Compositions and methods relating to tumor activated t cell engagers

Abstract

To provide a method for extending the half-life of a T cell engager and improving its toxicity profile.SOLUTION: The method comprises a step of binding a half-life extending molecule-linked inhibitory peptide mask to a T cell engager via a cleavable linker, in order to produce a modified T cell engager having reduced toxicity in healthy tissue as compared to an unmodified T cell engager. The cleavable linker is cleaved in the tumor microenvironment. The half-life extending molecule-linked inhibitory peptide mask is released from the modified T cell engager in order to produce a cleaved T cell engager. The modified T cell engager has a longer half-life than both the unmodified T cell engager and the cleaved T cell engager.SELECTED DRAWING: Figure 11

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 858,254, filed Jun. 6, 2019, and U.S. Provisional Patent Application No. 62 / 978,662, filed Feb. 19, 2020, which are hereby incorporated by reference herein in their entirety.

[0002] Sequence Listing This application includes a sequence listing, which is submitted electronically in ASCII format and is hereby incorporated by reference herein in its entirety. The ASCII copy created on Jun. 4, 2020, is named 52426-716_601_SL.txt and is 121,292 bytes in size.

Background Art

[0003] Protein-based therapies, including T cell engagers, have proven effective as treatments for various diseases. For any therapeutic class, it is necessary to improve the toxicity and side effects of such treatments, along with the half-life of the therapeutic molecule.

Summary of the Invention

[0004] Modified T cell engagers can be used for the selective destruction of individual cells or cell types, such as cancer cells of a tumor. Such modified T cell engagers induce an immune response against the tumor to remove the tumor. However, current treatments using modified T cell engagers can be toxic and ineffective. Further, such modified T cell engagers may have poor pharmacokinetic properties (PK). Provided herein are modified T cell engagers that have improved PK properties and efficacy in removing tumors while reducing toxicity in healthy tissue and thus improving safety. In some embodiments, the modified T cell engagers described herein are linked to a peptide that blocks the interaction of the T cell engager with its target in healthy tissue, thereby reducing target-mediated drug disposition (TMDD). Modified T cell engagers as described herein are also linked to a half-life extending molecule, such as a single domain antibody, which improves the PK profile of the modified T cell engager as compared to an unmodified T cell engager.

[0005] Disclosed herein, in one embodiment, is a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to the following configuration:

[0006]

Chemical formula

[0007] Disclosed herein is, in certain embodiments, a pharmaceutical composition comprising (i) a polypeptide complex as described herein and (ii) a pharmaceutically acceptable excipient.

[0008] Disclosed herein is, in certain embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex as described herein.

[0009] Incorporation by reference All publications, patents, and patent applications mentioned herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which illustrates exemplary embodiments in which the principles of the present disclosure are utilized and the accompanying drawings:

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【Figure 30C]]Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 in the presence of human serum albumin (HSA) buffer, as determined by BLI. The kinetic binding of PC-2 to CD3 in the presence of HSA buffer is shown.

【Figure 30D]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 in the presence of human serum albumin (HSA) buffer, as determined by BLI. The kinetic binding of PC-2 treated with uPa to CD3 in the presence of HSA buffer is shown.

【Figure 30E]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 in the presence of human serum albumin (HSA) buffer, as determined by BLI. The kinetic binding of PC-3 to CD3 in the presence of HSA buffer is shown.

【Figure 30F]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 in the presence of human serum albumin (HSA) buffer, as determined by BLI. The kinetic binding of PC-3 treated with uPa to CD3 in the presence of HSA buffer is shown.

【Figure 30G]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 in the presence of human serum albumin (HSA) buffer, as determined by BLI. The kinetic binding of PC-4 to CD3 in the presence of HSA buffer is shown.

【Figure 30H]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 in the presence of human serum albumin (HSA) buffer, as determined by BLI. The kinetic binding of PC-4 treated with MTSP1 to CD3 in the presence of HSA buffer is shown.

【Figure 30I]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 in the presence of human serum albumin (HSA) buffer, as determined by BLI. The kinetic binding of PC-5 to CD3 in the presence of HSA buffer is shown.

【Figure 30J]]Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 by BLI in the presence of human serum albumin (HSA) buffer. The kinetic binding of PC - 5 treated with MTSP1 to CD3 in the presence of HSA buffer is shown.

【Figure 30K]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 by BLI in the presence of human serum albumin (HSA) buffer. The kinetic binding of PC - 7 to CD3 in the presence of HSA buffer is shown.

【Figure 30L]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 by BLI in the presence of human serum albumin (HSA) buffer. The kinetic binding of MTSP1 - treated PC - 7 to CD3 in the presence of HSA buffer is shown.

【Figure 30M]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 by BLI in the presence of human serum albumin (HSA) buffer. The kinetic binding of PC - 8 to CD3 in the presence of HSA buffer is shown.

【Figure 30N]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 by BLI in the presence of human serum albumin (HSA) buffer. The kinetic binding of MTSP1 - treated PC - 8 to CD3 in the presence of HSA buffer is shown.

【Figure 30O]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 by BLI in the presence of human serum albumin (HSA) buffer. The kinetic binding of PC - 9 to CD3 in the presence of HSA buffer is shown.

【Figure 30P]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 by BLI in the presence of human serum albumin (HSA) buffer. The kinetic binding of MTSP1 - treated PC - 9 to CD3 in the presence of HSA buffer is shown.

【Figure 30Q]]Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 by BLI in the presence of human serum albumin (HSA) buffer. The kinetic binding of PC - 6 to CD3 in the presence of HSA buffer is shown.

【Figure 30R]] Figures 30A - 30R show the kinetic binding of the polypeptide complex to CD3 by BLI in the presence of human serum albumin (HSA) buffer. A blank is shown.

【Figure 31]] The equilibrium binding of the polypeptide complex to HER2 in buffer containing bovine serum albumin (BSA) or human albumin (HSA) is shown by ELISA.

【Figure 32A]] Figures 32A - 32C show the equilibrium binding of the polypeptide complex to EGFR in buffer containing BSA or HSA before and after protease treatment by ELISA. The equilibrium binding of the polypeptide complex to EGFR in buffer containing HSA before and after uPa treatment is shown.

【Figure 32B]] Figures 32A - 32C show the equilibrium binding of the polypeptide complex to EGFR in buffer containing BSA or HSA before and after protease treatment by ELISA. The equilibrium binding of the polypeptide complex to EGFR in buffer containing BSA before and after uPa treatment is shown.

【Figure 32C]] Figures 32A - 32C show the equilibrium binding of the polypeptide complex to EGFR in buffer containing BSA or HSA before and after protease treatment by ELISA. The equilibrium binding of the polypeptide complex to EGFR in buffer containing BSA or HSA before and after MTSP1 treatment is shown.

【Figure 33A]] Figures 33A - 33D show the equilibrium binding of the polypeptide complex to CD3 in buffer containing BSA or HSA before and after protease treatment by ELISA. The equilibrium binding of the polypeptide complex to CD3 in buffer containing HSA before and after uPa treatment is shown by ELISA.

【Figure 33B]]Figures 33A - 33D show the polypeptide complex equilibrium binding CD3 in buffer containing BSA or HSA before and after protease treatment by ELISA. The polypeptide complex equilibrium binding CD3 in buffer containing BSA before and after MTSP1 treatment is shown by ELISA.

【Figure 33C]] Figures 33A - 33D show the polypeptide complex equilibrium binding CD3 in buffer containing BSA or HSA before and after protease treatment by ELISA. The polypeptide complex equilibrium binding CD3 in buffer containing HSA before and after MTSP1 treatment is shown by ELISA.

【Figure 33D]] Figures 33A - 33D show the polypeptide complex equilibrium binding CD3 in buffer containing BSA or HSA before and after protease treatment by ELISA. The polypeptide complex equilibrium binding CD3 in buffer containing BSA before and after MTSP1 treatment is shown by ELISA.

【Figure 34]] The cell surface CD3, polypeptide complex, and EGFR tetramer ternary complex composition of human T cells are shown by flow cytometry.

【Figure 35]] The cell surface EGFR, polypeptide complex, and CD3 tetramer ternary complex composition of HCT116 cells are shown by flow cytometry. [[ID=1 The polypeptide complex - mediated cytotoxicity against tumor target cells HCC1569 is shown by the LDH - Glo assay. ​ The polypeptide complex - mediated T cell activation against tumor target cells HCC1569 is shown by IFNγ ELISA. ​ The polypeptide complex - mediated HCC1569 tumor cell death is shown using a real - time cell analyzer (RTCA).

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Modes for Carrying Out the Invention

[0011] Preferred embodiments of the present disclosure have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will be envisioned by those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be utilized in practicing the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be embraced thereby. Specific

[0012] Definitions The terms used herein are for the purpose of describing particular cases only and are not intended to limit the present invention. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Further, to the extent that the terms "including", "includes", "having", "has", "with" or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be construed in a manner similar to the term "comprising".

[0013] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations of any value in practice. Where a particular value is set forth in the present application and claims, the term "about" is assumed to mean within an acceptable error range of the particular value unless otherwise specified.

[0014] As used herein, "fragment" refers to a peptide or polypeptide that contains an amino acid sequence less than full length.

[0015] As used herein, "antigen-binding site" refers to the region of a polypeptide that interacts with an antigen. The antigen-binding site includes the antigen and the amino acid residues that interact directly with the antigen, as well as amino acid residues that are in proximity to the antigen but may not interact directly with the antigen.

[0016] Polypeptide or polypeptide complex Disclosed herein are, in some embodiments, polypeptides or polypeptide complexes that include a half-life extending molecule. In some embodiments, the polypeptide or polypeptide complex includes an antibody or an antibody fragment. In some embodiments, the polypeptide or polypeptide complex binds to a tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex binds to an effector cell antigen.

[0017] In further embodiments, the polypeptides or polypeptide complexes described herein have an optimal molecular weight for enhanced tissue penetration and distribution. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of about 80 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of about 90 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of about 100 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of about 110 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of about 120 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of about 130 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of less than about 80 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of less than about 90 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of less than about 100 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of less than about 110 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of less than about 120 kDa. In some embodiments, the polypeptide or polypeptide complex has a molecular weight of less than about 130 kDa.

