b) having a sequence and molecular weight different from all other fragments that can be released from the set of first polypeptides. In some embodiments, the set of second polypeptides lacks the barcode fragment of the set of first polypeptides (e.g., due to cleavage). In some embodiments, both the set of first polypeptides and the set of second polypeptides each include a reference fragment that is common to both the set of first polypeptides and the set of second polypeptides and (b) can be released by protease digestion. In some embodiments, the ratio of the set of first polypeptides to the polypeptide containing the reference fragment is greater than 0.70. In some embodiments, the ratio of the set of first polypeptides to the polypeptide containing the reference fragment is greater than 0.80, 0.90, 0.95, or 0.98. In some embodiments, a reference fragment occurs no more than once in each polypeptide of the first set of polypeptides and the second set of polypeptides. In some embodiments, the protease is a protease that cleaves at the C-terminal side of the glutamic acid residue. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, polypeptides of varying lengths include polypeptides containing at least one ELNN, such as any of those described herein. In some embodiments, the first set of polypeptides includes a full-length polypeptide, and the barcode fragment is part of the full-length polypeptide. In some embodiments, the full-length polypeptide is any of those described above herein or any other part of this specification (fusion) polypeptide. In some embodiments, polypeptides of varying lengths in the mixture differ from one another due to N-terminal cleavage, C-terminal cleavage, or both N-terminal and C-terminal cleavage of the full-length polypeptide.In some embodiments, the first set of polypeptides and the second set of polypeptides may differ in one or more pharmacological properties.

[0303] The disclosure also provides a method for evaluating the relative amount of a set of first polypeptides to a set of second polypeptides in a mixture containing polypeptides of varying lengths, wherein (1) each polypeptide in the set of first polypeptides shares a barcode fragment that occurs only once and only once in the polypeptide, and (2) each polypeptide in the set of second polypeptides lacks a barcode fragment shared by the set of first polypeptides, and each individual polypeptide in both the set of first polypeptides and the set of second polypeptides contains a reference fragment. In some embodiments, the method comprises contacting the mixture with a protease to produce a plurality of proteolytic fragments resulting from the cleavage of the set of first polypeptides and the set of second polypeptides, wherein the plurality of proteolytic fragments contain a plurality of reference fragments and a plurality of barcode fragments. In some embodiments, the method may further comprise determining the ratio of the amount of barcode fragments to the amount of reference fragments, thereby evaluating the relative amount of the set of first polypeptides to the set of second polypeptides. In some embodiments, the barcode fragment occurs once or less in each polypeptide of the set of first polypeptides. In some embodiments, a reference fragment is generated once or less for each polypeptide in the first set of polypeptides and the second set of polypeptides. In some embodiments, multiple proteolytic fragments include multiple reference fragments and multiple barcode fragments. In some embodiments, the protease cleaves the first and second sets of polypeptides (or polypeptides of varying lengths) at the C-terminal side of a glutamic acid residue that is not followed by a proline residue. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, the step of determining the ratio of the amount of barcode fragments to the amount of reference fragments includes identifying the barcode fragments and reference fragments from the mixture after contact with the protease. In some embodiments, the barcode fragments and reference fragments are identified based on their respective masses. In some embodiments, the barcode fragments and reference fragments are identified by mass spectrometry.

[0304] In some embodiments, the barcode fragment and the reference fragment are identified via 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 spiking the mixture with one or both of the isotopically labeled reference fragment and the isotopically labeled barcode fragment. In some embodiments, polypeptides of varying lengths include polypeptides comprising at least one ELNN described above or elsewhere herein. In some embodiments, the ELNN 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 (iii) comprising at least four different types of amino acids, which are G, A, S, T, E, or P. In some embodiments, the barcode fragment, if present, is part of the ELNN. In some embodiments, the mixture of polypeptides of varying lengths includes polypeptides described above or elsewhere in this specification. In some embodiments, the polypeptides of varying lengths include full-length polypeptides and their cleavage fragments. In some embodiments, the polypeptides of varying lengths consist mainly of full-length polypeptides and their cleavage fragments. In some embodiments, the polypeptides of varying lengths in the mixture differ from one another due to N-terminal cleavage, C-terminal cleavage, or both N-terminal and C-terminal cleavage of the full-length polypeptide. In some embodiments, the full-length polypeptide is a polypeptide described above or elsewhere in this specification. In some embodiments, the ratio of barcode fragments to reference fragments is greater than 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, or 0.99.

[0305] Quantification based on isobaric labeling of peptides In some embodiments, isobaric labeling can be used to determine the ratio of a barcode fragment to a reference fragment. Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics in which a peptide or protein (or part thereof) is labeled with various chemical groups that are isobaric (identical in mass) but vary in terms of the distribution of heavy isotopes around their structure. In some embodiments, these tags, commonly referred to as tandem mass tags, are designed so that during high-energy collision-induced dissociation (CID) in tandem mass spectrometry, the mass tag is cleaved at a specific linker region, thereby producing reporter ions of different masses. Some of the most common isobaric tags are amine-reactive tags.

[0306] Exemplary barcoded ELNN polypeptide This specification includes an ELNN that contains a barcode fragment, which is part of the ELNN.

[0307] Table 3a shows the amino acid sequences of exemplary barcoded ELNNs containing one barcode (e.g., SEQ ID NOs. 8002-8003, 8005-8009, and 8013-8022), two barcodes (e.g., SEQ ID NOs. 8001, 8004, and 8012), or three barcodes (e.g., SEQ ID NO. 8011). In some embodiments, of these exemplary barcoded ELNNs, 12 (SEQ ID NOs. 8001-8003, 8008-8009, 8011, 8015-8019, and 8022) are fused to a biologically active protein (such as TCE) at the C-terminus of a biologically active protein, and 10 (SEQ ID NOs. 8004-8007, 8010, 8012-8014, 8020, and 8021) are fused to the N-terminus of a biologically active protein. In some embodiments, ELNN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequences identified herein by sequence numbers 8001-8022 in Table 3a.

[0308] [Table 13-1]

[0309] [Table 13-2]

[0310] [Table 13-3]

[0311] [Table 13-4]

[0312] [Table 13-5]

[0313] Table 13-6

[0314] Table 13-7

[0315] In some embodiments, barcoded ELNNs can be obtained by introducing one or more mutations into an existing ELNN, such as any of those listed in Table 3b, according to one or more of the following criteria: minimizing sequence changes in the ELNN, minimizing changes in amino acid composition in 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 one of sequence numbers 601-659 listed in Table 3b. In some embodiments, ELNN sequences 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 to any of sequence numbers 601-659 listed in Table 3b are obtained by one or more mutations from the corresponding sequences in Table 3b (e.g., mutations less than 10, less than 8, less than 6, less than 5, less than 4, less than 3, or less than 2). In some embodiments, one or more mutations include deletion of a glutamate residue, insertion of a glutamate residue, substitution of a glutamate residue, substitution with a glutamate residue, or any combination thereof. In some embodiments, if the ELNN sequence is different from any one of sequence numbers 601-659 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, then any difference of at least 80%, at least 90%, at least 95%, at least 97%, or about 100% between the ELNN sequence and the corresponding sequence in Table 3b includes deletion of glutamate residues, insertion of glutamate residues, substitution of glutamate residues, substitution with glutamate residues, 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 includes substitutions of glutamate residues, or substitutions with glutamate residues, or both.

[0316] As used herein, “substitution of the first amino acid” refers to the replacement of a first amino acid residue with a second amino acid residue, where the second amino acid residue occupies the position of the substitution in the resulting sequence. For example, “substitution of glutamate” refers to the replacement of a glutamate (E) residue with a non-glutamate residue (e.g., serine (S)).

[0317] [Table 14-1]

[0318] [Table 14-2]

[0319] [Table 14-3]

[0320] [Table 14-4]

[0321] [Table 14-5]

[0322] [Table 14-6]

[0323] [Table 14-7]

[0324] [Table 14-8]

[0325] [Table 14-9]

[0326] In some embodiments, amino acid mutations are performed on ELNNs of intermediate length to those in Table 3b, as well as on ELNNs longer than those in Table 3b, such as those in which one or more dodecamer motifs from Table 1 are added to the N-terminus or C-terminus of the general-purpose ELNNs in Table 3b, in order to construct the barcoded ELNN sequence.

[0327] Additional examples of existing ELNNs that can be used in accordance with this disclosure are U.S. Patent Publications 2010 / 0239554(A1), 2010 / 0323956(A1), 2011 / 0046060(A1), 2011 / 0046061(A1), 2011 / 0077199(A1), or 2011 / 0172146(A1), Alternatively, they are disclosed in International Patent Publication Nos. 2010091122(A1), 2010144502(A2), 2010144508(A1), 2011028228(A1), 2011028229(A1), 2011028344(A2), 2014 / 011819(A2), or 2015 / 023891.

[0328] In some embodiments, a barcoded ELNN fused into the polypeptide chain adjacent to the N-terminus of a polypeptide chain ("N-terminal ELNN") may be bound to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus to facilitate the purification of the fused polypeptide. In some embodiments, a barcoded ELNN fused into the polypeptide chain at the C-terminus of a polypeptide chain ("C-terminal ELNN") may contain or be bound to the sequence EPEA at the C-terminus to facilitate the purification of the fused 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 bound at its N-terminus to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49), and the C-terminal barcoded ELNN is bound at its C-terminus to the sequence EPEA, thereby facilitating the purification of the fusion polypeptide to, for example, a purity of 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 matrices, and other such methods.

[0329] The barcode fragments described herein may be fused in a cleavable manner within an ELNN and may be released from the ELNN during protease digestion of the polypeptide (i.e., may be configured to be released). In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease cleaves at the C-terminal side of glutamic acid residues that are not followed by proline. In some embodiments, the barcoded ELNN (an ELNN containing a 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 may result in diverse cleavage patterns during Glu-C digestion. Therefore, when a Glu-C protease is used for barcode release, the barcoded ELNN or barcode fragment may not contain a Glu-Glu(EE) sequence. Additionally, if a dipeptide Glu-Pro(EP) sequence is present in the fused polypeptide, it may not be cleaved by the Glu-C protease during the barcode release process.

[0330] Structural configuration of activatable TCEs In some embodiments, the fusion protein comprises a single BsAb in the form of a TCE and a single ELNN. In some embodiments, such a fusion protein may have at least the following configurations: (TCE)-(ELNN), (ELNN)-(TCE), (TCE)-(linker)-(ELNN), (ELNN)-(linker)-(TCE), each listed in the direction from the N-terminus to the C-terminus.

[0331] In some embodiments, the fusion protein includes a C-terminal ELNN and optionally a linker between the ELNN and TCE (such as those listed in Table C, for example). In some embodiments, such a fusion protein is represented by formula I (shown from the N-terminus to the C-terminus): (TCE)-(linker)-(ELNN)(I) It can be represented by the formula, where TCE is as described herein, linker is a linker sequence (such as those described herein, for example, in Table C) comprising 1 to about 50 amino acid residues which may optionally include a TCE release segment (as described herein), and ELNN may be any ELNN as described herein.

[0332] In some embodiments, the fusion protein includes an N-terminal ELNN and optionally a linker between the ELNN and the TCE (such as those listed in Table C, for example). In some embodiments, such a fusion protein is represented by formula II (shown from the N-terminus to the C-terminus): (ELNN)-(linker)-(TCE)(II) It can be represented by the formula, where TCE is as described herein, linker is a linker sequence (such as those described herein, for example, in Table C) comprising 1 to about 50 amino acid residues which may optionally include a TCE release segment (as described herein), and ELNN may be any ELNN as described herein.

[0333] In some embodiments, the fusion protein includes both an N-terminal ELNN and a C-terminal ELNN. In some embodiments, such a fusion protein is given by formula III: (ELNN)-(linker)-(TCE)-(linker)-(ELNN)(III) It can be represented by the formula, where TCE is as described herein, each linker is a linker sequence (such as those described herein, for example, in Table C) having 1 to about 50 amino acid residues which may individually and optionally include a TCE release segment (as described herein), and each ELNN may individually be any ELNN as described herein.

[0334] This disclosure provides BsAb (e.g., TCE) containing one or more sequences of the disclosure in any one of Tables 5a to 5f of this specification.

[0335] Of particular interest are BsAbs (e.g., TCEs) for which increased pharmacokinetic parameters, increased solubility, increased stability, masking of activity, or some other improvement in pharmaceutical properties are desired, or BsAbs (e.g., TCEs) for which an increase in terminal phase half-life improves efficacy and / or safety. Therefore, paTCE fusion protein compositions are prepared with various objectives in mind, including, for example, improving the therapeutic efficacy of TCE by increasing in vivo exposure or increasing the length for which TCE remains within the therapeutic window when administered to a subject, compared to TCE not linked to any ELNN.

[0336] For example, it will be understood that variants can be created by making various amino acid substitutions (specifically, conserved amino acid substitutions) in the bispecific sequence without departing from the spirit of this disclosure with respect to the biological activity or pharmacological properties of TCE. Examples of conserved amino acid substitutions in polypeptide sequences are shown in Table 4. In addition, variants may also include polypeptides in which one or more amino acid residues are added to or deleted from the N-terminus or C-terminus of the full-length native amino acid sequence of TCE, for example, while retaining at least some of the biological activity of the native peptide.

[0337] 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 target paTCE. In some embodiments, TCEs found to retain a suitable level of activity can be linked to one or more ELNN polypeptides having at least about 80% sequence identity to the sequences from Tables 3a-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).

[0338] [Table 15]

[0339] This disclosure provides an ELLN-modified TCE (such as paTCE) that targets 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 the other portion of the bispecific TCE.

[0340] In some embodiments, the ELLN-modified TCE comprises (1) a first portion including a first binding domain and a second binding domain, (2) a second portion including an release segment, and (3) a third portion including an unstructured polypeptide mask (which may also be referred to herein as a masking portion).

[0341] In some embodiments, the ELNN-modified TCE has the stereochemistry of formula Ia (shown from the N-terminus to the C-terminus): (Part 1) - (Part 2) - (Part 3) (Ia) The formula comprises, wherein the first part is a bispecific antibody domain comprising the two antigen-binding domains described above, the first binding domain having a specific binding affinity to EGFR (e.g., expressed on cancer cells), the second binding domain having a specific binding affinity to CD3 (e.g., expressed on effector cells), the second part comprising a release segment (RS) that can be cleaved by a mammalian protease, and the third part being a masking portion that functions to mask the biological properties of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment that can be cleaved by proteases present in the tumor microenvironment.

[0342] In some embodiments, the first part comprises two binding domains, each containing VL and VH, the binding domains of the first part may be in the order (VL-VH)1-(VL-VH)2, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2 (wherein "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (for example, as described herein).

[0343] In some embodiments, the domain that binds to EGFR is scFv, which includes VH and VL.

[0344] In some embodiments, the first partial binding domain comprises sequences provided in Tables 5a-5f, where Tables 5a-5e represent sequences that bind to CD3, and Table 5f represents sequences that bind to EGFR; the RS sequence comprises sequences provided in Tables 7a-7b (e.g., those 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 respect to sequences comprising the group of sequences described in Tables 3a-3b. In some embodiments, the composition is a recombinant fusion protein. In some embodiments, the portions are linked by chemical conjugation.

[0345] In some embodiments, the fusion protein has the conformation of formula IIa (shown from the N-terminus to the C-terminus): (Part 3) - (Part 2) - (Part 1) (IIa) The formula comprises, wherein the first part is bispecific and comprises two antigen-binding domains, the first binding domain having a specific binding affinity to EGFR (e.g., expressed on cancer cells), the second binding domain having a specific binding affinity to CD3 (e.g., expressed on effector cells), the second part comprises a release segment (RS) that can be cleaved by a mammalian protease, and the third part is a masking portion that functions to mask the biological properties of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment that can be universally cleaved in the tumor microenvironment.

[0346] In some embodiments, the first part comprises two binding domains, each containing VL and VH, the binding domains of the first part may be in the order (VL-VH)1-(VL-VH)2, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2 (wherein "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (for example, as described herein).

[0347] In some embodiments, the domain that binds to EGFR is scFv, which includes VH and VL.

[0348] In some embodiments, the first partial binding domain comprises sequences provided in Tables 5a-6f, where Tables 5a-e represent sequences that bind to CD3, and Table 5f represents sequences that bind to EGFR; the RS sequence comprises sequences provided in Tables 7a-7b (e.g., those 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 respect to sequences comprising the group of sequences described in Tables 3a-3b. In some embodiments, the composition is a recombinant fusion protein. In some embodiments, the portions are linked by chemical conjugation.

[0349] In some embodiments, the paTCE composition has the stereochemistry of formula Illa (shown from the N-terminus to the C-terminus): (Part 5) - (Part 4) - (Part 1) - (Part 2) - (Part 3) (IIIa) The formula comprises, wherein the first part is bispecific and comprises two antigen-binding domains, the first binding domain having a specific binding affinity to EGFR (e.g., expressed on cancer cells), the second binding domain having a specific binding affinity to CD3 (e.g., expressed on effector cells), the second part comprises a release segment (RS) that can be cleaved by a mammalian protease, the third part is a masking portion that functions to mask the biological properties of the bispecific antibody domain, the fourth part comprises a release segment (RS) that can be cleaved by a mammalian protease which may be the same as or different from the second part, and the fifth part is a masking portion which may be the same as or different from the third part.

[0350] In some embodiments, the first part comprises two binding domains, each containing VL and VH, the binding domains of the first part may be in the order (VL-VH)1-(VL-VH)2, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2 (wherein "1" and "2" represent the first and second binding domains, respectively), and the paired binding domains are linked by a polypeptide linker (for example, as described herein).

[0351] In some embodiments, the domain that binds to EGFR is scFv, which includes VH and VL.

[0352] In some embodiments, the first partial binding domain comprises sequences provided in Tables 5a-5f, where Tables 5a-5e represent sequences that bind to CD3, and Table 5f represents sequences that bind to EGFR. Each RS sequence comprises, individually, sequences provided in Tables 7a-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 respect to sequences comprising the group of sequences described in Tables 3a-3b. In some embodiments, paTCE is a recombinant fusion protein. In some embodiments, one or more portions of paTCE are linked by chemical conjugation.

[0353] Provided herein are compositions that advantageously provide an EGFR-targeted bispecific therapeutic agent having higher selectivity, a longer half-life, lower toxicity and fewer side effects when cleaved by proteases found in target tissue or disease-induced unhealthy tissue, and as a result the composition has an improved therapeutic index compared to bispecific antibody compositions known in the art. Such compositions are useful for the treatment of cancer. In some embodiments, when paTCE is in proximity to target tissue or cells carrying or secreting a protease capable of cleaving RS, the bispecific binding domain is released from ELNN by the action of the protease, removing the steric hindrance barrier and freeing TCE to exert its pharmacological effect. This property is particularly advantageous in treating immunologically cold tumors that express EGFR. In some embodiments, the paTCE provided herein is activated in target tissue, where the target tissue is a solid tumor of an organ or system.

[0354] Joint domain In some embodiments, the binding domains provided herein comprise one or more full-length antibodies or one or more antigen-binding fragments thereof. Antibody antigen-binding fragments comprise any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide comprising some or more portions of an antibody that specifically binds to an antigen. Antibody antigen-binding fragments may be derived from a full antibody molecule using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques, including manipulation and expression of DNA encoding an antibody variable domain and optionally a constant domain. The terms binding domain and antibody domain are used interchangeably herein.

[0355] In some embodiments, single-chain binding domains are used that are capable of binding to effector cells and ligands or receptors associated with antigens of diseased tissues or cells, such as cancer, tumors, or other malignant tissues, including, but not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single-domain antibodies, VHH, single-chain antibody molecules (scFv), and diabodies.

[0356] In some embodiments, the binding domain is a bispecific antibody domain, which 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 an ISVD such as VHH), and the second antigen-binding domain is a second antigen-binding fragment (e.g., scFv, or an ISVD such as VHH).

[0357] In some embodiments, the antigen-binding fragment (AF) (e.g., a first antigen-binding fragment (AF1) and / or a second antigen-binding fragment (AF2)) may be (each independently) a chimeric, humanized, or human antigen-binding fragment. The antigen-binding fragment (AF) (e.g., a first antigen-binding fragment (AF1) and / or a second antigen-binding fragment (AF2)) may be (each independently) Fv, Fab, Fab', Fab'-SH, a linear antibody, VHH, or scFv.

[0358] In some embodiments, one or both antigen-binding fragments (e.g., the first and / or second antigen-binding fragments) may be configured as (Fab')2 or single-chain diabodies. In some embodiments, a bispecific antibody comprises a first binding domain having binding specificity to a cancer cell marker and a second binding domain having binding specificity to an effector cell antigen. In some embodiments, the binding domain to a tumor cell target is a variable domain of a T cell receptor engineered to bind to MHC, filled with a peptide fragment of a protein overexpressed by tumor cells.

[0359] In some embodiments, paTCEs are designed to provide a broad therapeutic window by considering the location of the target tissue protease, the presence of the same protease in healthy tissue not intended to be targeted, and the presence of the target ligand in healthy tissue but in greater abundance in unhealthy target tissue. The “therapeutic window” refers to the difference between the minimum effective dose and the maximum permissible dose for a given therapeutic composition. In some embodiments, to help achieve a broad therapeutic window for the TCE, the binding domain of the TCE is shielded by the proximity of a masking (e.g., ELNN) moiety such that the binding affinity of the intact composition to one or both ligands is reduced compared to the composition cleaved by the mammalian protease, thereby releasing the first portion from the shielding effect of the masking moiety.

[0360] In some embodiments, the complete antigen recognition and binding site comprises a dimer of one heavy chain variable domain (VH) and one light chain variable domain (VL). Within each VH and VL chain, there are three complementarity-determining regions (CDRs) that interact to define the antigen-binding site on the surface of the VH-VL dimer, and the six CDRs of the binding domain confer antigen-binding specificity to the antibody or single-chain binding domain. Framework sequences adjacent to the CDRs have a tertiary structure that is mainly conserved in native immunoglobulins across species, and framework residues (FRs) function to hold the CDRs in their proper orientation. In some embodiments, constant domains are not required for binding function but can help stabilize the VH-VL interaction. In some embodiments, the binding site may be a pair of VH-VL, VH-VH, or VL-VL domains from the same or different immunoglobulins; however, it is generally preferred to construct the single-chain binding domain using the respective 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 limited, as long as the VH and VL domains are positioned so that the antigen-binding site can be properly folded. Therefore, in some embodiments, a single-chain binding domain containing VH and VL (for example, in scFv) may have VH and VL arranged as VL-VH or VL-VH.

[0361] In some embodiments, the arrangement of the V chains is VH(cancer cell surface antigen)-VL(cancer cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), VH(cancer cell surface antigen)-VL(cancer cell surface antigen)-VH(effector cell antigen)-VL(effector cell antigen), VL(cancer cell surface antigen)-VH(cancer cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), VL(cancer cell surface antigen)-VH(cancer cell surface antigen) )-VH(effector cell antigen)-VL(effector cell antigen), VHH(cancer cell surface antigen)-VH(effector cell antigen)-VL(effector cell antigen), VHH(cancer cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), VL(cancer cell surface antigen)-VH(cancer cell surface antigen)-VHH(effector cell antigen), or VH(cancer cell surface antigen)-VL(cancer cell surface antigen)-VHH(effector cell antigen).

[0362] In some embodiments, the following sequence is used: VH(effector cell antigen)-VL(effector cell antigen)-VL(cancer cell surface antigen)-VH(cancer cell surface antigen), VH(effector cell antigen)-VL(effector cell antigen)-VH(cancer cell surface antigen)-VL(cancer cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen)-VL(cancer cell surface antigen)-VH(cancer cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen) The following combinations are possible: (cell antigen)-VH(cancer cell surface antigen)-VL(cancer cell surface antigen), VHH(effector cell antigen)-VH(cancer cell surface antigen)-VL(cancer cell surface antigen), VHH(effector cell antigen)-VL(cancer cell surface antigen)-VH(cancer cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen)-VHH(cancer cell surface antigen), or VH(effector cell antigen)-VL(effector cell antigen)-VHH(cancer cell surface antigen).

[0363] As used herein, “at the N-terminus” or “at the C-terminus,” and their grammatical variations, refer to relative positions within the primary amino acid sequence, rather than absolute N-terminus or C-terminus placement of a bispecific single-chain antibody. Therefore, as a non-limiting example, “the first binding domain located at the C-terminus relative to the second binding domain” indicates that the first binding is located on the carboxyl side of the second binding domain within the bispecific single-chain antibody, without prejudice that additional sequences, such as a linker and / or another compound, including an ELNN, His tag, or radioisotope, may be located at the C-terminus of the bispecific single-chain antibody.

[0364] In some embodiments, paTCE comprises a first portion including a first binding domain and a second binding domain, each of which is an scFv, and each scFv includes one VL and one VH. In some embodiments, the first binding domain is an scFv that binds to CD3, and the second binding domain is an scFv that binds to EGFR. In some embodiments, the paTCE composition comprises a first portion including a first binding domain and a second binding domain, one of which is an scFV and the other binding domain is a VHH. In some embodiments, paTCE comprises a first portion including a first binding domain and a second binding domain, where the binding domains are in a diabody configuration, one domain includes one VL region and one VH region, and the other domain includes one VL region and one 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.