[0018] Disclosed herein, in some embodiments, is a polypeptide or polypeptide complex according to Formula I: A2-A1-L1-P1-H1 (Formula I) wherein A1 comprises a first antigen recognition molecule that binds to a first target antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen recognition molecule that binds to a second target antigen.

[0019] Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula I: A2 - A1 - L1 - P1 - H1 (Formula I) Wherein, A1 is a first antigen - recognizing molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor - specific protease, H1 is a half - life extending molecule, and A2 is a second antigen - recognizing molecule that binds to a second target antigen.

[0020] Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula I: A2 - A1 - L1 - P1 - H1 (Formula I) Wherein, A1 comprises a first antigen - recognizing molecule that binds to a first target antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor - specific protease, H1 comprises a half - life extending molecule, and A2 comprises a second antigen - recognizing molecule that binds to a second target antigen.

[0021] Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula I: A2 - A1 - L1 - P1 - H1 (Formula I) In the formula, A1 is a first antigen recognition molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen recognition molecule that binds to a second target antigen. In some embodiments, the first target antigen includes a tumor cell antigen, and the second target antigen includes an effector cell antigen. In some embodiments, the first target antigen includes an effector cell antigen, and the second target antigen includes a tumor cell antigen. In some embodiments, when L1 is cleaved by a tumor-specific protease, the polypeptide or polypeptide complex of formula I binds to a target cell. In some embodiments, when L1 is cleaved by a tumor-specific protease, the polypeptide of formula I binds to an effector cell.

[0022] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex according to formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) In the formula, A1 includes a first antigen recognition molecule that binds to a first target antigen, P1 includes a peptide that binds to A1, L1 includes a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 includes a half-life extending molecule, A2 includes a second antigen recognition molecule that binds to a second target antigen, P2 includes a peptide that binds to A2, and L2 includes a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0023] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex according to formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) In the formula, A1 is a first antigen recognition molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, A2 is a second antigen recognition molecule that binds to a second target antigen, P2 is a peptide that binds to A2, and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0024] Disclosed herein are, in some embodiments, polypeptides or polypeptide complexes comprising formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) In the formula, A1 comprises a first antigen recognition molecule that binds to a first target antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extending molecule, A2 comprises a second antigen recognition molecule that binds to a second target antigen, P2 comprises a peptide that binds to A2, and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0025] Disclosed herein are, in some embodiments, polypeptides or polypeptide complexes comprising formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) In the formula, A1 is a first antigen recognition molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, A2 is a second antigen recognition molecule that binds to a second target antigen, P2 is a peptide that binds to A2, and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0026] In some embodiments, the polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or modified non-natural amino acid comprises a post-translational modification.

[0027] Further disclosed herein are, in some embodiments, polypeptides or polypeptide complexes according to Formula II: L 1a -P 1a -H 1a (Formula II) Wherein L 1a , when not cleaved, comprises a tumor-specific protease-cleavable linker moiety that connects P 1a to an antigen recognition molecule that binds to a target antigen, and P 1a , when L 1a is not cleaved, comprises a peptide that binds to the antigen recognition molecule, and H 1a comprises a half-life extending molecule.

[0028] Further disclosed herein are, in some embodiments, polypeptides or polypeptide complexes according to Formula II: L 1a -P 1a -H 1a (Formula II) Wherein L 1a , when not cleaved, is a tumor-specific protease-cleavable linker moiety that connects P 1a to an antigen recognition molecule that binds to a target antigen, and P 1a , when L 1a is not cleaved, is a peptide that binds to the antigen recognition molecule, and H 1a is a half-life extending molecule.

[0029] Further disclosed herein are, in some embodiments, polypeptides or polypeptide complexes comprising Formula II: L 1a -P 1a-H 1a (Formula II) In the formula, L 1a , when not cleaved, is connected to an antigen recognition molecule that binds to the target antigen, and contains a tumor-specific protease-cleavable linker moiety, and P 1a , when not cleaved, contains a peptide that binds to the antigen recognition molecule, and H 1a , when L 1a is not cleaved, contains a peptide that binds to the antigen recognition molecule, and H 1a contains a half-life extending molecule.

[0030] Further disclosed herein is, in some embodiments, a polypeptide or polypeptide comprising Formula II: L 1a -P 1a -H 1a (Formula II) In the formula, L 1a , when not cleaved, is a tumor-specific protease-cleavable linker moiety that connects P 1a to an antigen recognition molecule that binds to the target antigen, and P 1a , when L 1a is not cleaved, is a peptide that binds to the antigen recognition molecule, and H 1a is a half-life extending molecule. In some embodiments, the antigen recognition molecule comprises an antibody or antibody fragment. In some embodiments, the target antigen is an anti-CD3 effector cell antigen.

[0031] Antigen recognition molecule (A1) Disclosed herein is, in some embodiments, a polypeptide or polypeptide complex in which the first target antigen comprises an effector cell antigen and the second target antigen comprises a tumor cell antigen. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, the tumor cell antigen comprises EGFR, HER2, mesothelin, or CEACAM5.

[0032] In some embodiments, A1 comprises an antibody or antibody fragment. In some embodiments, A1 comprises a human or humanized antibody or antibody fragment. In some embodiments, L1 is attached to the N-terminus of the antibody or antibody fragment. In some embodiments, L1 is attached to the N-terminus of the antibody or antibody fragment, and A2 is attached to the other N-terminus of the antibody or antibody fragment. In some embodiments, A2 is attached to the C-terminus of the antibody or antibody fragment. In some embodiments, L1 is attached to the C-terminus of the antibody or antibody fragment. In some embodiments, A2 is attached to the N-terminus of the antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment. In some embodiments, A1 is a single-chain variable fragment (scFv). In some embodiments, the scFv comprises an scFv heavy-chain polypeptide and an scFv light-chain polypeptide. In some embodiments, A1 is a single-domain antibody. In some embodiments, A1 is a Fab fragment. In some embodiments, A1 comprises an anti-CD3e single-chain variable fragment. In some embodiments, A1 comprises an anti-CD3e single-chain variable fragment having a KD binding to CD3 of 1 μM or less on CD3-expressing cells. In some embodiments, A1 comprises a variable light chain and a variable heavy chain, each of which can specifically bind to human CD3.In some embodiments, A1 comprises a complementarity determining region (CDR) selected from the group consisting of muromonab-CD3 (OKT3), otrexup mab (TRX4), teprotumumab (MGA031), bisilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19.

[0033] In some embodiments, when L1 is cleaved by a tumor-specific protease, the polypeptide or polypeptide complex of formula I binds to an effector cell. In some embodiments, when L1 is cleaved by a tumor-specific protease and A1 binds to an effector cell, the polypeptide or polypeptide complex of formula I binds to an effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, A1 binds to a polypeptide that is part of the TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3ε. In some embodiments, the effector cell antigen comprises CD3 and the scFv comprises an amino acid sequence according to SEQ ID NO: 64, 65, or 66.

[0034] Disclosed herein is, in some embodiments, a polypeptide or polypeptide complex wherein the first target antigen comprises a tumor cell antigen and the second target antigen comprises an effector cell antigen. In some embodiments, the tumor cell antigen comprises EGFR, HER2, mesothelin, or CEACAM5. In some embodiments, the effector cell antigen comprises CD3.

[0035] In some embodiments, antigen recognition A1 comprises an antibody or an antibody fragment. In some embodiments, A1 comprises a human or humanized antibody or antibody fragment. In some embodiments, L1 is bound to the N-terminus of the antibody or antibody fragment. In some embodiments, A2 is bound to the C-terminus of the antibody or antibody fragment. In some embodiments, L1 is bound to the C-terminus of the antibody or antibody fragment. In some embodiments, A2 is bound to the N-terminus of the antibody or antibody fragment. In some embodiments, the antibody or its antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab. In some embodiments, the antibody or its antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain of a single-domain antibody derived from camelids (VHH). In some embodiments, the antibody or its antibody fragment is humanized or human.

[0036] In some embodiments, A1 is a Fab. In some embodiments, the Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide, and the Fab light chain polypeptide of A1 is bound to the C-terminus of a single-chain variable fragment (scFv) of A2. In some embodiments, the Fab heavy chain polypeptide of A1 is bound to the C-terminus of a single-chain variable fragment (scFv) of A2. In some embodiments, the Fab light chain polypeptide of A1 is bound to the N-terminus of a single-chain variable fragment (scFv) of A2. In some embodiments, the Fab heavy chain polypeptide of A1 is bound to the N-terminus of a single-chain variable fragment (scFv) of A2. In some embodiments, the Fab heavy chain polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2, and L1 is bound to the Fab light chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2, and L1 is bound to the Fab heavy chain polypeptide of A1. In some embodiments, the Fab heavy chain polypeptide of A1 is bound to the scFv light chain polypeptide of A2, and L1 is bound to the Fab light chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A1 is bound to the scFv light chain polypeptide of A2, and L1 is bound to the Fab heavy chain polypeptide of A1. In some embodiments, A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 comprises a linker that connects A2 to P2 and is a substrate for a tumor-specific protease. In some embodiments, the Fab heavy chain polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2, and L1 is bound to the Fab light chain polypeptide of A1, and L2 is bound to the scFv light chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2, and L1 is bound to the Fab light chain polypeptide of A1, and L2 is bound to the scFv light chain polypeptide of A2, and the polypeptide complex comprises the amino acid sequences according to SEQ ID NO:72 and SEQ ID NO:71.In some embodiments, the Fab light chain polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2, and L1 is bound to the Fab heavy chain polypeptide of A1, and L2 is bound to the scFv light chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A1 is bound to the scFv light chain polypeptide of A2, and L1 is bound to the Fab light chain polypeptide of A1, and L2 is bound to the scFv heavy chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A1 is bound to the scFv light chain polypeptide of A2, and L1 is bound to the Fab heavy chain polypeptide of A1, and L2 is bound to the scFv heavy chain polypeptide of A2.

[0037] In some embodiments, the antibody or antibody fragment thereof comprises an epidermal growth factor receptor (EGFR) binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a mesothelin binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a carcinoembryonic antigen-related cell adhesion molecule CEACAM5 binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a HER2 binding domain. In some embodiments, the tumor cell antigen comprises EGFR, and the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 56 or SEQ ID NO: 57. In some embodiments, the tumor cell antigen comprises EGFR, and the Fab heavy chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 59 or SEQ ID NO: 60. In some embodiments, the tumor cell antigen comprises HER2, and the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 61. In some embodiments, the tumor cell antigen comprises HER2, and the Fab heavy chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 62 or SEQ ID NO: 63.