[0365] In non-limiting examples, TCEs may include sequences exhibiting at least about 80% sequence identity to the antibody sequences identified herein, or alternatively, sequences exhibiting 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 comprises a bispecific sequence (e.g., BsAb) including a first binding domain and a second binding domain, wherein the first binding domain has specific binding affinity to a tumor-specific marker or cancer cell antigen and has at least about 80% sequence identity to the VL and VH sequences of the anti-EGFR antibodies disclosed in Table 5f, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 The second binding domain exhibits 8%, 99%, or 100% sequence identity, and the second binding domain has specific binding affinity to effector cells, exhibiting at least about 80% sequence identity to the paired VL and VH sequences of the anti-CD3 antibodies disclosed in any of Tables 5a to 5e of this Specified, 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.

[0366] In some embodiments, the TCE may include a binding domain (e.g., VH and / or VL amino acid sequence) 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 to 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 each of which are incorporated herein by reference. Additional, non-limiting examples of anti-CD3 antibody sequences include those of pasotaxizumab (also known as AMG-212) and acapatamab (also known as AMG-160).

[0367] In some embodiments, the TCE may include a binding domain of or derived from an anti-EGFR antibody (e.g., VH and / or VL amino acid sequences). Non-limiting examples of anti-EGFR antibody sequences include those of panitumumab and cetuximab.

[0368] This disclosure provides an antigen-binding domain that binds to EGFR. This disclosure provides scFv that binds to EGFR (e.g., scFv having pairs of VH and VL as shown in Table 5f). This disclosure further provides nucleic acids encoding antigen-binding domains (e.g., scFv) or polypeptides, as well as vectors, hosts, and methods for producing these antigen-binding domains or polypeptides. A multispecific polypeptide comprising an EGFR-binding antigen-binding domain according to this disclosure and at least one CD3-binding domain including paTCE is also provided. This disclosure includes therapeutic methods using the antigen-binding domain or polypeptide.

[0369] Also provided are nucleic acid molecules or vectors containing nucleic acids that encode the antigen-binding domain (e.g., scFv) or polypeptide of the present disclosure.

[0370] This disclosure also relates to non-human host or host cells transformed or transfected with nucleic acids or vectors encoding antigen-binding domains (e.g., scFv) or polypeptides disclosed herein.

[0371] This disclosure further relates to compositions comprising antigen-binding domains (e.g., scFv) or polypeptides disclosed herein, such as pharmaceutical compositions.

[0372] This specification includes methods for producing antigen-binding domains (e.g., scFv) or polypeptides as disclosed herein, comprising the following steps: a. A step of expressing a nucleic acid sequence encoding an antigen-binding domain (e.g., scFv) or polypeptide in a host cell or host organism, or in another expression system; thereafter optionally, b. A step of isolating and / or purifying an antigen-binding domain (e.g., scFv) or polypeptide.

[0373] Compositions and polypeptides comprising an antigen-binding domain (e.g., scFv) for use as pharmaceuticals are provided herein. In some embodiments, the polypeptide or composition is for use in the treatment of proliferative disorders. In some embodiments, the proliferative disorder is cancer.

[0374] This disclosure also provides a therapeutic method comprising the step of administering a composition or polypeptide containing an antigen-binding domain (e.g., scFv) to a subject requiring it. In some embodiments, the therapeutic method is for treating a proliferative disorder. In some embodiments, the proliferative disorder is cancer.

[0375] This specification includes compositions and polypeptides comprising antigen-binding domains (e.g., scFv) for use in the preparation of pharmaceuticals. In some embodiments, the pharmaceuticals are used in the treatment of proliferative disorders. In some embodiments, the proliferative disorder is cancer.

[0376] In some embodiments, the structure of each VH or VL of an antigen-binding domain (e.g., scFv) sequence can be considered to consist of four framework regions ("FR"), referred to herein in the art as "framework region 1" ("FR1"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), and the framework regions are interrupted by three complementarity-determining regions ("CDR"), referred herein and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3"), respectively.

[0377] In some embodiments, the techniques provided herein utilize an antigen-binding domain (e.g., scFv) capable of binding to EGFR. In the context of this technique, "binding to a particular target molecule" has the usual meaning in the art as understood in the context of antibodies and their respective antigens.

[0378] As will be apparent from the above and further description herein, the antigen-binding domains of the Technology (e.g., scFv) can be used as “building blocks” for forming polypeptides of the Technology, which combine one or more desirable properties or biological functions within a single molecule, for example, by suitably combining them with other groups, residues, moieties, or binding units to form the compounds or fusion proteins of the Technology described herein (but not limited to, divalent / trivalent / tetravalent / polyvalent and double / triple / quadrivalent / multispecific polypeptides of the Technology described herein).

[0379] The terms “specificity,” “specific binding,” or “specific binding” refer 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,” “specific binding,” or “specific binding” are used herein interchangeably with “selectivity,” “selective binding,” or “selective binding.” Binding units such as scFv preferably bind specifically to their designated targets.

[0380] The specificity / selectivity of bonding units can be determined based on affinity. Affinity indicates the strength or stability of molecular interactions. Affinity is generally expressed in units of moles / liter (or M) in K. D It is given by.

[0381] Affinity is a measure of the binding strength between a part and a binding site on a target molecule, K D The lower the value, the stronger the binding strength between the target molecule and the targeting site.

[0382] Typically, the bonding units used in this technology (such as scFv) are 10 -5 ~10 -12 moles / liter or less, preferably 10 -7 ~10 -12 moles / liter or less, more preferably 10 -8 ~10 -12 K in moles / liter D It then binds to those targets.

[0383] In some embodiments, 10 -4 K for moles / liter D The value is considered nonspecific. In some embodiments, 10 -4 K₀ (moles / liter) D The value is considered unique.

[0384] K is a key factor in biological interactions such as the binding of antibody sequences to antigens, which are considered specific. DTypically, this ranges from 10,000 nM or 10 μM to 0.001 nM or 1 μM or less.

[0385] Therefore, specific / selective binding can be measured using the same method, e.g., SPR, where the binding unit (or polypeptide containing it) is 10 -5 ~10 -12 K is less than or equal to moles / liter D Binds to EGFR by value, 10 -4 K for moles / liter D This could mean binding to different targets based on their values.

[0386] Specific binding from a particular species to a particular target does not preclude the binding unit from also specifically binding to similar targets from different species. For example, specific binding to human EGFR does not preclude the binding unit (or polypeptide containing it) from also specifically binding to EGFR from cynomolgus monkeys.

[0387] The specific binding of the binding unit to the specified target can be determined in any preferred manner that is essentially known, including, for example, Scatchard analysis, and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assay, as well as different variations thereof that are essentially known in the art; and other techniques referred to herein.

[0388] The dissociation constant may be, for example, an actual or apparent dissociation constant, as will be obvious to those skilled in the art. Methods for determining the dissociation constant will be obvious to those skilled in the art, and include, for example, the techniques mentioned below.

[0389] The affinity of molecular interactions between two molecules can be measured through various techniques that are essentially known, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, e.g., Ober et al. 2001, Intern. Immunology 13:1551-1559). As used herein, the term “surface plasmon resonance” refers to an optical phenomenon that enables real-time analysis of biomolecular-specific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule is k on , k off Measured value, therefore K D The substance passes over immobilized molecules under flowing conditions that yield the desired value. This can be carried out, for example, using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further information, 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).

[0390] Another well-known biosensor technique for determining the affinity of biomolecular interactions is bio-layer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377:209-217). As used herein, the terms “bio-layer interferometry” or “BLI” refer to an unlabeled optical technique that analyzes the interference patterns of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on a biosensor chip (signal beam). A change in the number of molecules bound to the biosensor chip causes 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 biosensor chip surface. Since the interactions can be measured in real time, the association and dissociation rates, as well as affinity, can be determined. BLI can be performed, for example, using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0391] Alternatively, affinity can be measured in the Kinetic Exclusion Assay (KinExA) (e.g., Drake et al. 2004, Anal. Biochem., 328:35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). 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 the antibody / antigen complex is passed through a column containing beads pre-coated with the antigen (or antibody) to bind the free antibody (or antigen) to the coated molecule. Detection of the thus captured antibody (or antigen) is achieved using a fluorescently labeled protein that binds to the antibody (or antigen).

[0392] The GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5:1765-74).

[0393] In some embodiments, paTCE includes a first binding domain which is an scFv and a second binding domain which is an scFv. In some embodiments, the first scFv includes a VL domain and a VH domain which specifically bind to an effector cell antigen (e.g., CD3), and the second scFv specifically binds to a cancer cell antigen (e.g., EGFR). In some embodiments, the scFv includes six CDRs. In some embodiments, the scFv includes VH and VL regions containing amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the VL and VH sequences of the paired anti-CD3 antibody identified in Table 5a. In some embodiments, the scFv includes the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions of the paired anti-CD3 antibody VL and VH sequences identified in Table 5a. In some embodiments, the scFv is derived from an anti-EGFR antibody identified as the antibody shown in Table 5f. In some embodiments, the scFv includes the VH and VL regions, which contain amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the VH and VL sequences disclosed in Table 5f. In some embodiments, the VH and VL include the CDR-1, CDR-2, and CDR-3 regions of the VH and VL sequences in Table 5f.

[0394] In some embodiments, paTCE comprises a first binding domain which is an scFv and a second binding domain which is also an scFv. In some embodiments, the scFv comprises VL and VH domains, respectively, derived from a monoclonal antibody having binding specificity to a tumor-specific marker or cancer cell antigen and an effector cell antigen. In some embodiments, the first and second binding domains each comprise six CDRs, respectively, derived from a cancer cell marker such as a tumor-specific marker and a monoclonal antibody having binding specificity to an effector cell antigen. In some embodiments, the first and second binding domains of the first portion of the composition in question may have three, four, five, or six CDRs within each binding domain. In some embodiments, paTCE comprises a first binding domain and a 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, each of which is derived from a monoclonal antibody capable of binding to a tumor-specific marker or cancer cell antigen and an effector cell antigen, respectively.

[0395] In some embodiments, the second binding domain includes VH and VL regions derived from a monoclonal antibody capable of binding to human CD3. In some embodiments, the second binding domain includes an scFv containing the VH and VL regions, each VH and VL region exhibiting or being identical to at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VL and VH sequences of a paired anti-CD3 antibody identified in Table 5a. In some embodiments, the second domain includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions, each region derived from a monoclonal antibody identified herein as the antibody listed in Table 5a. In some embodiments, the VH and / or VL domains may be configured as an scFv or a diabody.

[0396] In some embodiments, paTCE includes a first binding domain which is a diabody and a second binding domain which is a diabody. In some embodiments, the diabody includes VL and VH domains derived from a monoclonal antibody having binding specificity to a tumor-specific marker or cancer cell antigen and an effector cell antigen, respectively.

[0397] In some embodiments, the Disclosure provides a paTCE composition in which the second binding domain of the diabody comprises a VH region and a VL region, and each of the VH and VL regions exhibits or is identical to at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the VL and VH sequences of the huUCHT1 antibody shown in Table 5a. In some embodiments, the second domain of the diabody of the composition is derived from an anti-CD3 antibody as described herein. In some embodiments, the anti-CD3 diabody is linked to an anti-EGFR binding scFv sequence as disclosed herein.

[0398] Methods for measuring the binding affinity and / or other biological activity of an antigen-binding domain may be those disclosed herein or methods generally known in the art. For example, K DThe binding affinity of a binding pair (e.g., antibody and antigen) shown as can be determined by, but is not limited to, radioactive binding assays, non-radioactive binding assays such as fluorescence resonance energy transfer and surface plasmon resonance (SPR, Biacore), and various suitable assays including enzyme-linked immunosorbent assay (ELISA), kinetic elimination assay (KinExA®), or as described in the examples. An increase or decrease in binding affinity, for example, an increase in binding affinity of a TCE cleaved to remove a masking moiety compared to a paTCE with a bound masking moiety, can be determined by measuring the binding affinity of TCEs with and without a masking moiety to their target binding partner.

[0399] The half-life of a target chimeric assembly can be measured by a variety of suitable methods. For example, the half-life of a substance can be determined by administering the substance to a target and periodically sampling a biological sample (e.g., blood, or plasma, or a biological fluid such as 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-pressure liquid chromatography and high-performance protein liquid chromatography. In some cases, the substance may be labeled with a detectable tag, such as a radioactive tag or a fluorescent tag, which can be used to determine the concentration of the substance in the sample (e.g., a blood or plasma sample). Various pharmacokinetic parameters can then be determined from the results, which can be done using a software package such as SoftMax Pro software or by manual calculations known in the art.

[0400] In addition, the physicochemical properties of the paTCE composition can be measured to confirm the degree of solubility, structure, and stability retention. Affinity and binding constant ((K D , k on and koff ), the half-life of dissociation of the ligand-receptor complex, and the activity (IC 50 value) of the binding domain that inhibits the biological activity of the blocked ligand compared to the free ligand, to enable determination of the binding characteristics of the binding domain to the ligand. An assay of the subject composition is performed. The term "EC 50 " refers to the concentration required to achieve half of the maximum biological response of an active substance and is generally determined by ELISA or cell-based assays, including the methods of the examples described herein.