[0038] In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for tumor cell antigens as compared to the binding affinity of a polypeptide or polypeptide complex that does not have P1 or L1 for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for tumor cell antigens, which is at least 5-fold (5X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for tumor cell antigens, which is at least 8-fold (8X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for tumor cell antigens, which is at least 10-fold (10X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for tumor cell antigens, which is at least 15-fold (15X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for tumor cell antigens, which is at least 20-fold (20X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for tumor cell antigens, which is at least 25-fold (2:5X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex that does not have P1 or L1.In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 30-fold (30X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 35-fold (35X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 40-fold (40X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 45-fold (45X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 50-fold (50X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 55-fold (55X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 60-fold (60X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1.In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 65-fold (65X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 70-fold (70X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 75-fold (75X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 80-fold (80X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 85-fold (85X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 90-fold (90X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 95-fold (95X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1.In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 100-fold (100X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 120-fold (120X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 1000-fold (1000X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex that does not have P1 or L1.

[0039] In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen as compared to the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 5-fold (5X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 8-fold (8X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 10-fold (10X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 15-fold (15X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 20-fold (20X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 25-fold (25X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen.In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 30-fold (30X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 35-fold (35X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 40-fold (40X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 45-fold (45X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 50-fold (50X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 55-fold (55X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 60-fold (60X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen.In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 65-fold (65X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 70-fold (70X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 75-fold (75X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 80-fold (80X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 85-fold (85X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 90-fold (90X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the tumor cell antigen, which is at least 95-fold (95X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen.In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for a tumor cell antigen, which is at least 100-fold (100X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for a tumor cell antigen, which is at least 120-fold (120X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for a tumor cell antigen, which is at least 1000-fold (1000X) higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen.

[0040] In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay compared to the EC of the IFNγ release T cell activation assay of the polypeptide or polypeptide complex that does not have P1 or L1. 50 In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay. 50 In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay, which is at least 10-fold (10X) higher than the EC of the IFNγ release T cell activation assay in the form of the polypeptide or polypeptide complex that does not have P1 or L1. 50 In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay, which is at least 10-fold (10X) higher than the EC of the IFNγ release T cell activation assay in the form of the polypeptide or polypeptide complex that does not have P1 or L1. 50 In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay, which is at least 1×10-fold (10X) higher than the EC of the IFNγ release T cell activation assay in the form of the polypeptide or polypeptide complex that does not have P1 or L1. 50 In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay, which is at least 10-fold (10X) higher than the EC of the IFNγ release T cell activation assay in the form of the polypeptide or polypeptide complex that does not have P1 or L1. 50At least 20 times (20X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay 50 and it is the EC of the IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 At least 30 times (30X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay 50 and it is the EC of the IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 At least 40 times (40X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay 50 and it is the EC of the IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 At least 50 times (50X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay 50 and it is the EC of the IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 At least 60 times (60X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay 50 and it is the EC of the IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 At least 70 times (70X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of the IFNγ release T cell activation assay 50 and it is the EC of the IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex that does not have P1 or L150 In some embodiments, the polypeptide or polypeptide complex has an increased EC20 activity in an IFNγ release T cell activation assay that is at least 80 times (80X) higher than that of the control. 50 EC of IFNγ release T cell activation assay of polypeptides or polypeptide complexes having P1 or L1 or not. 50 In some embodiments, the polypeptide or polypeptide complex has an increased EC2 of an IFNγ release T cell activation assay that is at least 90 times (90X) higher than 50 EC of IFNγ release T cell activation assay of polypeptides or polypeptide complexes having P1 or L1 or not. 50 In some embodiments, the polypeptide or polypeptide complex has an increased EC value of 100× greater than ... 50 EC of IFNγ release T cell activation assay of polypeptides or polypeptide complexes having P1 or L1 or not. 50 At least 1000 times (1000X) higher than

[0041] In some forms, the polypeptide or polypeptide complex comprises a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease, the EC of the polypeptide or polypeptide complex in an IFNγ release T cell activation assay. 50 Compared to the IFNγ release T cell activation assay, the EC 50 In some embodiments, the polypeptide or polypeptide complex has an increased EC 50 and that the EC of the IFNγ-releasing T cell activation assay of a polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. 50 In some embodiments, the polypeptide or polypeptide complex has an increased EC value in an IFNγ release T cell activation assay that is at least 10-fold (10X) higher than 50and it is at least 20-fold (20X) higher than the EC of the IFNγ-releasing T cell activation assay of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 In some embodiments, the polypeptide or polypeptide complex has an even increased EC of the IFNγ-releasing T cell activation assay 50 and it is at least 30-fold (30X) higher than the EC of the IFNγ-releasing T cell activation assay of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 In some embodiments, the polypeptide or polypeptide complex has an even increased EC of the IFNγ-releasing T cell activation assay 50 and it is at least 40-fold (40X) higher than the EC of the IFNγ-releasing T cell activation assay of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 In some embodiments, the polypeptide or polypeptide complex has an even increased EC of the IFNγ-releasing T cell activation assay 50 and it is at least 50-fold (50X) higher than the EC of the IFNγ-releasing T cell activation assay of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 In some embodiments, the polypeptide or polypeptide complex has an even increased EC of the IFNγ-releasing T cell activation assay 50 and it is at least 60-fold (60X) higher than the EC of the IFNγ-releasing T cell activation assay of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 In some embodiments, the polypeptide or polypeptide complex has an even increased EC of the IFNγ-releasing T cell activation assay 50 and it is at least 20-fold (20X) higher than the EC of the IFNγ-releasing T cell activation assay of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50is at least 70-fold (70X) higher. In some embodiments, the polypeptide or polypeptide complex has an increased EC in an IFNγ release T cell activation assay 50 than the EC in an IFNγ release T cell activation assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 is at least 80-fold (80X) higher. In some embodiments, the polypeptide or polypeptide complex has an increased EC in an IFNγ release T cell activation assay 50 than the EC in an IFNγ release T cell activation assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 is at least 90-fold (90X) higher. In some embodiments, the polypeptide or polypeptide complex has an increased EC in an IFNγ release T cell activation assay 50 than the EC in an IFNγ release T cell activation assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 is at least 100-fold (100X) higher. In some embodiments, the polypeptide or polypeptide complex has an increased EC in an IFNγ release T cell activation assay 50 than the EC in an IFNγ release T cell activation assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 is at least 1000-fold (1000X) higher.

[0042] In some embodiments, the polypeptide or polypeptide complex has an increased EC in a T cell cytotoxicity assay than the EC in a T cell cytotoxicity assay of a polypeptide or polypeptide complex that does not have P1 or L1 50 and has an increased EC in a T cell cytotoxicity assay 50 In some embodiments, the polypeptide or polypeptide complex has an increased EC in a T cell cytotoxicity assay 50has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 is at least 10-fold (10X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytotoxicity assay 50 has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 is at least 20-fold (20X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytotoxicity assay 50 has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 is at least 30-fold (30X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytotoxicity assay 50 has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 is at least 40-fold (40X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytotoxicity assay 50 has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 is at least 50-fold (50X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytotoxicity assay 50 has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 is at least 60-fold (60X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytotoxicity assay 50and it has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 which is at least 70-fold (70X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex has an even increased EC of a T cell cytotoxicity assay 50 and it has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 which is at least 80-fold (80X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex has an even increased EC of a T cell cytotoxicity assay 50 and it has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 which is at least 90-fold (90X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex has an even increased EC of a T cell cytotoxicity assay 50 and it has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 which is at least 100-fold (100X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex has an even increased EC of a T cell cytotoxicity assay 50 and it has an EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex that does not have P1 or L1 50 which is at least 1000-fold (1000X) higher than 50 .

[0043] In some embodiments, the polypeptide or polypeptide complex has an even increased EC of a T cell cytotoxicity assay as compared to the EC of a T cell cytotoxicity assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 and has an even increased EC of a T cell cytotoxicity assay 50 In some embodiments, the polypeptide or polypeptide complex has an even increased EC of a T cell cytotoxicity assay 50having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 10-fold (10X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytolysis assay 50 having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 20-fold (20X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytolysis assay 50 having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 30-fold (30X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytolysis assay 50 having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 40-fold (40X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytolysis assay 50 having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 50-fold (50X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytolysis assay 50 having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 60-fold (60X) higher than. In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytolysis assay 50has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 70-fold (70X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytolysis assay 50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 80-fold (80X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytolysis assay 50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 90-fold (90X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytolysis assay 50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 100-fold (100X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex has an increased EC of a T cell cytolysis assay 50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease 50 that is at least 1000-fold (1000X) higher than 50 .

[0044] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens compared to the binding affinity of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 10-fold (10X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 50-fold (50X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 75-fold (75X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 100-fold (100X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 120-fold (120X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2.In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 200-fold (200X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 300-fold (300X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 400-fold (400X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 500-fold (500X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 600-fold (600X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2.In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 700-fold (700X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 800-fold (800X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 900-fold (900X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 1000-fold (1000X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 10,000-fold (10,000X) higher than the binding affinity for tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2.

[0045] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens compared to the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 10-fold (10X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 50-fold (50X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 75-fold (75X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 100-fold (100X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease for tumor cell antigens.In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 120-fold (120X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 200-fold (200X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 300-fold (300X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 400-fold (400X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 500-fold (500X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens.In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 600-fold (600X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 700-fold (700X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 800-fold (800X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 900-fold (900X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 1000-fold (1000X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens.In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity for tumor cell antigens, which is at least 10,000-fold (10,000X) higher than the binding affinity of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases for tumor cell antigens.