[0401] Anti-CD3 binding domain Also provided are anti-CD3 antibodies, fragments thereof, and fusion proteins comprising such antibodies and / or fragments.

[0402] In some embodiments, the Disclosure provides a paTCE composition comprising a first portion of a binding domain having binding affinity to T cells. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody that binds to CD3. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody against CD3 epsilon and / or CD3 delta. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody against CD3 epsilon. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody against CD3 delta. Exemplary, non-limiting examples of VL and VH sequences of monoclonal antibodies against CD3 are presented in Table 5a. In some embodiments, the Disclosure provides a paTCE comprising a binding domain having binding affinity to CD3, comprising the anti-CD3 VL and VH sequences described in Table 5a. In some embodiments, the Disclosure provides a paTCE comprising a first portion of a binding domain having binding affinity to CD3 epsilon, comprising the anti-CD3 epsilon VL and VH sequences described in Table 5a. In some embodiments, the Disclosure provides a paTCE composition in which a first partial binding domain comprises an scFv including a VH region and a VL region, and each VH region exhibits or is identical to at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the paired VL and VH sequences of the huUCHT1 anti-CD3 antibody in Table 5a. In some embodiments, the Disclosure provides a paTCE composition comprising a binding domain having binding affinity to CD3, comprising a CDR-L1 region, a CDR-L2 region, a CDR-L3 region, a CDR-H1 region, a CDR-H2 region, and a CDR-H3 region (each derived from the respective anti-CD3 VL and VH sequences described in Table 5a).In some embodiments, the present disclosure provides a paTCE composition comprising a binding domain having binding affinity to CD3, including 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, the present disclosure provides a paTCE composition comprising a binding domain having binding affinity to CD3, including 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).

[0403] The CD3 complex is a group of cell surface molecules that associate with T-cell antigen receptors (TCRs) and function in the cell surface expression of TCRs and in the signaling cascade from which peptide:MHC ligands originate when they bind to TCRs. While not bound by any scientific theory, typically, when an antigen binds to a T-cell receptor, CD3 sends a signal across the cell membrane to the cytoplasm inside the T cell. This triggers the activation of T cells, which rapidly divide and produce new T cells sensitized to attack the specific antigen that the TCR has exposed. The CD3 complex consists of the CD3 epsilon molecule and four other membrane-bound polypeptides (CD3-gamma, -delta, and / or -zeta). In humans, CD3-epsilon is encoded by the CD3E gene on chromosome 11. Each intracellular domain of the CD3 chain contains an immunoreceptor tyrosine-based activation motif (ITAM), which functions as a nucleation site of the intracellular signaling mechanism during T-cell receptor engagement.

[0404] Many therapeutic strategies modulate T cell immunity by targeting TCR signaling, particularly anti-human CD3 monoclonal antibodies (mAbs) that are widely used clinically in immunosuppressive regimens. The CD3-specific mouse mAb OKT3 was the first mAb approved for use in humans (Sgro, C. Side-effects of a monoclonal antibody, muromonab CD3 / orthoclone OKT3: bibliographic review. Toxicology 105:23-29, 1995), transplantation (Chatenoud, Clin. Transplant 7:422-430, (1993), Chatenoud, Nat. Rev. Immunol. 3:123-132 (2003), Kumar, Transplant. Proc. 30:1351-1352 (1998)), and is widely used clinically as an immunosuppressant in type 1 diabetes and psoriasis. Importantly, anti-CD3 mAbs can induce partial T cell signaling and clonal anergy (Smith, JA, Nonmitogenic Anti-CD3 Monoclonal Antibodies Deliver a 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 potent 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 with target cells, and that neither FcR-mediated ADCC nor complement fixation was required for bivalent anti-CD3 mAbs to lyse target cells.

[0405] OKT3 exhibits both pro-mitotic and T-cell-killing activity in a time-dependent manner. Following early activation of T cells that result in cytokine release, subsequent administration of OKT3 subsequently blocks all known T-cell functions. It is this subsequent blockade of T-cell function that has led to OKT3 finding such widespread use as an immunosuppressant in therapeutic regimens for reducing or even eliminating allograft tissue rejection. Other antibodies specific to the CD3 molecule are disclosed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50; International Publications 2005 / 118635 and 2007 / 033230 describe anti-human monoclonal CD3 epsilon antibodies; U.S. Patent No. 5,821,337 describes the mouse anti-CD3 monoclonal Ab UCHT1 (muxCD3), Shalaby et al. Med. 175, 217-225 (1992) and the VL and VH sequences of the humanized variant of this antibody (hu UCHT1); and U.S. Patent Application No. 20120034228 discloses a binding domain capable of binding to the epitopes of the CD3 epsilon chain in human and non-chimpanzee primates.

[0406] In some embodiments, the anti-CD3 antibody domain includes a VH region containing the sequence EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 311) or its CDR, and a VL region containing the sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361) or its CDR.

[0407] In some embodiments, the anti-CD3 antibody domain includes a VH region containing the sequence EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126) or its CDR, and a VL region containing the sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127) or its CDR.

[0408] [Table 16-1]

[0409] [Table 16-2]

[0410] [Table 16-3] * The underlined sequences, if present, are CDRs within VL and VH.

[0411] In some embodiments, the disclosure relates to antigen-binding fragments (AFs) having specific binding affinity to effector cell antigens.

[0412] Effector cell antigens, particularly various AFs that bind to CD3 on T cells, have a particular utility in pairing with antigen-binding fragments having binding affinity to EGFR antigens associated with diseased cells or tissues in a compositional format in order to mobilize and bring about effector cell-mediated cytotoxicity of diseased cells or tissues.

[0413] The binding specificity to the target antigen can be determined by the complementarity-determining region or by a CDR such as a light chain CDR or a heavy chain CDR. In many cases, the binding specificity is determined by the light chain CDR and the heavy chain CDR. A given combination of heavy chain CDR and light chain CDR provides a given binding pocket that confers higher affinity and / or specificity to the effector cell antigen compared to other reference antigens. A bispecific composition having a first antigen-binding fragment for EGFR linked by a short, flexible peptide linker to a second antigen-binding fragment having binding specificity to the effector cell antigen, which on the one hand binds to the effector cell antigen and on the other hand binds to the antigen on diseased cells or tissues, is bispecific, and each antigen-binding fragment has a specific binding affinity to its respective ligand.

[0414] In such compositions, AF directed towards EGFR in diseased tissue is used in combination with AF directed towards effector cell markers to bring effector cells into proximity to the diseased tissue cells to induce cytolysis of the diseased tissue cells. Furthermore, the first antigen fragment (AF1) and the second antigen fragment (AF2) are incorporated into a specially designed polypeptide containing a cleavable release segment and an ELNN segment to confer inert characteristics to the composition, which is activated by the release of fused AF1 and AF2 upon cleavage of the release segment when the diseased tissue has a protease capable of cleaving the release segment at one or more positions in the release segment sequence.

[0415] In some embodiments, the AF2 of the subject composition has binding affinity for an effector cell antigen 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 a member of the CD3 complex, which includes all known CD3 subunits of the CD3 complex, in individual or combined forms; for example, CD3 epsilon, CD3 delta, CD3 gamma, and CD3 zeta. In some embodiments, AF2 has binding affinity for CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta.

[0416] In some embodiments, the present disclosure provides an antigen-binding domain (e.g., an antibody or an antigen-binding fragment thereof) that binds to the differentiation antigen group 3 T cell receptor (CD3), comprising the following CDRs: RSSX1GAVTX2SNYAN (where X1 corresponds to T or N and X2 corresponds to T or S), with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to an amino acid sequence containing a VL domain CDR1; GTNKRAP, with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to an amino acid sequence containing a VL domain CDR2; ALWYX4NLWV (where X4 corresponds to S or P), with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to an amino acid sequence containing a VL domain CDR3; GFTFX8TYAMN (where X8 corresponds to S or N), with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to an amino acid sequence containing a VH domain CDR1; RIRX 10 KX 11 NX 12 YATYYADSVKX 13 (where X 10This corresponds to T or S, and X 11 This corresponds to R or Y, and X 12 This corresponds to D or N, and X 13 VH domain CDR2;HX containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to (corresponding to G or D). 14 NFGNSYVSWFAX 15 (In the formula, X 14 This corresponds to E or G, and X 15 The VH domain CDR3 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to H or Y.

[0417] In some embodiments, an antigen-binding domain (e.g., an antibody or its antigen-binding fragment) that binds to the differentiated antigen group 3 T cell receptor (CD3) is a VL region CDR1;GTNKRAP having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDR:RSSNGAVTSSNYAN. In contrast, VL region CDR2;ALWYPNLWV containing amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, contains amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity. VL region CDR3;GFTFSTYAMN containing an amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or VH region CDR1;RIRTKRNDYATYYADSVKG containing an amino acid sequence with 100% identity has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% identity. The present invention provides an antigen-binding domain comprising: a VH region CDR2 containing an amino acid sequence having 97%, 98%, or 99% identity, or 100% identity; and a VH region CDR3 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity with respect to HENFGNSYVSWFAH.

[0418] In some embodiments, the antigen-binding domain has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with the VL region FR1;WVQQKPGQAPRGLIG, which includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the following FR:ELVVTQEPSLTVSPGGTVTLTC. An amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to VL region FR2;GTPARFSGSLLEGKAALTLSGVQPEDEAVYYC, or 100% identity with respect to VL region FR3;FGGGTKLTVL, which contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 92;GTPARFSGSLLEGKAALTLSGVQPEDEAVYYC, or 100% identity with respect to VL region FR3;FGGGTKLTVL VH region FR1 contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to VL region FR4;EVQLVESGGGIVQPGGSLRLSCAAS; VH region FR2 contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to VH region FR1;WVRQAPGKGLEWVG VH region FR3 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to ;RFTISRDDSKNTLYLQMNSLKTEDTAVYYCVR; and VH region FR4 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to WGQGTLVTVSS.

[0419] In some embodiments, the present disclosure relates to an antigen-binding domain (e.g., an antibody or its antigen-binding fragment) that binds to CD3, wherein the VL region comprises three VL CDRs, and the three VL CDRs have the following amino acid sequence: The antigen-binding domain is provided, comprising CDR1, CDR2, and CDR3 of the VL containing ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLEGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 127), the VH region containing three VH CDRs, the three VH CDRs containing CDR1, CDR2, and CDR3 of the VH region containing the following amino acid sequence: EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNDYATYYADSVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (SEQ ID NO: 126).

[0420] In some embodiments, an antigen-binding domain (e.g., an antibody or its antigen-binding fragment) that binds to the differentiated antigen group 3 T cell receptor (CD3) is a VL region CDR1;GTNKRAP having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDR:RSSNGAVTSSNYAN. In contrast, VL region CDR2;ALWYPNLWV containing amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, contains amino acid sequences having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity. VL region CDR3;GFTFSTYAMN containing an amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or VH region CDR1;RIRTKRNNYATYYADSVKG containing an amino acid sequence with 100% identity has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, or 96% identity. The present invention provides an antigen-binding domain comprising: a VH region CDR2 containing an amino acid sequence having 97%, 98%, or 99% identity, or 100% identity; and a VH region CDR3 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity with respect to HENFGNSYVSWFAH.

[0421] In some embodiments, the antigen-binding domain has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with the VL region FR1;WVQQKPGQAPRGLIG, which includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to the following FR:ELVVTQEPSLTVSPGGTVTLTC. An amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to VL region FR2;GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC, or 100% identity with respect to VL region FR3;FGGGTKLTVL, which contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 92;GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC, or 100% identity with respect to VL region FR3;FGGGTKLTVL VH region FR1 contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to VL region FR4;EVQLVESGGGIVQPGGSLRLSCAAS; VH region FR2 contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to VH region FR1;WVRQAPGKGLEWVG VH region FR3 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR; and VH region FR4 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to WGQGTLVTVSS.

[0422] In some embodiments, the present disclosure relates to an antigen-binding domain (e.g., an antibody or its antigen-binding fragment) that binds to CD3, wherein the VL region comprises three VL CDRs, and the three VL CDRs have the following amino acid sequence: The VH region CDR1, CDR2, and CDR3 include ELVVTQEPSLTVSPGGTVTLTCRSSX1GAVTX2SNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX3YYCALWYX4NLWVFGGGTKLTVL (wherein 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 the VH region contains three VH CDRs, and the three VH CDRs have the following amino acid sequence: EVQLX5ESGGGX6VQPGGSLX7LSCAASGFTFX8TYAMNWVRQAPGKGLEWVX9RIRX 10 KX 11 NNYATYYADSVKX 12 RFTISRDDSKNTX 13 YLQMNX 14 LKTEDTAVYYCVRHX 15 NFGNSYVSWFAX 16 WGQGTLVTVSS (wherein 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 However, it corresponds to T or S, X 11 However, it corresponds to R or Y, and X 12 However, it corresponds to G or D, and X 13 However, it corresponds to V or A, X 14 However, it corresponds to S or N, and X 15 However, it corresponds to E or G, and X 16 The present invention provides an antigen-binding domain comprising CDR1, CDR2, and CDR3 of the VH region (corresponding to H or Y).

[0423] In some embodiments, the present disclosure relates to an antigen-binding domain (e.g., an antibody or its antigen-binding fragment) that binds to CD3, wherein the VL region comprises three VL CDRs, and the three VL CDRs have the following amino acid sequence: The VL region contains CDR1, CDR2, and CDR3, which include ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), and the VH region contains three VH CDRs, the three VH CDR regions having the following amino acid sequence: The present invention provides an antigen-binding domain comprising CDR1, CDR2, and CDR3 of a VH domain containing EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS (Sequence ID 311).

[0424] In some embodiments, the present disclosure relates to an antigen-binding domain (e.g., an antibody or its antigen-binding fragment) that binds to CD3, and includes 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 / 757 Includes pairs of VL domain amino acid sequence numbers / VH domain amino acid sequence numbers selected from the group consisting of 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.

[0425] In some embodiments, the Disclosure provides antigen-binding fragments (e.g., AF1 or AF2) that bind to CD3 protein complexes with improved stability compared to CD3-binding antibodies or antigen-binding fragments known in the Art. In some embodiments, the CD3 antigen-binding fragments of the Disclosure are designed to confer a greater degree of stability to the chimeric bispecific antigen-binding fragment composition into which they are incorporated, resulting in improved expression and recovery of the fusion protein, increased shelf life, and improved stability upon administration to a subject. In some embodiments, the anti-CD3 AFs of the Disclosure have a greater degree of thermal stability compared to certain CD3-binding antibodies and antigen-binding fragments known in the Art. In some embodiments, the anti-CD3 AFs of the Disclosure have a greater degree of thermal stability compared to SP34 or its antigen-binding fragment. In some embodiments, the anti-CD3 AFs of the Disclosure have a greater degree of thermal stability compared to CD3.9 and / or CD3.23 disclosed in PCT International Patent Application Publication No. 2021263058, the entirety of which is incorporated herein by reference. In some embodiments, the anti-CD3 AF of this disclosure is less immunogenic in humans than certain CD3-binding antibodies and antigen-binding fragments known in the art. In some embodiments, the anti-CD3 AF of this disclosure is less immunogenic than SP34 or its antigen-binding fragment. In some embodiments, the anti-CD3 AF of this disclosure is less immunogenic than CD3.9 and / or CD3.23 disclosed in PCT International Patent Application Publication No. 2021263058, the entirety of which is incorporated herein by reference.In some embodiments, the degree to which AF is immunogenic is determined by immunogenicity prediction methods such as TEPITOPpan (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 NetMHC IIpan-4.0 (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-CD3 AFs, utilized as components of chimeric bispecific antigen-binding fragment compositions into which they are incorporated, exhibit desirable pharmaceutically acceptable properties, including high thermal stability and low aggregation tendency, resulting in improved expression and recovery during manufacturing and storage, as well as promoting a longer serum half-life. Biophysical properties such as thermal stability are often limited by antibody variable domains with significantly different inherent properties. High thermal stability is often associated with other desirable properties, including high expression levels and low agglutination (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 defined as the "melting temperature" (T), which is the temperature at which half of the molecule denatures. m This is determined by measuring the melting temperature of each heterodimer, which indicates its thermal stability. The following examples include the method described below. mIn vitro assays for determining the melting point of the heterodimer are known in the art. The melting point of the heterodimer can be measured using techniques such as differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60, Ghirlando et al (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of the heterodimer can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9), or as described in the following examples.

[0426] In some embodiments of the polypeptides of this disclosure, the antigen-binding fragment (e.g., AF1 or AF2) can exhibit higher thermal stability than the anti-CD3-binding fragment consisting of the sequence of SEQ ID NO: 206 (see Table 5e), and in an in vitro assay, the higher melting temperature (T) of the first antigen-binding fragment is compared to that of the anti-CD3-binding fragment. m ), or when incorporating the first antigen-binding fragment into the test bispecific antigen-binding domain, a higher Tm of the test bispecific antigen-binding domain compared to the Tm of the control bispecific antigen-binding domain. m As demonstrated, the test bispecific antigen-binding domain comprises a first antigen-binding fragment and a reference antigen-binding fragment that binds to antigens other than CD3, while the control bispecific antigen-binding domain comprises an anti-CD3-binding fragment consisting of the sequence of Sequence ID No. 206 (see Table 5e) and a reference antigen-binding fragment. In some embodiments, the melting temperature (T) of the first antigen-binding fragment is m ) is the T of the anti-CD3 binding fragment consisting of the sequence of sequence number 206. m It may be 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).

[0427] In some embodiments, any polypeptide among the embodiments of the subject composition 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 CD3 complex subunits identified herein as CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta units of CD3. The antigen-binding fragment (AF) can bind to the CD3 epsilon fragment of CD3. In some embodiments, the antigen-binding fragment (AF) can be determined in an in vitro antigen-binding assay containing a human CD3 antigen to have a binding affinity (K) of about 10 nM to about 400 nM, or about 50 nM to about 350 nM, or about 100 nM to about 300 nM. D It can specifically bind to human CD3 at a constant of (K). In some embodiments, any polypeptide among the embodiments of the subject compositions described herein can be determined in an in vitro antigen binding assay to have 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 contains an antigen-binding fragment (AF) that specifically binds to human CD3. For clarity, 400 K D The antigen-binding fragment (AF) containing 10 nM K D It binds to its ligand more weakly than those having a higher binding affinity (K) in an in vitro antigen-binding assay. In some embodiments, any polypeptide among the embodiments of the subject compositions described herein has a binding affinity (K) D The antigen-binding fragment (AF) is determined by ) and contains 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 fragment consisting of the amino acid sequences in Tables 5a to e.

[0428] In some embodiments, this disclosure relates to the binding affinity (K) of each antigen in an in vitro antigen binding assay. DThe present invention provides a bispecific polypeptide comprising an antigen-binding fragment (AF) (anti-CD3 AF) that exhibits a binding affinity to CD3 that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or at least 1000 times weaker than the binding affinity of the embodiment of anti-EGFR AF described herein incorporated into the target polypeptide, as determined by [the specified method].

[0429] The binding affinity of the target composition to the target ligand can be evaluated using, for example, a binding assay such as the Biacore assay using a chip-binding receptor or binding protein, or a competitive binding assay, or an ELISA assay as described in U.S. Patent No. 5,534,617, the assay described in the examples herein, a radioreceptor assay, or other assays known in the art. The binding affinity constant can then be determined using a standard method such as the Scatchard analysis described in van Zoelen, et al., Trends Pharmacol Sciences (1998) 19) 12): 487, or other methods known in the art.

[0430] In some embodiments, the Disclosure provides antigen-binding fragments (AFs) (anti-CD3 AFs) that bind to CD3 and are incorporated into chimeric bispecific polypeptide compositions designed to have an isoelectric point (pI) that imparts improved stability to the composition compared to corresponding compositions containing CD3-binding antibodies or antigen-binding fragments known in the Art. In some embodiments, any polypeptide among the embodiments of the Subject Compositions described herein comprises a CD3-binding AF (anti-CD3 AF), wherein the anti-CD3 AF exhibits a pI of 6.0 to 6.6 (inclusive). In some embodiments, any polypeptide among the embodiments of the Subject Compositions 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 unit lower than the pI of a reference antigen-binding fragment (e.g., consisting of the sequence shown in SEQ ID NO: 206 (see Table 5e)). In some embodiments, any polypeptide among the embodiments of the subject composition described herein comprises a CD3-binding AF (anti-CD3 AF) fused to another AF (anti-EGFR AF) that binds to the EGFR antigen, wherein the anti-CD3 AF exhibits a pI of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units of the pI of the AF that binds to the EGFR antigen or its epitope. In some embodiments, any polypeptide among the embodiments of the subject composition described herein comprises a CD3-binding AF (anti-CD3 AF) fused to an AF (anti-EGFR AF) that binds to the EGFR antigen, wherein the AF exhibits a pI of 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.The fusion antigen-binding fragments resulting from such a design, in which the pIs of the two antigen-binding fragments are within such a range, are specifically intended to confer a higher degree of stability to the chimeric bispecific antigen-binding fragment composition into which they are incorporated, resulting in improved expression and recovery of the soluble, non-aggregated fusion protein, increased shelf life of the formulated chimeric bispecific polypeptide composition, and improved 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 may allow for the selection of a buffer or other solution 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 6.6 or less. In some embodiments, the antigen-binding fragments (AFs) may exhibit an isoelectric point (pI) of 6.0 to 6.6 (inclusive of both ends). 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 unit lower than the isoelectric point (pI) of the reference antigen-binding fragment consisting 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) of approximately 10 nM to approximately 400 nM (determined by an in vitro antigen-binding assay including human CD3 antigen, etc.). D It can specifically bind to human CD3 at a constant of K. In some embodiments, the antigen-binding fragment (AF) has a binding affinity (K) of less than approximately 10 nM, or less than approximately 50 nM, or less than approximately 100 nM, or less than approximately 150 nM, or less than approximately 200 nM, or less than approximately 250 nM, or less than approximately 300 nM, or less than approximately 350 nM, or less than approximately 400 nM (determined by an in vitro antigen-binding assay, etc.). D ) can specifically bind to human CD3. In some embodiments, the antigen-binding fragment (AF) is (each binding affinity (K) in an in vitro antigen-binding assay. D(As determined by, etc.) it 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 compared to the binding affinity of the antigen-binding fragment consisting of the amino acid sequence of SEQ ID NO: 206 (see Table 5e).

[0431] In some embodiments, the antigen-binding fragments VL and VH are fused by a relatively long linker consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids, which have a flexible characteristic when joined together. In some embodiments, the VL and VH of any of the scFv embodiments described herein are linked by a relatively long linker having the sequence SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the VL and VH of any of the scFv embodiments described herein are linked by a relatively long linker of hydrophilic amino acids 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 short linkers of hydrophilic amino acids having 3, 4, 5, 6, or 7 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, the disclosure provides compositions comprising single-chain diabodies in which, after folding, a first domain (VL or VH) pairs with a last domain (VH or VL) to form one scFv, two central domains pair to form another scFv, the first and second domains, as well as the third and last domains, are fused together by one of the aforementioned short linkers, and the second and third variable domains are fused by one of the aforementioned relatively long linkers. In some embodiments, the selection of short and relatively long linkers is intended to prevent incorrect pairing of adjacent variable domains, thereby facilitating the formation of a single-chain configuration including the VL and VH of the first and second antigen-binding fragments.

[0432] [Table 17]

[0433] [Table 18]

[0434] [Table 19]

[0435] [Table 20-1]

[0436] [Table 20-2]

[0437] anti-EGFR binding domain Also provided are anti-EGFR antibodies, fragments thereof, and fusion proteins containing such antibodies and / or fragments.

[0438] In some embodiments, the present disclosure provides a paTCE composition comprising a first partially binding domain having binding affinity to the tumor-specific marker EGFR and a second binding domain that binds to an effector cell antigen such as the CD3 antigen.

[0439] In some embodiments, the first partial binding domain is an scFv domain containing a VH domain and a VL domain. Non-restrictive examples of VH and VL domain sequences are shown in Table 5f. In some embodiments, the binding domain having binding affinity to the tumor-specific marker EGFR is an scFv domain containing the VH domain and VL domain listed in Table 5f. In some embodiments, the binding domain having binding affinity to EGFR is an scFv domain containing three CDRs derived from the VH domain and three CDRs derived from the VL domain listed in Table 5f.

[0440] In some embodiments, the present disclosure provides a paTCE composition comprising a first partially binding domain having binding affinity to the tumor-specific marker EGFR, including the anti-EGFR VH and VL sequences listed in Table 5f. In some embodiments, binding is determined in an in vitro binding assay, and is approximately 10 -10 ~10 -7 M's K D It has a value. In some embodiments, binding is determined in an in vitro binding assay, with a K value of about 1-10 nM. D It has a value. In some embodiments, binding is determined in an in vitro binding assay, with a K value of about 2 nM. D The paTCE composition is specifically intended to include any one of the binding domains disclosed herein or a sequence variant thereof, insofar as the variant exhibits binding specificity to the described antibody.