[0046] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an EC of the IFNγ release T cell activation assay of the polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 and has an increased EC of the IFNγ release T cell activation assay compared to 50 it. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of the IFNγ release T cell activation assay 50 and it is at least 10-fold (10X) higher than the EC of the IFNγ release T cell activation assay in the form of the polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. 50 In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of the IFNγ release T cell activation assay 50 and it is at least 50-fold (50X) higher than the EC of the IFNγ release T cell activation assay in the form of the polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. 50 In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of the IFNγ release T cell activation assay 50 and it is at least 100-fold (100X) higher than the EC of the IFNγ release T cell activation assay in the form of the polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. 50is at least 75 - fold (75X) higher. In some embodiments, the polypeptide or polypeptide complex P2 - L2 - A2 - A1 - L1 - P1 - H1 (Formula Ia) has a greater increased EC 50 in the IFNγ - releasing T - cell activation assay, which is the EC of the IFNγ - releasing T - cell activation assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 is at least 100 - fold (100X) higher. In some embodiments, the polypeptide or polypeptide complex P2 - L2 - A2 - A1 - L1 - P1 - H1 (Formula Ia) has a greater increased EC 50 in the IFNγ - releasing T - cell activation assay, which is the EC of the IFNγ - releasing T - cell activation assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 is at least 200 - fold (200X) higher. In some embodiments, the polypeptide or polypeptide complex P2 - L2 - A2 - A1 - L1 - P1 - H1 (Formula Ia) has a greater increased EC 50 in the IFNγ - releasing T - cell activation assay, which is the EC of the IFNγ - releasing T - cell activation assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 is at least 300 - fold (300X) higher. In some embodiments, the polypeptide or polypeptide complex P2 - L2 - A2 - A1 - L1 - P1 - H1 (Formula Ia) has a greater increased EC 50 in the IFNγ - releasing T - cell activation assay, which is the EC of the IFNγ - releasing T - cell activation assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 is at least 400 - fold (400X) higher. In some embodiments, the polypeptide or polypeptide complex P2 - L2 - A2 - A1 - L1 - P1 - H1 (Formula Ia) has a greater increased EC 50in the form of a polypeptide or polypeptide complex of formula Ia that has no P1, L1, P2, or L2, the EC of the IFNγ release T cell activation assay 50 is at least 500-fold (500X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an even more increased EC of the IFNγ release T cell activation assay 50 in the form of a polypeptide or polypeptide complex of formula Ia that has no P1, L1, P2, or L2, the EC of the IFNγ release T cell activation assay 50 is at least 600-fold (600X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an even more increased EC of the IFNγ release T cell activation assay 50 in the form of a polypeptide or polypeptide complex of formula Ia that has no P1, L1, P2, or L2, the EC of the IFNγ release T cell activation assay 50 is at least 700-fold (700X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an even more increased EC of the IFNγ release T cell activation assay 50 in the form of a polypeptide or polypeptide complex of formula Ia that has no P1, L1, P2, or L2, the EC of the IFNγ release T cell activation assay 50 is at least 800-fold (800X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an even more increased EC of the IFNγ release T cell activation assay 50 in the form of a polypeptide or polypeptide complex of formula Ia that has no P1, L1, P2, or L2, the EC of the IFNγ release T cell activation assay 50is at least 900-fold (900X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of the IFNγ release T cell activation assay 50 than the EC of the IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 is at least 1000-fold (1000X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of the IFNγ release T cell activation assay 50 than the EC of the IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 is at least 10,000-fold (10,000X) higher.

[0047] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of the T cell cytolysis assay of a polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 compared to the EC of the T cell cytolysis assay 50 In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of the T cell cytolysis assay 50 than the EC of the T cell cytolysis assay of a polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 is at least 10-fold (10X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of the T cell cytolysis assay 50and which has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 50-fold (50X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and which has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 75-fold (75X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and which has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 100-fold (100X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and which has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 200-fold (200X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and which has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 300-fold (300X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50and has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 which is at least 400-fold (400X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 which is at least 500-fold (500X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 which is at least 600-fold (600X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 which is at least 700-fold (700X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 which is at least 800-fold (800X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 900-fold (900X) higher than that of 50 . In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 1000-fold (1000X) higher than that of 50 . In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 10,000-fold (10,000X) higher than that of 50 .

[0048] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay compared to the EC of a T cell cytolysis assay of a polypeptide or polypeptide complex that does not have P1, L1, P2, or L2 50 and has an increased EC of a T cell cytolysis assay. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 has an EC that is a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia that does not have P1, L1, P2, or L2 50 and has an increased EC of a T cell cytolysis assay. 50At least 10 times (10X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of a T cell cytotoxicity assay 50 which is at least 50 times (50X) higher than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 At least 50 times (50X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of a T cell cytotoxicity assay 50 which is at least 75 times (75X) higher than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 At least 75 times (75X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of a T cell cytotoxicity assay 50 which is at least 100 times (100X) higher than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 At least 100 times (100X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of a T cell cytotoxicity assay 50 which is at least 200 times (200X) higher than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 At least 200 times (200X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC of a T cell cytotoxicity assay 50 which is at least 200 times (200X) higher than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50It is at least 300 times (300X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a greater increased EC in a T cell cytotoxicity assay 50 than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 It is at least 400 times (400X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a greater increased EC in a T cell cytotoxicity assay 50 than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 It is at least 500 times (500X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a greater increased EC in a T cell cytotoxicity assay 50 than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 It is at least 600 times (600X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a greater increased EC in a T cell cytotoxicity assay 50 than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 It is at least 700 times (700X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a greater increased EC in a T cell cytotoxicity assay 50 than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50is at least 800-fold (800X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC in a T cell cytotoxicity assay 50 than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 is at least 900-fold (900X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC in a T cell cytotoxicity assay 50 than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 is at least 1000-fold (1000X) higher. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC in a T cell cytotoxicity assay 50 than the EC of a T cell cytotoxicity assay in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2 50 [[ID=A]]is at least 10,000-fold (10,000X) higher.

[0049] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC in a T cell cytotoxicity assay of a polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 compared to an increased EC in a T cell cytotoxicity assay 50 In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC in a T cell cytotoxicity assay 50and it has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 which is at least 10-fold (10X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and it has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 which is at least 50-fold (50X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and it has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 which is at least 75-fold (75X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and it has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 which is at least 100-fold (100X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 and it has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 which is at least 200-fold (200X) higher than. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 300-fold (300X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 400-fold (400X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 500-fold (500X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 600-fold (600X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 having an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 700-fold (700X) higher than 50 . In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 800-fold (800X) higher than that. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 900-fold (900X) higher than that. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 1000-fold (1000X) higher than that. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (formula Ia) has an increased EC of a T cell cytolysis assay 50 has an EC of a T cell cytolysis assay of a polypeptide or polypeptide complex of formula Ia in which L1 and L2 are cleaved by a tumor-specific protease 50 that is at least 10,000-fold (10,000X) higher than that.

[0050] Antigen recognition molecule (A2) In some embodiments, A2 comprises an antibody or an antibody fragment. In some embodiments, the antibody or its antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab. In some embodiments, the antibody or its antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain of a single-domain antibody derived from camelids (VHH). In some embodiments, the antibody or its antibody fragment is humanized or human. In some embodiments, A2 is a Fab. In some embodiments, the Fab comprises (a) a Fab light-chain polypeptide and (b) a Fab heavy-chain polypeptide. In some embodiments, the antibody or its antibody fragment comprises an epidermal growth factor receptor (EGFR) binding domain. In some embodiments, the antibody or its antibody fragment comprises a mesothelin binding domain. In some embodiments, the antibody or its antibody fragment comprises a carcinoembryonic antigen-related cell adhesion molecule CEACAM5 binding domain. In some embodiments, the antibody or its antibody fragment comprises a carcinoembryonic antigen-related cell adhesion molecule HER2 binding domain. In some embodiments, the tumor cell antigen comprises EGFR, and the Fab light-chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 56 or SEQ ID NO: 57. In some embodiments, the tumor cell antigen comprises EGFR, and the Fab heavy-chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 59 or SEQ ID NO: 60. In some embodiments, the tumor cell antigen comprises HER2, and the Fab light-chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 61. In some embodiments, the tumor cell antigen comprises HER2, and the Fab heavy-chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 62 or SEQ ID NO: 63.

[0051] In some embodiments, the Fab light chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab light chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and the polypeptide complex comprises the amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 76. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and the polypeptide complex comprises the amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 78. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and the polypeptide complex comprises the amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 73. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and the polypeptide complex comprises the amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 74. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1. In some embodiments, A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 comprises a linker that connects A2 to P2 and is a substrate for a tumor-specific protease. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2.In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2, and the polypeptide complex comprises the amino acid sequences of SEQ ID NO: 70 and SEQ ID NO: 73. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2, and the polypeptide complex comprises the amino acid sequences of SEQ ID NO: 80 and SEQ ID NO: 81. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2.

[0052] In some embodiments, A2 comprises an anti-CD3e single-chain variable fragment. In some embodiments, A2 comprises a single-chain variable fragment of anti-CD3e that has a KD binding of 1 μM or less to CD3 on CD3-expressing cells. In some embodiments, A2 comprises a variable light chain and a variable heavy chain, each of which can specifically bind to human CD3. In some embodiments, A2 comprises a complementarity-determining region (CDR) selected from the group consisting of muromonab-CD3 (OKT3), otrexup (TRX4), teprotumumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19. In some embodiments, the polypeptide or polypeptide complex of Formula I binds to an effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, A2 binds to a polypeptide that is part of the TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3ε. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68.

[0053] Peptide (P1 and P2 and P 1a ) In some embodiments, P1 impairs the binding of A1 to the first target antigen. In some embodiments, P1 binds to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, and H-bonding interactions, or combinations thereof. In some embodiments, P1 binds to A1 at or near the antigen-binding site. In some embodiments, P1 dissociates from A1 when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to the first target antigen. In some embodiments, P1 has less than 70% sequence identity to the first target antigen. In some embodiments, P1 has less than 75% sequence identity to the first target antigen. In some embodiments, P1 has less than 80% sequence identity to the first target antigen. In some embodiments, P1 has less than 85% sequence identity to the first target antigen. In some embodiments, P1 has less than 90% sequence identity to the first target antigen. In some embodiments, P1 has less than 95% sequence identity to the first target antigen. In some embodiments, P1 has less than 98% sequence identity to the first target antigen. In some embodiments, P1 has less than 99% sequence identity to the first target antigen. In some embodiments, P1 comprises a de novo amino acid sequence that shares less than 10% sequence identity with the first target antigen.

[0054] In some embodiments, P2 impairs the binding of A2 to the second target antigen. In some embodiments, P2 binds to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, pi-stacking interactions, and H-bonding interactions, or combinations thereof. In some embodiments, P2 binds to A2 at or near the antigen-binding site. In some embodiments, P2 dissociates from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to the second target antigen. In some embodiments, P2 has less than 70% sequence identity to the second target antigen. In some embodiments, P2 has less than 75% sequence identity to the second target antigen. In some embodiments, P2 has less than 80% sequence identity to the second target antigen. In some embodiments, P2 has less than 85% sequence identity to the second target antigen. In some embodiments, P2 has less than 90% sequence identity to the second target antigen. In some embodiments, P2 has less than 95% sequence identity to the second target antigen. In some embodiments, P2 has less than 98% sequence identity to the second target antigen. In some embodiments, P2 has less than 99% sequence identity to the second target antigen. In some embodiments, P2 comprises a de novo amino acid sequence that shares less than 10% sequence identity with the second target antigen.

[0055] In some embodiments, P 1a impairs the binding of the antigen recognition molecule to the target antigen when L 1a is not cleaved. In some embodiments, the antigen recognition molecule comprises an antibody or an antibody fragment. In some embodiments, the target antigen is an anti-CD3 effector cell antigen. In some embodiments, the target antigen is a tumor cell antigen. In some embodiments, the tumor cell antigen is EGFR, HER2, mesothelin, or CEACAM5. In some embodiments, P 1a has less than 70% sequence identity to the target antigen. In some embodiments, P 1ahas less than 75% sequence identity to the target antigen. In some embodiments, P 1a has less than 80% sequence identity to the target antigen. In some embodiments, P 1a has less than 85% sequence identity to the target antigen. In some embodiments, P 1a has less than 90% sequence identity to the target antigen. In some embodiments, P 1a has less than 95% sequence identity to the target antigen. In some embodiments, P 1a has less than 98% sequence identity to the target antigen. In some embodiments, P 1a has less than 99% sequence identity to the target antigen. In some embodiments, P 1a contains a de novo amino acid sequence that shares less than 10% sequence identity to a second target antigen.

[0056] In some embodiments, P1, P2, or P 1a contains a peptide sequence that is at least 5 amino acids in length. In some embodiments, P1, P2, or P 1a contains a peptide sequence that is at least 6 amino acids in length. In some embodiments, P1, P2, or P 1a contains a peptide sequence that is at least 10 amino acids in length. In some embodiments, P1, P2, or P 1a contains a peptide sequence that is at least 10 amino acids in length and at most 20 amino acids in length. In some embodiments, P1, P2, or P 1a contains a peptide sequence that is at least 16 amino acids in length. In some embodiments, P1, P2, or P 1a contains a peptide sequence that is at most 40 amino acids in length. In some embodiments, P1, P2, or P 1a contains at least two cysteine - amino acid residues. In some embodiments, P1, P2, or P 1a contains a cyclic peptide or a linear peptide. In some embodiments, P1, P2, or P 1acontains a cyclic peptide. In some embodiments, P1, P2, or P 1a contains a linear peptide. In some embodiments, the tumor cell antigen comprises EGFR, and P1 or P2 comprises peptide-1, peptide-2, peptide-3, peptide-4, peptide-5, peptide-6, or peptide-7. In some embodiments, the tumor cell antigen comprises EGFR, and P1 or P2

[0057]

Chemical formula

[0058]

Chemical formula

[0059]

Chemical formula

[0060] In some embodiments, P1, P2, or P 1a , or P1, P2, and P 1aIt includes a modified amino acid, or a non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or modified non-natural amino acid includes a post-translational modification. In some embodiments, P1, P2, or P 1a , or P1, P2, and P 1a includes a modification, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moiety, covalent attachment of nucleotide or nucleotide derivative, covalent attachment of lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-linking, formation of cystine, formation of pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and other tRNA-mediated addition of amino acids to proteins including but not limited to ubiquitination. The modification is made at any location of P1, P2, or P 1a , or P1, P2, and P 1a , including the peptide backbone, amino acid side chains, and termini.

[0061] In some embodiments, P1, P2, or P 1a does not include albumin or albumin fragment. In some embodiments, P1, P2, or P 1a does not include an albumin binding domain.

[0062] Linker portions (L1, L2, L3, and L 3a ) In some embodiments, L1, L2, L3, or L 3a is a peptide sequence having at least 5 to a maximum of 50 amino acids. In some embodiments, L1, L2, L3, or L 3ais a peptide sequence having at least 10 and at most 30 amino acids. In some embodiments, L1, L2, L3, or L 3a is a peptide sequence having at least 10 amino acids. In some embodiments, L1, L2, L3, or L 3a is a peptide sequence having at least 18 amino acids. In some embodiments, L1, L2, L3, or L 3a is a peptide sequence having at least 26 amino acids. In some embodiments, L1, L2, L3, or L 3a has a formula including (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 29). In some embodiments, (G2S) n has a formula including wherein n is an integer of at least 1. In some embodiments, L1, L2, L3, or L 3a is L1, L2, L3, or L 3a has a formula including (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 30), (GGGS) n (SEQ ID NO: 31), (GGGGS) n (SEQ ID NO: 32), and (GSSGGS) n (SEQ ID NO: 33) and has a formula selected from the group consisting of wherein n is an integer of at least 1. In some embodiments, the tumor-specific protease is selected from the group consisting of metalloprotease, serine protease, cysteine protease, threonine protease, and aspartic protease. In some embodiments, L1, L2, L3, or L 3a includes a urokinase-cleavable amino acid sequence, a matriptase-cleavable amino acid sequence, a legumain-cleavable amino acid sequence, or a matrix metalloprotease-cleavable amino acid sequence.

[0063] In some embodiments, L1, L2, L3, or L 3ais Linker-1, Linker-2, Linker-3, Linker-4, Linker-5, Linker-6, Linker-7, Linker-8, Linker-9, Linker-10, Linker-11, Linker-12, Linker-13, Linker-14, Linker-15, Linker-16, Linker-17, Linker-18, Linker-19, or Linker 20. In some embodiments, L1 or L2 is

[0064] [Chemical Formula] comprises an amino acid sequence selected from the group consisting of. In some embodiments, L1 or L2 is an amino acid sequence,

[0065] [Chemical Formula] comprises. In some embodiments, L3 or L 3a comprises the amino acid sequence GGGGSGGGS (SEQ ID NO: 51).

[0066] In some embodiments, L1 is attached to the N-terminus of A1. In some embodiments, L1 is attached to the C-terminus of A1. In some embodiments, L2 is attached to the N-terminus of A2. In some embodiments, L2 is attached to the C-terminus of A2. In some embodiments, P1 becomes unbound from A1 when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to the first target antigen. In some embodiments, P2 becomes unbound from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to the second target antigen.

[0067] In some embodiments, L1, L2, L3, or L 3a , or, L1, L2, L3, and L 3aIt includes a modified amino acid, or a non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid includes a post-translational modification. In some embodiments, L1, L2, L3, or L 3a , or L1, L2, L3, and L 3a includes a modification, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moiety, covalent attachment of nucleotide or nucleotide derivative, covalent attachment of lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-linking, formation of cystine, formation of pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, etc., including but not limited to the tRNA-mediated addition of amino acids to proteins, and ubiquitination. The modification is made at any location of L1, L2, L3, or L 3a , or L1, L2, L3, and L 3a .

[0068] Half-life extension molecules (H1 and H 1a ) In some embodiments, H1 does not prevent A1 from binding to the first target antigen. In some embodiments, H1 includes a linking moiety (L3) that connects H1 to P1. In some embodiments, H 1a does not prevent the antigen recognition molecule from binding to the target antigen. In some embodiments, H 1a is H 1a connected to P 1a by a linking moiety (L3). In some embodiments, the half-life extension molecule (H1 or H 1a ) does not have a binding affinity for the antigen recognition molecule. In some embodiments, the half-life extension molecule (H1 or H1a ) does not have binding affinity for the target antigen. In some embodiments, the half-life extending molecule (H1 or H 1a ) does not shield the antigen recognition molecule from the target antigen. In some embodiments, the half-life extending molecule (H1 or H 1a ) is not directly linked to the antigen recognition molecule.

[0069] In some embodiments, H1 or H 1a comprises an amino acid sequence having a repetitive sequence motif. In some embodiments, H1 or H 1a comprises an amino acid sequence having a highly ordered secondary structure. As used in this context, "highly ordered secondary structure" means that at least about 50%, or about 70%, or about 80%, or about 90% of the amino acid residues of H1 or H 1a contribute to the secondary structure, and they are measured or determined by means including, but not limited to, spectrophotometric measurements (e.g., circular dichroism spectroscopy in the "far-ultraviolet" spectral region (190 - 250 nm)), and algorithms such as the Chou-Fasman algorithm and the Garnier-Osguthorpe-Robson ("GOR") algorithm.

[0070] In some embodiments, H1 or H 1a comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 or H 1a comprises albumin. In some embodiments, H1 or H 1a comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H1 or H 1aIt contains a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or a fragment thereof, a blood immunoglobulin or a fragment thereof, or CD35 / CR1. In some embodiments, the serum protein includes thyroxine-binding protein, transthyretin, alpha1-glycoprotein, transferrin, transferrin receptor or its transferrin-binding portion, fibrinogen, or albumin. In some embodiments, the blood immunoglobulin molecule includes IgG1, IgG2, IgG3, IgG4, sIgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody includes a single-domain antibody, a single-chain variable fragment, or a Fab. In some embodiments, the single-domain antibody includes a single-domain antibody that binds to albumin. In some embodiments, the antibody is a human antibody or a humanized antibody. In some embodiments, the antibody is selected from the group consisting of 645gH1gL1, 645dsgH5gL4, 23-13-A01-sc02, A10m3 or a fragment thereof, DOM7r-31, DOM7h-11-15, Alb-1, Alb-8, Alb-23, 10G, 10GE, and SA21. In some embodiments, the single-domain antibody is 10G, and the single-domain antibody has an amino acid sequence,

[0071]

Chemical formula

[0072] In some embodiments, H1 or H 1a or, H1 and H 1a includes a modified amino acid, or a non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid includes a post-translational modification. In some embodiments, H1 or H 1aAlternatively, H1 and H 1a include modifications such as acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moiety, covalent attachment of nucleotide or nucleotide derivative, covalent attachment of lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-linking, formation of cystine, formation of pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids such as arginylation to proteins, and ubiquitination, but not limited thereto. The modifications are made at any location of the peptide backbone, amino acid side chain, and termini of H1 or H 1a Alternatively, H1 and H 1a can be made at any location of.