[0441] [Table 21-1]

[0442] [Table 21-2]

[0443] [Table 21-3]

[0444] Table 21-4

[0445] Table 21-5

[0446] Table 21-6

[0447] Table 21-7

[0448] Table 21-8

[0449] Table 21-9

[0450] Table 21-10

[0451] Table 21-11

[0452] Table 21-12

[0453] In certain 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 a 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 It contains the amino acid sequence 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 This corresponds to I or L, and X 11 This corresponds to T or S, and X 12 This corresponds to K or Q, and X 13 This corresponds to K or R, and X 14 This corresponds to V or I, and X 15 This corresponds to S, D, or A, and X 16 This corresponds to F or L, and X 17 This corresponds to S or R, and X 18 This corresponds to Q or E, and X 19 corresponds to I or F, X 20 This corresponds to T or V (sequence number 577), Each EGFR antibody listed in Table 5f contains the following CDR sequence: HCDR1-GGSVSSGDYYWT (Sequence ID 562) HCDR2-HIYYSGNTNYNPSLKS (Sequence ID 563) HCDR3-DRVTGAFDI (Sequence ID 564) LCDR1-QASQDISNYLN (Sequence ID 565) LCDR2-DASNLET (Sequence ID 566) LCDR3-QHFDHLPLA (Sequence ID 567)

[0454] In some embodiments, the present disclosure relates to an anti-EGFR antibody VH region comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the following CDR:GGSVSSGDYYWT VH region CDR1;HIYYSGNTNYNPSLKS VH region. The present invention provides an anti-EGFR antibody VH region comprising: VH region CDR2 containing an amino acid sequence having 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity; and VH region CDR3 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity with respect to DRVTGAFDI.

[0455] In some embodiments, the present disclosure relates to an anti-EGFR antibody VL region having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the following CDR:QASQDISNYLN, or at least 85%, 90%, 91%, 92%, 93%, 94% identity with respect to VL region CDR1;DASNLET, The present invention provides an anti-EGFR antibody VH region comprising a VL region CDR2 containing an amino acid sequence having 95%, 96%, 97%, 98%, or 99% identity, or 100% identity; and a VL region CDR3 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity with respect to QHFDHLPLA.

[0456] In some embodiments, the anti-EGFR antibody VH region has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with the VH region FR1;WIRQPPGKGLEWIG, which includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with the following framework region (FR):QVQLQESGPGLVKPSETLSLTCTVS. The VH region FR3 contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to VH region FR2 containing an amino acid sequence; and VH region FR4 contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to WGQGTLVTVSS.

[0457] In some embodiments, the anti-EGFR antibody VL region has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with the following framework region (FR):DIQMTQSPSSLSASVGDRVTITC VL region FR1;WYQQKPGKAPKLLIY It includes VL region FR2 containing a amino acid sequence; VL region FR3 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC; and VL region FR4 containing an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, with respect to FGQGTKVEIK.

[0458] In some embodiments, the present disclosure is an array The present invention provides an EGFR antibody VH region containing the sequence QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS (SEQ ID NO: 468), or its CDR, and an anti-EGFR antibody VL region containing the sequence DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQGTKVEIK (SEQ ID NO: 469), or its CDR.

[0459] In some embodiments, this disclosure is, The present invention provides an anti-EGFR binding domain (e.g., scFv) containing a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or 100% identity with respect to DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQHFDHLPLAFGQGTKVEIKSESATPESGPGTSPGATPESGPGTSESATPQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQPPGKGLEWIGHIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRVTGAFDIWGQGTLVTVSS (Sequence ID 449).

[0460] In some embodiments, the antigen-binding fragments VL and VH (e.g., Table 5f) are fused by a relatively long linker consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids, which have a flexible characteristic when joined together. In some embodiments, the VL and VH of any of the scFv embodiments described herein (e.g., Table 5f) are linked by a relatively long linker having the sequence SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the VL and VH of any of the scFv embodiments described herein are linked by a relatively long linker of hydrophilic amino acids 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 short linkers of hydrophilic amino acids having 3, 4, 5, 6, or 7 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, the disclosure provides compositions comprising single-chain diabodies in which, after folding, a first domain (VL or VH) pairs with a last domain (VH or VL) to form one scFv, two central domains pair to form another scFv, the first and second domains, as well as the third and last domains, are fused together by one of the aforementioned short linkers, and the second and third variable domains are fused by one of the aforementioned relatively long linkers. In some embodiments, the selection of short and relatively long linkers is intended to prevent incorrect pairing of adjacent variable domains, thereby facilitating the formation of a single-chain configuration including the VL and VH of the first and second antigen-binding fragments.

[0461] In some embodiments, the Disclosure provides EGFR-binding antigen-binding fragments (e.g., AF1 or AF2) that have improved stability compared to EGFR-binding antibodies or antigen-binding fragments known in the Art. In some embodiments, the EGFR antigen-binding fragments of the Disclosure are designed to confer a greater degree of stability to the chimeric bispecific antigen-binding fragment composition into which they are incorporated, resulting in improved expression and recovery of the fusion protein, increased shelf life, and improved stability when administered to a subject. In some embodiments, the anti-EGFR AFs of the Disclosure have a greater degree of thermal stability compared to certain EGFR-binding antibodies and antigen-binding fragments known in the Art. In some embodiments, the anti-EGFR AFs of the Disclosure have a greater degree of thermal stability compared to antigen-binding fragments including panitumumab VH and VL. In some embodiments, the anti-EGFR AFs of the Disclosure have a greater degree of thermal stability compared to EGFR 2, as disclosed in PCT International Patent Application Publication No. 2020 / 264208. In some embodiments, the anti-EGFR AF of this disclosure is less immunogenic in humans compared to certain EGFR-binding antibodies and antigen-binding fragments known in the art. In some embodiments, the anti-EGFR AF of this disclosure is less immunogenic than antigen-binding fragments including panitumumab VH and VL. In some embodiments, the anti-EGFR AF of this disclosure is less immunogenic than EGFR 2, as disclosed in PCT International Patent Application Publication No. 2020 / 264208.In some embodiments, the degree to which AF is immunogenic is determined by immunogenicity prediction methods such as TEPITOPpan (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 NetMHC IIpan-4.0 (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, utilized as components of chimeric bispecific antigen-binding fragment compositions into which they are incorporated, exhibit desirable pharmaceutically acceptable properties, including high thermal stability and low aggregation tendency, resulting in improved expression and recovery during manufacturing and storage, as well as promoting a longer serum half-life. Biophysical properties such as thermal stability are often limited by antibody variable domains with significantly different inherent properties. High thermal stability is often associated with other desirable properties, including high expression levels and low agglutination (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 defined as the "melting temperature" (T), which is the temperature at which half of the molecule denatures. m This is determined by measuring the melting temperature of each heterodimer, which indicates its thermal stability. The following examples include the method described below. mIn vitro assays for determining the melting point of the heterodimer are known in the art. The melting point of the heterodimer can be measured using techniques such as differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60, Ghirlando et al (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of the heterodimer can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9), or as described in the following examples.

[0462] In some embodiments of the polypeptides of this disclosure, the antigen-binding fragment (e.g., AF1 or AF2) has a higher melting temperature (Tm) than the anti-EGFR-binding fragment. m As demonstrated in in vitro assays, it may exhibit higher thermal stability than anti-EGFR binding fragments containing VH of SEQ ID NO: 450 and VL of SEQ ID NO: 451 (see Table 5f). Alternatively, if the first antigen-binding fragment is incorporated into the test bispecific antigen-binding domain, the T of the test bispecific antigen-binding domain may be higher. m The Tm of the test bispecific antigen-binding domain is higher compared to that of the control bispecific antigen-binding domain, and the test bispecific antigen-binding domain comprises a first antigen-binding fragment and a reference antigen-binding fragment that binds to antigens other than EGFR, while the control bispecific antigen-binding domain comprises an anti-EGFR-binding fragment (see Table 5f) including VH of SEQ ID NO: 450 and VL of SEQ ID NO: 451, and a reference antigen-binding fragment. In some embodiments, the melting temperature (Tm) of the first antigen-binding fragment is higher. m ) is an anti-EGFR binding fragment containing VH of SEQ ID NO: 450 and VL of SEQ ID NO: 451 m It may be 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 of the first antigen-binding fragment (T m ) is an anti-EGFR binding fragment containing VH of SEQ ID NO: 450 and VL of SEQ ID NO: 451 mIt may be 2°C to 15°C higher, 3°C to 15°C higher, 4°C to 15°C higher, or 5°C to 15°C higher (see Table 5f).

[0463] In some embodiments, any polypeptide among the embodiments of the subject composition 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) is determined in an in vitro antigen-binding assay containing a human EGFR antigen to have a binding affinity (K) of about 10 nM to about 400 nM, or about 50 nM to about 350 nM, or about 100 nM to about 300 nM. D It can specifically bind to human EGFR at a constant (K). In some embodiments, any polypeptide among the embodiments of the subject compositions described herein can be determined in an in vitro antigen binding assay to have 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 ) contains an antigen-binding fragment (AF) that specifically binds to human EGFR. To clarify, K D The antigen-binding fragment (AF) with a value of 400 is K D It binds to its ligand more weakly than one with a binding affinity of 10 nM. In some embodiments, any polypeptide among the embodiments of the subject compositions described herein has a binding affinity (K) of the respective polypeptide in an in vitro antigen binding assay. D The antigen-binding fragment (AF) is determined by ) and contains 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 sequence in Table 5f.

[0464] In some embodiments, this disclosure relates to the binding affinity (K) of each antigen in an in vitro antigen binding assay. DThe present invention provides a bispecific polypeptide comprising an antigen-binding fragment (AF) (anti-EGFR AF) that exhibits a binding affinity to EGFR that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or at least 1000 times weaker than the binding affinity of the embodiment of anti-EGFR AF described herein incorporated into the target polypeptide, as determined by [the specified method].

[0465] The binding affinity of the target composition to the target ligand can be evaluated using, for example, a binding assay such as the Biacore assay using a chip-binding receptor or binding protein, or a competitive binding assay, or an ELISA assay as described in U.S. Patent No. 5,534,617, the assay described in the examples herein, a radioreceptor assay, or other assays known in the art. The binding affinity constant can then be determined using a standard method such as the Scatchard analysis described in van Zoelen, et al., Trends Pharmacol Sciences (1998) 19) 12): 487, or other methods known in the art.

[0466] In some embodiments, the Disclosure provides antigen-binding fragments (AFs) (anti-EGFR AFs) that bind to EGFR and are incorporated into chimeric bispecific polypeptide compositions designed to have an isoelectric point (pI) that imparts improved stability to the composition compared to corresponding compositions containing EGFR-binding antibodies or antigen-binding fragments known in the Art. In some embodiments, any polypeptide among the embodiments of the Subject Compositions described herein comprises an EGFR-binding AF (anti-EGFR AF), wherein the anti-EGFR AF exhibits a pI of 6.0 to 6.6 (inclusive). In some embodiments, any polypeptide among the embodiments of the Subject Compositions 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 unit lower than the pI of the reference antigen-binding fragment. In some embodiments, any polypeptide among the embodiments of the subject composition described herein comprises an EGFR-binding AF (anti-EGFR AF) fused to another AF (anti-CD3 AF) that binds to the CD3 antigen, wherein the anti-EGFR AF exhibits a pI of at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units of the pI of the AF that binds to the CD3 antigen or its epitope. In some embodiments, any polypeptide among the embodiments of the subject composition described herein comprises an EGFR-binding AF (anti-EGFR AF) fused to an AF (anti-CD3 AF) that binds to the CD3 antigen, wherein the AF exhibits a pI of 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.The fusion antigen-binding fragments resulting from such a design, in which the pIs of the two antigen-binding fragments are within such a range, are specifically intended to confer a higher degree of stability to the chimeric bispecific antigen-binding fragment composition into which they are incorporated, resulting in improved expression and recovery of the soluble, non-aggregated fusion protein, increased shelf life of the formulated chimeric bispecific polypeptide composition, and improved 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 may allow for the selection of a buffer or other solution in which both AFs (anti-EGFR AF and anti-CD3 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 6.6 or less. In some embodiments, the antigen-binding fragments (AFs) may exhibit an isoelectric point (pI) of 6.0 to 6.6 (inclusive of both ends).

[0467] Unless otherwise specified, the numbering of amino acid residues in the variable domains of antibody domains, antigen-binding domains, or fragments thereof described herein follows the Kabat numbering scheme. The Kabat numbering for EGFR.2 VH (SEQ ID NO: 450) and VL (SEQ ID NO: 451) is provided below.