[0073] In some embodiments, H1 includes a linking moiety (L3) that connects H1 to P1. In some embodiments, L3 is a peptide sequence having at least 5 to a maximum of 50 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to a maximum of 30 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 is (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 30), (GGGS) n (SEQ ID NO: 31), (GGGGS) n (SEQ ID NO: 32), and (GSSGGS) nIt has a formula selected from the group consisting of (SEQ ID NO: 33), wherein n is an integer of at least 1. In some embodiments, L3 includes the amino acid sequence, GGGGSGGGS (SEQ ID NO: 51).

[0074] In some embodiments, H 1a is H 1a connected to P 1a by a linking moiety (L 3a ). In some embodiments, L 3a is a peptide sequence having at least 5 to a maximum of 50 amino acids. In some embodiments, L 3a is a peptide sequence having at least 10 to a maximum of 30 amino acids. In some embodiments, L 3a is a peptide sequence having at least 10 amino acids. In some embodiments, L 3a is a peptide sequence having at least 18 amino acids. In some embodiments, L 3a is a peptide sequence having at least 26 amino acids. In some embodiments, L 3a has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 30), (GGGS) n (SEQ ID NO: 31), (GGGGS) n (SEQ ID NO: 32), and (GSSGGS) n (SEQ ID NO: 33), wherein n is an integer of at least 1. In some embodiments, L 3a includes the amino acid sequence, GGGGSGGGS (SEQ ID NO: 51).

[0075] Disclosed herein are, in some embodiments, a polypeptide or a polypeptide complex, which polypeptide or polypeptide complex includes a structural arrangement according to Configuration 1,

[0076]

Chemical Formula

[0077] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 2:

[0078]

Chemical formula

[0079] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 3:

[0080]

Chemical Formula

[0081] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 4:

[0082]

Chemical Formula

[0083] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 5:

[0084] [Chemical formula] Here, the polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, where the scFv is linked to a peptide (P1) that impairs the binding of the scFv to the effector cell antigen, and P1 is linked to the N-terminus of the heavy-chain variable domain of the scFv by a linker portion (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extending molecule, and comprises a Fab that binds to a tumor cell antigen, where the Fab comprises a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, where the Fab heavy-chain polypeptide is linked to the C-terminus of the light-chain variable domain of the scFv, and where the Fab is linked to P2 and L2, where P2 comprises a peptide that impairs the binding to the tumor cell antigen, and L2 comprises a linker portion that connects the Fab light-chain polypeptide to P2 and is a substrate for a tumor-specific protease.

[0085] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex, the polypeptide or polypeptide complex including a structural arrangement according to Configuration 6:

[0086]

Chem.

[0087] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex, the polypeptide or polypeptide complex including a structural arrangement according to Configuration 7:

[0088]

Chem.

[0089] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 8:

[0090]

Chemical formula

[0091] Disclosed herein are, in some embodiments, polypeptides or polypeptide complexes, which are polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 9:

[0092]

Chemical formula

[0093] Disclosed herein are, in some embodiments, polypeptides or polypeptide complexes, which are polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 10:

[0094]

Chemical formula

[0095] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 11:

[0096] [Chemical formula] Here, the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (P1) that impairs the binding of the Fab to the tumor cell antigen, and P1 is linked to the N-terminus of the Fab heavy chain polypeptide by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extending molecule and comprises a single-chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to the N-terminus of the Fab light chain polypeptide, wherein the scFv is further linked to P2 and L2, wherein P2 comprises a peptide that impairs the binding of the scFv to the effector cell antigen, and L2 connects the light chain variable domain of the scFv to P2 and comprises a linking moiety that is a substrate for a tumor-specific protease.

[0097] Disclosed herein are, in some embodiments, a polypeptide or a polypeptide complex, the polypeptide or polypeptide complex including a structural arrangement according to Configuration 12:

[0098] [Chemical formula] Here, the polypeptide or polypeptide complex includes a Fab that binds to a tumor cell antigen, the Fab including a Fab light chain polypeptide and a Fab heavy chain polypeptide, where the Fab is linked to a peptide that impairs the binding of the Fab to the tumor cell antigen, and the peptide is linked to the N-terminus of the Fab heavy chain polypeptide by a linker that is a substrate for a tumor-specific protease, and the peptide is further linked to a half-life extending molecule and includes a single-chain variable fragment (scFv) that binds to an effector cell antigen, the scFv including a light chain variable domain and a heavy chain variable domain, where the heavy chain variable domain of the scFv is linked to the N-terminus of the Fab light chain polypeptide.

[0099] Disclosed herein are, in some embodiments, a polypeptide or a polypeptide complex, the polypeptide or polypeptide complex including a structural arrangement according to Configuration 13:

[0100] [Chemical formula] Here, the polypeptide or polypeptide complex comprises a Fab that binds to a tumor cell antigen, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, where the Fab is linked to a peptide (P1) that impairs the binding of the Fab to the tumor cell antigen, and P1 is linked to the N-terminus of the Fab light chain polypeptide by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extending molecule and comprises a single-chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, where the light chain variable domain of the scFv is linked to the N-terminus of the Fab heavy chain polypeptide, where the scFv is linked to P2 and L2, where P2 comprises a peptide that impairs the binding of the scFv to the effector cell antigen, and L2 connects the heavy chain variable domain of the scFv to P2 and comprises a linking moiety that is a substrate for a tumor-specific protease.

[0101] Disclosed herein are, in some embodiments, a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 14:

[0102]

Chemical Structure

[0103] Disclosed herein are, in some embodiments, polypeptides or polypeptide complexes, the polypeptides or polypeptide complexes being polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 15:

[0104]

Chemical formula

[0105] Disclosed herein are, in some embodiments, polypeptides or polypeptide complexes, the polypeptides or polypeptide complexes being polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 16:

[0106]

Chemical formula

[0107] A polynucleotide encoding a polypeptide or polypeptide complex Disclosed herein are, in some embodiments, isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex as disclosed herein. In some embodiments, the polypeptide or polypeptide complex comprises an antibody or antibody fragment. In some embodiments, the polypeptide or polypeptide complex comprises a Fab and a single-chain variable fragment (scFv).

[0108] Disclosed herein are, in some embodiments, isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex according to Formula I. A2 - A1 - L1 - P1 - H1 (Formula I) Wherein A1 comprises a first antigen recognition molecule that binds to a first target antigen, P1 comprises a peptide that binds to A1, L1 comprises a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extending molecule, and A2 comprises a second antigen recognition molecule that binds to a second target antigen.

[0109] Disclosed herein are, in some embodiments, isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex according to Formula I. A2 - A1 - L1 - P1 - H1 (Formula I) In the formula, A1 is a first antigen - recognizing molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor - specific protease, H1 contains a half - life extending molecule, and A2 is a second antigen - recognizing molecule that binds to a second target antigen.

[0110] Disclosed herein are, in some embodiments, isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex comprising Formula I. A2 - A1 - L1 - P1 - H1 (Formula I) In the formula, A1 comprises a first antigen - recognizing molecule that binds to a first target antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor - specific protease, H1 contains a half - life extending molecule, and A2 contains a second antigen - recognizing molecule that binds to a second target antigen.

[0111] Disclosed herein are, in some embodiments, isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex comprising Formula I. A2 - A1 - L1 - P1 - H1 (Formula I) In the formula, A1 is a first antigen - recognizing molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor - specific protease, H1 is a s a half - life extending molecule, and A2 is a second antigen - recognizing molecule that binds to a second target antigen.

[0112] Disclosed herein are, in some embodiments, isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex according to Formula Ia. P2-L2-A2-A1-L1-P1-H1 (Formula Ia) In the formula, A1 contains a first antigen recognition molecule that binds to a first target antigen, P1 contains a peptide that binds to A1, L1 contains a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 contains a half-life extending molecule, A2 contains a second antigen recognition molecule that binds to a second target antigen, P2 contains a peptide that binds to A2, and L2 contains a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0113] Disclosed herein, in some embodiments, is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex according to Formula Ia. P2-L2-A2-A1-L1-P1-H1 (Formula Ia) In the formula, A1 is a first antigen recognition molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, A2 is a second antigen recognition molecule that binds to a second target antigen, P2 is a peptide that binds to A2, and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0114] Disclosed herein, in some embodiments, is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising Formula Ia. P2-L2-A2-A1-L1-P1-H1 (Formula Ia) In the formula, A1 contains a first antigen recognition molecule that binds to a first target antigen, P1 contains a peptide that binds to A1, L1 contains a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 contains a half-life extending molecule, A2 contains a second antigen recognition molecule that binds to a second target antigen, P2 contains a peptide that binds to A2, and L2 contains a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0115] Disclosed herein are, in some embodiments, isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex comprising Formula Ia. P2-L2-A2-A1-L1-P1-H1 (Formula Ia) In the formula, A1 is a first antigen recognition molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, A2 is a second antigen recognition molecule that binds to a second target antigen, P2 is a peptide that binds to A2, and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0116] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 1:

[0117] [Chemical formula] Here, the polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, where the scFv is linked to a peptide (P1) that impairs the binding of the scFv to an effector cell antigen, and where P1 is linked to the N-terminus of the light-chain variable domain of the scFv by a linker portion (L1) that is a substrate for a tumor-specific protease, and where P1 is further linked to a half-life extending molecule, and comprises a Fab that binds to a tumor cell antigen, where the Fab comprises a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, where the Fab heavy-chain polypeptide is linked to the C-terminus of the heavy-chain variable domain of the scFv, and where, here, the Fab is linked to P2 and L2, where P2 comprises a peptide that impairs the binding of the Fab to a tumor cell antigen, and where L2 connects the Fab light-chain polypeptide to P2 and comprises a linker portion that is a substrate for a tumor-specific protease.

[0118] Disclosed herein, in some embodiments, is an isolated nucleic acid molecule encoding a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 2:

[0119]

Chemical formula

[0120] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 3:

[0121]

Chem.