[0468] [Table 22-1]

[0469] [Table 22-2]

[0470] [Table 22-3]

[0471] Linkers and spacers between antibody regions in bispecific antibodies In some embodiments of the polypeptides of this disclosure, pairs of light chain variable regions (VL) and heavy chain variable regions (VH) of an antigen-binding fragment may be linked by a linker or a long linker (e.g., a hydrophilic amino acid). 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., an scFv such as an anti-CD3 scFv) are linked by a linker or a long linker (e.g., a hydrophilic amino acid). In some embodiments, the linker connecting the light chain variable region (VL) and the heavy chain variable region (VH) of an antigen-binding fragment (e.g., a first antigen-binding fragment (AF1) and / or a second antigen-binding fragment (AF2)) may (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 respect to the sequences listed in Table A. In some embodiments, the linker connecting the light chain variable region (VL) and the heavy chain variable region (VH) of an antigen-binding fragment (e.g., a first antigen-binding fragment (AF1) and / or a second antigen-binding fragment (AF2)) may (each independently) contain the same amino acid sequence with respect to the sequences listed in Table A. In some embodiments of the polypeptides of this disclosure, two antigen-binding fragments (e.g., a first and a second antigen-binding fragment) are fused together by a peptide linker or a short linker. In some embodiments, the peptide linker linking two antigen-binding fragments (e.g., the first and second antigen-binding fragments) 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 respect to the sequences listed in Table B. In some embodiments, the peptide linker linking two antigen-binding fragments (e.g., the first and second antigen-binding fragments) may contain the same amino acid sequence with respect to the sequences listed 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 a peptide linker.In some embodiments of the polypeptides of this disclosure, the linker used to link the scFv of a first antigen-binding fragment (e.g., anti-EGFR scFv) and the linker used to link the VL and VH of a second antigen-binding fragment (e.g., anti-CD3 scFv) may be GGGGSGGGS (SEQ ID NO: 125) in Table A. In other embodiments, the linker used to link the VL and VH of an antigen-binding fragment (e.g., anti-CD3 scFv) may be SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments of this disclosure, after folding, the first domain (VL or VH) pairs with the last domain (VH or VL) to form one scFv, the two central domains pair with each other to form another scFv, and the first and second domains, as well as the third and last domains, The present invention provides a polypeptide comprising a single-chain diabody, fused to one another by short linkers of hydrophilic amino acids identified by the sequences listed in Table B, with second and third variable domains fused to each other by long linkers identified by the sequences listed in Table A. In some embodiments, the selection of short and long linkers is to prevent mispairing of adjacent variable domains, thereby promoting the formation of a single-chain structure including the VL and VH of the first and second binding sites.

[0472] [Table 23]

[0473] [Table 24]

[0474] Spacer and TCE emission segment This specification includes fusion proteins comprising a TCE component that becomes biologically active or has increased biological activity upon release from ELNN to, for example, the fusion protein described herein, by cleaving an optional cleavage sequence incorporated within an optional spacer sequence.

[0475] In some embodiments, spacers may be provided to reduce steric hindrance in order to improve the expression of fusion proteins from host cells and / or to allow TCE components to assume their desired tertiary structure and / or to interact appropriately with their target molecules. For methods of identifying spacers and 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 having a length of 1 to 50 amino acid residues, or about 1 to 25 residues, or about 1 to 10 residues. The spacer sequence may contain any of 20 native L amino acids, excluding the cleavage site, and preferably, but not limited to, sterically unhindrant hydrophilic amino acids, including 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 of combinations mainly of glycine and alanine residues. In some embodiments, the spacer polypeptide, excluding the cleavage sequence, substantially lacks 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, and which may be subjected to protease action to release TCE from the fusion protein.

[0476] [Table 25]

[0477] In some embodiments of the polypeptides of this disclosure, release segments (RSs) (e.g., a first release segment (RS1), a second release segment (RS2), etc.) may be fused to a bispecific antibody domain (BsAb) by a spacer. In some embodiments, the spacer may comprise at least four amino acids, each independently being glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), or proline (P). In some embodiments, the peptide of this disclosure may comprise a first release segment fused to the bispecific antibody domain via a first spacer, and a second release segment 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 comprise amino acid sequences having at least (about) 80%, at least (about) 90%, or 100% sequence identity with respect to the sequences listed in Table C. In some embodiments, the spacers (e.g., a first spacer, a second spacer, etc.) may (each independently) contain the same amino acid sequence as the sequence described in Table C.

[0478] In some embodiments, the incorporation of the cleavage sequence into the fusion protein is designed to enable the release of TCE that is activated or more activated upon release from one or more ELNNs. In some embodiments, the cleavage sequence is located sufficiently close to the TCE sequence, generally within 18, 12, 6, or 2 amino acids of the TCE sequence terminus, so that any remaining residues bound to the TCE after cleavage do not significantly interfere with the activity of the TCE (e.g., receptor binding) and provide sufficient access to a protease capable of further cleaving the cleavage sequence. In some embodiments, the cleavage site is a sequence that can be cleaved by an endogenous protease in the mammalian subject so that paTCE can be cleaved after administration to the subject. In such cases, paTCE can function as a circulating storage site for TCE. Examples of cleavage sites intended herein, but not limited to, include polypeptide sequences cleavable by mammalian endogenous proteases listed in Table 6.

[0479] In some embodiments, the paTCE fusion protein includes a spacer sequence containing one or more cleavage sequences configured to release TCE from the fusion protein upon action by a protease. In some embodiments, the spacer sequence does not contain any cleavage sequences. In some embodiments, 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 to the sequences from Table 7a or 7b.

[0480] In some embodiments, the Disclosure provides TCE-releasing segment polypeptides (or releasing segments (RS)) that are substrates for one or more mammalian proteases that associate with diseased tissue or cells found in close proximity to diseased tissue, or that are produced by them. Such proteases include, but are not limited to, the classes of proteases such as metalloproteinases, cysteine ​​proteases, aspartate proteases, and serine proteases, including, but are not limited to, the proteases listed in Table 6. The RS is particularly useful for incorporation into a target recombinant polypeptide and confers an inactive form that can be activated by cleavage of the RS by a mammalian protease. As described herein, the RS is incorporated into a target recombinant polypeptide composition, linking the incorporated binding site to an ELNN (an exemplary configuration thereof is described herein), and as a result, upon cleavage of the RS by the action of one or more proteases for which the RS is a substrate, the binding site and the ELNN are released from the composition and the binding site, and are no longer shielded by the ELNN, restoring their full potential to bind to their ligands.

[0481] [Table 26-1]

[0482] [Table 26-2]

[0483] In some embodiments, the Disclosure provides an activatable recombinant polypeptide comprising a first release segment (RS1) sequence having at least 88%, at least 94%, or 100% sequence identity when optimally aligned to the sequences identified in Table 7a, wherein RS1 is a substrate of one or more mammalian proteases. In some embodiments, RS is further manipulated to remove a legmine cleavage site. In some embodiments, the Disclosure provides an activatable recombinant polypeptide comprising an RS1 and a second release segment (RS2) sequence, each having at least 88%, at least 94%, or 100% sequence identity when optimally aligned to the sequences identified herein by the sequences listed in Table 7a, wherein RS1 and RS2 are each substrates of one or more mammalian proteases. In some embodiments, RS1 and RS2 do not function as substrates of legmine.

[0484] In some embodiments, the Disclosure provides an activatable recombinant polypeptide comprising a first RS (RS1) sequence having at least 90%, at least 93%, at least 97%, or 100% sequence identity when optimally aligned to the sequences identified in Table 7b, wherein RS1 is a substrate of one or more mammalian proteases. In some embodiments, the Disclosure provides an activatable recombinant polypeptide comprising an RS1 and a second release segment (RS2) sequence, each having at least 88%, at least 94%, or 100% sequence identity when optimally aligned to the sequences identified herein by the sequences listed in Table 7b, wherein RS1 and RS2 are each substrates of one or more mammalian proteases (e.g., 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.

[0485] This disclosure envisions a release segment that is a substrate for one, two, or three different classes of proteases, including the proteases in Table 6, which are metalloproteinases, cysteine ​​proteases, aspartate proteases, or serine proteases. In some embodiments, the paTCE includes RS (e.g., RS1 and RS2) that function as substrates for one or more proteases found in close association with or co-localizing with tumor or cancer cells. Otherwise, the binding moieties of paTCE that are shielded by the ELNN (and therefore have lower binding affinity to their respective ligands) are released from the ELNN upon cleavage of the RS, restoring their full potential to bind to target and effector cell ligands. In some embodiments, paTCE comprises RSs (e.g., RS1 and RS2), each comprising an amino acid sequence that is a substrate for one or more cellular proteases located within a targeted cell, including, but not limited to, the proteases listed in Table 6. In some embodiments, the RSs are substrates for two or three classes of proteases that cleave different portions of each RS. In some embodiments, each RS, being a substrate for two, three, or more classes of proteases, has two, three, or more distinct cleavage sites; nevertheless, cleavage by a single protease results in the release of the binding moiety from the ELNN.

[0486] In some embodiments, the RS of this disclosure for incorporation into a fusion protein (such as paTCE) may include, but are not limited to, meprin, neprilysin (CD10), PSMA, BMP-1, disintegrin and metalloproteinase (ADAM), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM containing a thrombospongin motif (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3 (matrix Matrix metalloproteinase-3 (MMP-3, stromericin 1), matrix metalloproteinase-7 (matrix metalloproteinase-7, MMP-7, matrisin 1), matrix metalloproteinase-8 (matrix metalloproteinase-8, MMP-8, collagenase 2), matrix metalloproteinase-9 (matrix metalloproteinase-9, MMP-9, gelatinase B), matrix metalloproteinase-10 (matrix metalloproteinase-10, MMP-10, stromericin 2), matrix metalloproteinase-11 (matrix metalloproteinase-11, MMP-11, stromericin 3), matrix metalloproteinase-12 (matrix metalloproteinase-12, MMP-12, macrophage elastase), matrix metalloproteinase-13 (matrix metalloproteinase-13, MMP-13, collagenase-3), matrix metalloproteinase-14 (matrix metalloproteinase-14, MMP-14, MT1-MMP), matrix metalloproteinase-15 (matrix metalloproteinase-15, MMP-15, MT2-MMP),Matrix metalloproteinase-19 (MMP-19), Matrix metalloproteinase-23 (MMP-23, CA-MMP), Matrix metalloproteinase-24 (MT5-MMP), Matrix metalloproteinase-26 (Matrix 2, MMP-26, Matricin 2), Matrix metalloproteinase-27 (matrix Metalloproteinase-27 (MMP-27, CMMP), Regmine, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin X, Cathepsin D, Cathepsin E, Secretase, Urokinase (uPA), Tissue Plasminogen Activator (tPA), Plasmin, Thrombin, Prostate-Specific Antigen (PSA, KLK3), Human Neutrophil Elastase (HNE), Elastase, Tryptase, II RS is a substrate for one or more proteases, including type 1 transmembrane serine protease (TTSP), DESC1, hepsin (HPN), matryptase, matryptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast-activating protein (FAP), kallikrein-related peptidases (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14. In some embodiments, RS is a substrate for ADAM17. In some embodiments, RS is a substrate for BMP-1. In some embodiments, RS is a substrate for cathepsin. In some embodiments, RS is a substrate for HtrA1. In some embodiments, RS is a substrate for legmine. In some embodiments, RS is a substrate for MMP-1. In some embodiments, RS is a substrate for 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 isRS is a substrate of uPA. In some embodiments, RS is a substrate of matryptase. 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, RS of the recombinant polypeptide composition in question is a substrate of at least two proteases, including, but not limited to, legmine, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase. In some embodiments, RS of the recombinant polypeptide composition in question is a substrate of legmine, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matryptase. In certain embodiments, RS of the recombinant polypeptide composition in question is not a substrate of legmine. In some embodiments, the RS of the recombinant polypeptide composition in question is a substrate of uPA, matryptase (also known as MT-SP1 and ST14), MMP2, MMP7, MMP9, and MMP14. In some embodiments, the RS of the recombinant polypeptide composition in question is a substrate of uPA, matryptase, MMP2, MMP7, MMP9, and MMP14, but not a substrate of legmine.

[0487] [Table 27-1]

[0488] [Table 27-2]

[0489] [Table 27-3]

[0490] [Table 28-1]

[0491] Table 28-2

[0492] Table 28-3

[0493] Table 28-4

[0494] Table 28-5

[0495] Table 28-6

[0496] Table 28-7

[0497] Table 28-8

[0498] Table 28-9

[0499] In some embodiments, the paTCE comprises RS1 and RS2, which have different cleavage rates and different cleavage efficiencies for a plurality of proteases for which they are substrates. Since a given protease may be found at different concentrations in a tumor compared to healthy tissue or the circulatory system, the disclosure ensures that, when in proximity to cancer cells or tissue and their co-localized proteases, the paTCE is preferentially converted from an inactive form to an active form (i.e., by separation and release of the binding moiety and ELNN from the paTCE after cleavage of the RS) compared to the cleavage rate of the RS in healthy tissue or the circulatory system, and as a result, provides an RS having a higher or lower cleavage efficiency for a given protease, such that the released binding moiety of the TCE has a higher binding ability to the ligand in the tumor compared to the inactive form remaining in the circulatory system. Such selective design can improve the therapeutic index of the resulting composition and result in reduced side effects compared to conventional therapeutics that do not incorporate such site-specific activation.