[0122] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 4:

[0123]

Chem.

[0124] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 5:

[0125] [Chemical formula] Here, the polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, wherein the scFv is linked to a peptide (P1) that impairs the binding of the scFv to an effector cell antigen, and P1 is linked to the N-terminus of the heavy-chain variable domain of the scFv by a linker (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extending molecule, and comprises a Fab that binds to a tumor cell antigen, wherein the Fab comprises a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, wherein the Fab heavy-chain polypeptide is linked to the C-terminus of the light-chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs the binding of the Fab to a tumor cell antigen, and L2 comprises a linker that connects the Fab light-chain polypeptide to P2 and is a substrate for a tumor-specific protease.

[0126] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 6:

[0127]

Chemical formula

[0128] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 7:

[0129]

Chemical formula

[0130] Disclosed herein, in some embodiments, is an isolated nucleic acid molecule encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 8:

[0131]

Chemical formula

[0132] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 9:

[0133]

Chemical formula

[0134] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 10:

[0135]

Chemical formula

[0136] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 11:

[0137]

Chemical formula

[0138] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 12:

[0139]

Chemical formula

[0140] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 13:

[0141]

Chemical formula

[0142] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 14:

[0143]

Chemical formula

[0144] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 15:

[0145]

Chemical formula

[0146] Disclosed herein are, in some embodiments, isolated nucleic acid molecules encoding a polypeptide or polypeptide complex, wherein the polypeptide or polypeptide complex comprises a structural arrangement according to Configuration 16:

[0147]

Chemical formula

[0148] <Pharmaceutical composition> Disclosed herein are, in some embodiments, a pharmaceutical composition comprising (a) a polypeptide or polypeptide complex as disclosed herein and (b) a pharmaceutically acceptable excipient.

[0149] In some embodiments, the pharmaceutical composition is a (a) polypeptide or polypeptide complex according to formula I: A2-A1-L1-P1-H1 (Formula I) wherein A1 comprises a first antigen recognition molecule that binds to a first target antigen, P1 comprises a peptide that binds to A1, L1 comprises a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extending molecule, and A2 comprises a second antigen recognition molecule that binds to a second target antigen, and comprises (a) the polypeptide or polypeptide complex and (b) a pharmaceutically acceptable excipient.

[0150] In some embodiments, the pharmaceutical composition is a (a) polypeptide or polypeptide complex according to formula I: A2-A1-L1-P1-H1 (Formula I) wherein A1 is a first antigen recognition molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen recognition molecule that binds to a second target antigen, comprising: (a) a polypeptide or polypeptide complex; and (b) a pharmaceutically acceptable excipient.

[0151] In some embodiments, the pharmaceutical composition comprises a (a) polypeptide or polypeptide complex comprising Formula I: A2 - A1 - L1 - P1 - H1 (Formula I) wherein A1 comprises a first antigen recognition molecule that binds to a first target antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extending molecule, and A2 comprises a second antigen recognition molecule that binds to a second target antigen, comprising: (a) a polypeptide or polypeptide complex; and (b) a pharmaceutically acceptable excipient.

[0152] In some embodiments, the pharmaceutical composition comprises a (a) polypeptide or polypeptide complex comprising Formula I: A2 - A1 - L1 - P1 - H1 (Formula I) wherein A1 is a first antigen recognition molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen recognition molecule that binds to a second target antigen, comprising: (a) a polypeptide or polypeptide complex; and (b) a pharmaceutically acceptable excipient.

[0153] In some embodiments, the pharmaceutical composition comprises a (a) polypeptide or polypeptide complex according to Formula Ia: P2 - L2 - A2 - A1 - L1 - P1 - H1 (Formula Ia) wherein A1 comprises a first antigen recognition molecule that binds to a first target antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extending molecule, A2 comprises a second antigen recognition molecule that binds to a second target antigen, P2 comprises a peptide that binds to A2, and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease, and comprising (a) a polypeptide or polypeptide complex and (b) a pharmaceutically acceptable excipient.

[0154] In some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex according to Formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) wherein A1 is a first antigen recognition molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, A2 is a second antigen recognition molecule that binds to a second target antigen, P2 is a peptide that binds to A2, and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease, and comprising (a) a polypeptide or polypeptide complex and (b) a pharmaceutically acceptable excipient.

[0155] In some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising Formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) wherein A1 comprises a first antigen recognition molecule that binds to a first target antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extending molecule, A2 comprises a second antigen recognition molecule that binds to a second target antigen, P2 comprises a peptide that binds to A2, and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease, and (a) a polypeptide or polypeptide complex and (b) a pharmaceutically acceptable excipient are included.

[0156] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising Formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) wherein A1 is a first antigen recognition molecule that binds to a first target antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, A2 is a second antigen recognition molecule that binds to a second target antigen, P2 is a peptide that binds to A2, and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease, and (a) a polypeptide or polypeptide complex and (b) a pharmaceutically acceptable excipient are included.

[0157] Disclosed herein is that in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 1,

[0158]

Chemical formula

[0159] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising the structural arrangement according to Configuration 2,

[0160]

Chemical formula

[0161] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 3,

[0162]

Chemical formula

[0163] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 4

[0164]

Chemical formula

[0165] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising the structural arrangement according to Configuration 5,

[0166]

Chemical formula

[0167] Disclosed herein is, in some embodiments, a pharmaceutical composition that is (a) a polypeptide or polypeptide complex comprising the structural arrangement according to Configuration 6

[0168]

Chemical formula

[0169] Disclosed herein is, in some embodiments, a pharmaceutical composition that is (a) a polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 7

[0170] [Chemical formula] Here, the polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, where the scFv is linked to a peptide (P1) that impairs the binding of the scFv to the effector cell antigen, and P1 is linked to the N-terminus of the heavy-chain variable domain of the scFv by a linker moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extending molecule and comprises a Fab that binds to a tumor cell antigen, where the Fab comprises a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, where the Fab light-chain polypeptide is linked to the C-terminus of the light-chain variable domain of the scFv, and where the Fab is linked to P2 and L2, where P2 comprises a peptide that impairs the binding to the tumor cell antigen, and L2 connects the Fab heavy-chain polypeptide to P2 and comprises a linker moiety that is a substrate for a tumor-specific protease, (a) a polypeptide or polypeptide complex and (b) a pharmaceutically acceptable excipient.

[0171] Disclosed herein is, in some embodiments, a pharmaceutical composition that is (a) a polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 8

[0172]

Chemical formula

[0173] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising the structural arrangement according to Configuration 9,

[0174]

Chemical formula

[0175] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising the structural arrangement according to Configuration 10,

[0176]

Chemical formula

[0177] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising the structural arrangement according to Configuration 11,

[0178]

Chemical formula

[0179] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising the structural arrangement according to Configuration 12

[0180]

Chemical formula

[0181] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising the structural arrangement according to Configuration 13,

[0182]

Chemical formula

[0183] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising the structural arrangement according to Configuration 14,

[0184]

Chemical formula

[0185] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising the structural arrangement according to Configuration 15,

[0186]

Chemical formula

[0187] Disclosed herein, in some embodiments, the pharmaceutical composition is (a) a polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 16

[0188]

Chemical formula

[0189] In some embodiments, the polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety. In some embodiments, the detectable label comprises a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.

[0190] For administration to a subject, the polypeptide or polypeptide complex as disclosed herein may be provided in a pharmaceutical composition with one or more pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers can be formulated by conventional methods and administered to a subject in appropriate dosages. Preferably, the composition is sterile. These compositions may also contain adjuvants such as preservatives, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents.

[0191] The pharmaceutical composition may be in any suitable form (depending on the desired method of administration). It may be provided in unit dosage form, in a sealed container, or as part of a kit. Such a kit may include instructions for use. It may include a plurality of the aforementioned unit dosage forms.

[0192] The pharmaceutical composition may be adapted for administration by any suitable route, including parenteral (e.g., subcutaneous, intramuscular, or intravenous) routes. Such compositions may be prepared by any method known in the art of pharmacy, such as, for example, by mixing the active ingredient with a carrier or excipient under aseptic conditions.

[0193] The dosage of the substances of the present disclosure may vary within a wide range depending on the disease or disorder to be treated, the age and condition of the individual to be treated, etc., and the physician will ultimately determine the appropriate dosage to be used.

[0194] Table 1 provides the amino acid sequences of the constructs described herein.

[0195] [Table 1-1]

[0196] [Table 1-2]

[0197] [Table 1-3]

[0198] [Table 1-4]

[0199] [Table 1-5]

[0200]

Table 1-6

[0201]

Table 1-7

[0202]

Table 1-8

[0203]

Table 1-9

[0204]

Table 1-10

[0205]

Table 1-11

[0206] The polypeptide or polypeptide complex, in some embodiments, comprises the sequences defined in Table 1. In some embodiments, the sequence has at least, or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81. Optionally, the sequence has at least, or about 95% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81. Optionally, the sequence has at least, or about 97% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81.In some cases, the sequence contains at least, or about, 99% identity to sequence numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81. In some cases, the sequence contains at least, or about, 100% identity to sequence numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81. In some cases, the sequence contains at least, or about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400 or more amino acids of at least, or about, sequence numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or 81.

[0207] The term "sequence identity" means that two polynucleotide sequences are identical over a comparison window, i.e., on an individual nucleotide basis. The term "percent sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions at which the identical nucleic acid bases (e.g., A, T, C, G, U, or I) occur in both sequences, calculating the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to calculate the percent sequence identity. In general, techniques for determining sequence identity include comparing two nucleotide or amino acid sequences and determining their percent identity. Sequence comparisons for purposes such as evaluating identity may be performed by any suitable alignment algorithm including, but not limited to, the Needleman-Wunsch algorithm (e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / , optionally with default settings), the BLAST algorithm (e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi, optionally with default settings), and the Smith-Waterman algorithm (e.g., the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / , optionally with default settings). The optimal alignment may be evaluated using any appropriate parameters of the selected algorithm, including default parameters. "Percent identity," also referred to as "percent homology" between two sequences, may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using an advanced BLAST computer program, including version 2.2.9 available from the National Institutes of Health.The BLAST program is based on the sequence methods as discussed in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), and Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine the percent identity over the full length of the sequences being compared. Default parameters are provided, for example, by the blastp program to optimize searches using short query sequences. The program may also permit the use of a SEG filter for masked-off segments of the query sequence as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). High sequence identity generally includes the range of sequence identity from approximately 80% to 100%, and integer values therebetween.