[0500] In some embodiments, the cleavage efficiency is the log2 value of the ratio of the percentage of the test substrate containing cleaved RS to the percentage of the cleaved control substrate AC1611, when each is subjected to a protease enzyme in a biochemical assay where the initial substrate concentration is 6 μM, and the reaction is incubated at 37°C for 2 hours before being stopped by the addition of EDTA, and the amounts of digested products and uncleaved substrate are analyzed by unreduced SDS-PAGE to establish the ratio of cleaved percentages. The cleavage efficiency can be calculated as follows:

[0501]

number

[0502] In some embodiments, RS comprises the amino acid sequence EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), where X is any amino acid other than 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 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 legmine. In some embodiments, RS is not cleavable by legmine in human blood, plasma, or serum.

[0503] In some embodiments, RS is not cleavable during incubation with regmine at about 1 nM or less for about 20 hours. In some embodiments, RS is cleaved by regmine more slowly or inefficiently than RSR-2295 (EAGRSANHTPAGLTGP) is cleaved by regmine. In some embodiments, RS is cleaved by regmine at a rate less than about 50% of the rate at which regmine cleaves RSR-2295 (EAGRSANHTPAGLTGP). In some embodiments, RS is cleaved by regmine at a rate less than about 25% of the rate at which regmine cleaves RSR-2295. In some embodiments, RS is cleaved by regmine at a rate less than about 10% of the rate at which regmine cleaves RSR-2295. In some embodiments, RS is cleaved by regmine at a rate less than about 5% of the rate at which regmine cleaves RSR-2295. In some embodiments, RS is cut by the Regmine at a rate less than 2.5% of the rate at which the Regmine cuts RSR-2295.

[0504] In some embodiments, RS is cleaved by legmine at a rate less than 50% of the rate at which legmine cleaves RSR-2295 (EAGRSANHTPAGLTGP) in human plasma. In some embodiments, RS is cleaved by legmine at a rate less than 25% of the rate at which legmine cleaves RSR-2295 in human plasma. In some embodiments, RS is cleaved by legmine at a rate less than 10% of the rate at which legmine cleaves RSR-2295 in human plasma. In some embodiments, RS is cleaved by legmine at a rate less than 5% of the rate at which legmine cleaves RSR-2295 in human plasma. In some embodiments, RS is cleaved by legmine at a rate less than 2.5% of the rate at which legmine cleaves RSR-2295 in human plasma.

[0505] In some embodiments, the disclosure provides a paTCE comprising a plurality of RSs, each RS sequence being identified herein by the sequence group listed in Table 7a, and the RSs being 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 distinct from the first RS, each RS sequence being identified herein by the sequence listed in Table 7a, and the RSs being 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 distinct from the first RS, and a third RS distinct from the first and second RS, each sequence being identified herein by the sequence listed in Table 7a, and the first, second, and third RSs being linked to each other by 1 to 6 amino acids which are glycine, serine, alanine, and threonine. In some embodiments, multiple RSs of a paTCE are linked to form sequences that can be cleaved by multiple proteases at different cleavage rates or efficiencies. In some embodiments, the Disclosure provides a paTCE comprising RS1 and RS2, each having ELNNs (e.g., ELNN1 and ELNN2), such as those described in Table 7a or 7b and those described herein, wherein RS1 is fused between ELNN1 and the binding portion, and RS2 is fused between ELNN2 and the binding portion. In some embodiments, the paTCE is more readily cleaved in target tissue expressing multiple proteases (e.g., tumor tissue) compared to healthy tissue or in normal circulation, resulting in an improved ability of the resulting fragments carrying the binding portion to more readily penetrate target tissue, e.g., tumors, and to bind and link cancer cells and effector cells.

[0506] In some embodiments, paTCE includes a first release segment (RS1) located between a first ELNN and a bispecific antibody. In some embodiments, the polypeptide further includes a second release segment (RS2) located between a bispecific antibody and a second ELNN. In some embodiments, RS1 and RS2 are sequence-identical. In some embodiments, RS1 and RS2 are sequence-distinguishable. In some embodiments, RS1 includes an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequences or subsets thereof identified in Table 7a or 7b of this specification. 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 respect to the sequences or subsets thereof identified in Table 7a or 7b of this specification. In some embodiments, RS1 and RS2 are substrates for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence.

[0507] In some embodiments, paTCE further comprises one or more reference fragments (e.g., barcode fragments) that can be released from paTCE during protease digestion. In some embodiments, the one or more reference fragments are a single reference fragment that differs in sequence and molecular weight from all other peptide fragments that can be released from the polypeptide during protease digestion of the polypeptide.

[0508] Exemplary paTCE In some embodiments, paTCE comprises an amino acid sequence having at least (approximately) 80% sequence identity with respect to the sequences listed in Table D (SEQ ID NOs. 1000-1007) or a subset thereof. In some embodiments, paTCE comprises an amino acid sequence having at least (approximately) 81%, at least (approximately) 82%, at least (approximately) 83%, at least (approximately) 84%, at least (approximately) 85%, at least (approximately) 86%, at least (approximately) 87%, at least (approximately) 88%, at least (approximately) 89%, at least (approximately) 90%, at least (approximately) 91%, at least (approximately) 92%, at least (approximately) 93%, at least (approximately) 94%, at least (approximately) 95%, at least (approximately) 96%, at least (approximately) 97%, at least (approximately) 98%, at least (approximately) 99%, or (approximately) 100% sequence identity with respect to the sequences listed in SEQ ID NOs. 1000-1007 or a subset thereof. In some embodiments, paTCE comprises an amino acid sequence having at least (approximately) 90%, at least (approximately) 91%, at least (approximately) 92%, at least (approximately) 93%, at least (approximately) 94%, at least (approximately) 95%, at least (approximately) 96%, at least (approximately) 97%, at least (approximately) 98%, at least (approximately) 99%, or approximately 100% sequence identity with respect to the sequences or subsets thereof described in SEQ ID NOs. In some embodiments, paTCE comprises an amino acid sequence identical to the sequences described in SEQ ID NOs. 1000–1007. The compositions of this disclosure are specifically intended to include sequence variants of the amino acid sequences described in Table D, such as substituted or inserted linker sequences or purified tag sequences bound thereto, insofar as the variants exhibit substantially similar or identical biological activity and / or activation mechanisms.

[0509] [Table 29-1]

[0510] [Table 29-2]

[0511] [Table 29-3]

[0512] [Table 29-4]

[0513] Recombinant production Also provided are polynucleotides encoding any polypeptide disclosed herein and / or reverse complements of such polynucleotides.

[0514] The disclosure herein includes an expression vector comprising a polynucleotide sequence, such as one of those described in the preceding paragraphs, and a regulatory sequence operably ligated to the polynucleotide sequence.

[0515] The disclosure herein includes host cells containing expression vectors as described in any of 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.

[0516] In some embodiments, the Disclosure provides a method for producing the composition of interest. In some embodiments, such a method comprises culturing host cells containing a nucleic acid construct encoding a polypeptide described herein (e.g., paTCE) under conditions that promote polypeptide expression, and then recovering the polypeptide using a standard purification method (e.g., column chromatography, HPLC), such that 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 recovered and expressed polypeptide or the bound fragments of the paTCE fusion polypeptide are properly folded. In some embodiments of the method of production, the expressed polypeptide is recovered, and at least or 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.

[0517] In some embodiments, the Disclosure provides methods for producing polypeptides (such as paTCE fusion polypeptides) at high fermentation expression levels of functional proteins using E. coli or mammalian host cells, and for providing expression vectors encoding polypeptides useful in producing cytotoxic polypeptide compositions at high expression levels. In some embodiments, the method comprises: 1) preparing a polynucleotide encoding the polypeptide 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 levels of protein expression in a biological system; 3) transforming a suitable host cell with the expression vector; and 4) culturing the host cell in a conventional nutrient medium under conditions suitable for expression of the polypeptide composition. Preferably, the host cell is Escherichia coli (E. coli). As used herein, the term “correctly folded” means that the antigen-binding fragment components of the composition have the ability to specifically bind to their target ligands (e.g., upon activation). In some embodiments, the present disclosure provides a method for producing polypeptides, the method comprising culturing a host cell containing a polypeptide-encoding vector in a fermentation reaction under conditions effective for expressing a polypeptide product.

[0518] Pharmaceutical composition Disclosed herein are pharmaceutical compositions comprising a polypeptide (such as paTCE), such as any of those described herein, and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intra-arterial, intra-abdominal, intraperitoneal, intravitreous, intrathecal, or intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for intravenous injection. In some embodiments, the pharmaceutical composition is in liquid form or frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder that is reconstituted before administration.

[0519] The pharmaceutical composition may be administered for treatment via any preferred route. In some embodiments, the dose is administered intradermally, subcutaneously, intravenously, intra-arterially, intra-abdominally, intraperitoneally, intrathecally, or intramuscularly. In some embodiments, the subject is a mouse, rat, monkey, or human. In preferred embodiments, the subject is a human.

[0520] 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 dose. In some embodiments, the therapeutically effective dose results in an increased retention time of the fusion protein within the therapeutic window compared to the corresponding TCE of a fusion protein that is not linked to an ELNN and is administered to the subject in an equivalent dose.

[0521] 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 that is reconstituted before administration. In some embodiments, the composition may also be supplied in liquid form that can be administered directly to the subject, or it may be frozen.

[0522] Medical kit In some embodiments, the Disclosure provides a kit for facilitating the use of paTCE. In some embodiments, the kit comprises (a) a first container containing a pharmaceutically effective amount of paTCE in a lyophilized composition, and (b) a second container containing a diluent for reconstituting the lyophilized formulation. In some embodiments, the kit further comprises instructions for storing the kit, information on cancers treatable with paTCE, instructions for reconstituting the lyophilized formulation, and / or instructions for administration.

[0523] Treatment method Disclosed herein is the use of polypeptides, such as any of those described herein, in the preparation of a medicament for the treatment of a disease in a subject. In some embodiments, the specific disease to be treated depends on the selection of a biologically active protein. In some embodiments, the disease is cancer. Included herein is a paTCE polypeptide for use in the treatment of 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 a carcinoma, sarcoma, or melanoma. In some embodiments, the cancer is a carcinoma. In some embodiments, the cancer is a sarcoma. In some embodiments, the cancer is a melanoma.

[0524] EGFR is one of the most frequently altering oncogenes in solid tumors. EGFR activation promotes processes involved in 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 result in increased tyrosine kinase activity of EGFR. Overexpression of the EGFR protein may or may not be associated with EGFR gene amplification. Furthermore, wild-type EGFR protein is commonly overexpressed in many types of solid tumors and is often associated with poor prognosis. For example, alterations of EGFR in solid tumors known in the art, such as those 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 these is incorporated herein in whole). Current EGFR inhibitors, including tyrosine kinase inhibitors and monoclonal antibody inhibitors, have shown limited efficacy and are plagued by congenital and acquired resistance in clinical settings.

[0525] In some embodiments, cancer is associated with EGFR overexpression (e.g., compared to non-cancerous cells of the same tissue type). In some embodiments, cancer comprises cells expressing, on average, 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. In some embodiments, cancer comprises cells having one or more oncogenic mutations in the EGFR gene. In some embodiments, cancer comprises cells having EGFR gene amplification. In some embodiments, the cells include an increase of 2–5 times, 2–10 times, 2–15 times, 2–30 times, 2–50 times, 3–5 times, 3–10 times, 3–15 times, 3–30 times, 3–50 times, 5–10 times, 5–15 times, 5–30 times, or 5–50 times compared to non-cancerous cells of the same tissue type.

[0526] In some embodiments, 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 embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple-negative breast cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the brain cancer is glioblastoma.

[0527] In some embodiments, cancers include undifferentiated and medullary thyroid cancer, appendiceal cancer, masculinizing tumors, biliary tract cancer, bladder cancer, breast cancer, bile duct cancer, carcinoid tumors, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, intraepithelial peritoneal malignancy with malignant ascites, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, follicular cancer, gallbladder cancer, and gastric cancer. Cancer, gastrointestinal stromal tumor (GIST), G-GE junction cancer, urogenital tract cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocellular carcinoma, HR+ and HER2+ breast cancer, Haasle cell carcinoma, inflammatory breast cancer, Kaposi's 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 cancer, peritoneal carcinomatosis, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, stomach cancer This includes cancer, testicular cancer, thyroid cancer, triple-negative breast cancer, urothelial carcinoma, uterine cancer, serous uterine carcinoma, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0528] This disclosure includes methods for treating a disease in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition, such as any described herein, to a subject in need of treatment. 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 human.

[0529] In some embodiments, the EGFR-targeting bispecific composition of this disclosure (e.g., paTCE) may be combined with one or more checkpoint inhibitors....

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