[0208] Embodiment 1 includes a polypeptide or polypeptide complex according to Formula I, A2 - A1 - L1 - P1 - H1 (Formula I) wherein A1 includes a first antigen recognition molecule that binds to a first target antigen, P1 includes a peptide that binds to A1, L1 includes a linking moiety that connects A1 to P1 and is a substrate for a tumor - specific protease, H1 includes a half - life extending molecule, and A2 includes a second antigen recognition molecule that binds to a second target antigen.

[0209] Embodiment 2 includes the polypeptide or polypeptide complex of Embodiment 1, where the first target antigen includes an effector cell antigen, and the second target antigen includes a tumor cell antigen.

[0210] Embodiment 3 includes the polypeptide or polypeptide complex of any one of Embodiments 1 to 2, where the effector cell antigen includes CD3.

[0211] Embodiment 4 includes the polypeptide or polypeptide complex of any one of Embodiments 1 to 3, where the tumor cell antigen includes EGFR, HER2, mesothelin, or CEACAM5.

[0212] Embodiment 5 includes the polypeptide or polypeptide complex of any one of Embodiments 1 to 4, where A1 includes an antibody or antibody fragment.

[0213] Embodiment 6 includes the polypeptide or polypeptide complex of any one of Embodiments 1 to 5, where A1 includes an antibody or antibody fragment that is human or humanized.

[0214] Embodiment 7 includes the polypeptide or polypeptide complex of any one of Embodiments 1 to 6, where L1 is bound to the N-terminus of an antibody or antibody fragment.

[0215] Embodiment 8 includes the polypeptide or polypeptide complex of any one of Embodiments 1 to 7, where A2 is bound to the C-terminus of an antibody or antibody fragment.

[0216] Embodiment 9 includes the polypeptide or polypeptide complex of any one of Embodiments 1 to 8, where L1 is bound to the C-terminus of an antibody or antibody fragment.

[0217] Embodiment 10 includes the polypeptide or polypeptide complex of any one of Embodiments 1 to 9, where A2 is bound to the N-terminus of an antibody or antibody fragment.

[0218] Embodiment 11 includes any one of the polypeptides or polypeptide complexes of Embodiments 1 to 10, wherein the antibody or antibody fragment includes a single-chain variable fragment, a single-domain antibody, or a Fab fragment.

[0219] Embodiment 12 includes any one of the polypeptides or polypeptide complexes of Embodiments 1 to 11, wherein A1 is a single-chain variable fragment (scFv).

[0220] Embodiment 13 includes any one of the polypeptides or polypeptide complexes of Embodiments 1 to 12, wherein the scFv includes an scFv heavy-chain polypeptide and an scFv light-chain polypeptide.

[0221] Embodiment 14 includes any one of the polypeptides or polypeptide complexes of Embodiments 1 to 13, wherein A1 is a single-domain antibody.

[0222] Embodiment 15 includes any one of the polypeptides or polypeptide complexes of Embodiments 1 to 14, wherein A1 is a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain of a single-domain antibody derived from camelids (VHH).

[0223] Embodiment 16 includes any one of the polypeptides or polypeptide complexes of Embodiments 1 to 15, wherein A1 includes a single-chain variable fragment against CD3e.

[0224] Embodiment 17 includes any one of the polypeptides or polypeptide complexes of Embodiments 1 to 16, wherein A1 has a K of 1 μM or less for CD3 on CD3-expressing cells D and includes a single-chain variable fragment against CD3e.

[0225] Embodiment 18 includes any one polypeptide or polypeptide complex of Embodiments 1 to 17, wherein A1 includes a variable light chain and a variable heavy chain, each of which can specifically bind to human CD3.

[0226] Embodiment 19 includes any one polypeptide or polypeptide complex of Embodiments 1 to 18, wherein A1 includes a complementarity-determining region (CDR) selected from the group consisting of muromonab-CD3 (OKT3), otrexup (TRX4), teprotumumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19.

[0227] Embodiment 20 includes any one polypeptide or polypeptide complex of Embodiments 1 to 19, wherein when L1 is cleaved by a tumor-specific protease, the polypeptide or polypeptide complex of Formula I binds to effector cells.

[0228] Embodiment 21 includes any one polypeptide or polypeptide complex of Embodiments 1 to 20, wherein when L1 is cleaved by a tumor-specific protease and A1 binds to effector cells, the polypeptide or polypeptide complex of Formula I binds to effector cells.

[0229] Embodiment 22 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 21, wherein the effector cell is a T cell.

[0230] Embodiment 23 includes any one polypeptide or polypeptide complex of Embodiments 1 to 22, wherein A1 binds to a polypeptide that is part of the TCR-CD3 complex on effector cells.

[0231] Embodiment 24 includes any one polypeptide or polypeptide complex of Embodiments 1 to 23, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3ε.

[0232] Embodiment 25 includes any one polypeptide or polypeptide complex of Embodiments 1 to 24, wherein the effector cell antigen includes CD3, and the scFv includes the amino acid sequence according to SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68.

[0233] Embodiment 26 includes any one polypeptide or polypeptide complex of Embodiments 1 to 25, wherein A2 includes an antibody or an antibody fragment.

[0234] Embodiment 27 includes any one polypeptide or polypeptide complex of Embodiments 1 to 26, wherein the antibody or its antibody fragment includes a single-chain variable fragment, a single-domain antibody, or a Fab.

[0235] Embodiment 28 includes any one polypeptide or polypeptide complex of Embodiments 1 to 27, wherein the antibody or its antibody fragment includes a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain of a single-domain antibody derived from camelids (VHH).

[0236] Embodiment 29 includes any one polypeptide or polypeptide complex of Embodiments 1 to 28, wherein the antibody or its antibody fragment is humanized or human.

[0237] Embodiment 30 includes any one polypeptide or polypeptide complex of Embodiments 1 to 29, wherein A2 is Fab.

[0238] Embodiment 31 includes any one polypeptide or polypeptide complex of Embodiments 1 to 30, wherein the Fab includes (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide.

[0239] Embodiment 32 includes any one polypeptide or polypeptide complex of Embodiments 1 to 31, wherein the antibody or antibody fragment thereof includes an epidermal growth factor receptor (EGFR) binding domain.

[0240] Embodiment 33 includes any one polypeptide or polypeptide complex of Embodiments 1 to 32, wherein the antibody or antibody fragment thereof includes a mesothelin binding domain.

[0241] Embodiment 34 includes any one polypeptide or polypeptide complex of Embodiments 1 to 33, wherein the antibody or antibody fragment thereof includes a carcinoembryonic antigen-related cell adhesion molecule CEACAM5 binding domain.

[0242] Embodiment 35 includes any one polypeptide or polypeptide complex of Embodiments 1 to 34, wherein the tumor cell antigen includes EGFR, and the Fab light chain polypeptide includes the amino acid sequence according to SEQ ID NO: 56 or SEQ ID NO: 57.

[0243] Embodiment 36 includes any one polypeptide or polypeptide complex of Embodiments 1 to 35, wherein the tumor cell antigen includes EGFR, and the Fab heavy chain polypeptide includes the amino acid sequence according to SEQ ID NO: 59 or SEQ ID NO: 60.

[0244] Embodiment 37 includes any one polypeptide or polypeptide complex of Embodiments 1 to 36, wherein the tumor cell antigen includes HER2, and the Fab light chain polypeptide includes the amino acid sequence according to SEQ ID NO: 61.

[0245] Embodiment 38 includes any one polypeptide or polypeptide complex of Embodiments 1-37, wherein the tumor cell antigen includes HER2 and the Fab heavy chain polypeptide includes the amino acid sequence according to SEQ ID NO: 62 or SEQ ID NO: 63.

[0246] Embodiment 39 includes any one polypeptide or polypeptide complex of Embodiments 1-38, wherein the Fab light chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1.

[0247] Embodiment 40 includes any one polypeptide or polypeptide complex of Embodiments 1-39, wherein the Fab heavy chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1.

[0248] Embodiment 41 includes any one polypeptide or polypeptide complex of Embodiments 1-40, wherein the Fab light chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1.

[0249] Embodiment 42 includes any one polypeptide or polypeptide complex of Embodiments 1-41, wherein the Fab heavy chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1.

[0250] Embodiment 43 includes any one polypeptide or polypeptide complex of Embodiments 1-42, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1.

[0251] Embodiment 44 includes any one polypeptide or polypeptide complex of Embodiments 1-43, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and the polypeptide complex includes the amino acid sequences according to SEQ ID NO: 57 and SEQ ID NO: 76.

[0252] Embodiment 45 includes any one polypeptide or polypeptide complex of Embodiments 1 to 44, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and the polypeptide complex includes the amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 78.

[0253] Embodiment 46 includes any one polypeptide or polypeptide complex of Embodiments 1 to 45, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and the polypeptide complex includes the amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 73.

[0254] Embodiment 47 includes any one polypeptide or polypeptide complex of Embodiments 1 to 46, wherein the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1.

[0255] Embodiment 48 includes any one polypeptide or polypeptide complex of Embodiments 1 to 47, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1.

[0256] Embodiment 49 includes any one polypeptide or polypeptide complex of Embodiments 1 to 48, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and the polypeptide complex includes the amino acid sequences of SEQ ID NO: 57 and SEQ ID NO: 74.

[0257] Embodiment 50 includes any one polypeptide or polypeptide complex of Embodiments 1 to 49, wherein the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1.

[0258] Embodiment 51 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 50, wherein A2 further includes P2 and L2, where P2 includes a peptide that binds to A2, and L2 includes a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0259] Embodiment 52 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 51, wherein the polypeptide or polypeptide complex is of formula Ia. P2-L2-A2-A1-L1-P1-H1 (Formula Ia)

[0260] Embodiment 53 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 52, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2.

[0261] Embodiment 54 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 53, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2, and the polypeptide complex includes the amino acid sequences of SEQ ID NO: 70 and SEQ ID NO: 73.

[0262] Embodiment 55 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2, and the polypeptide complex includes the amino acid sequences of SEQ ID NO: 80 and SEQ ID NO: 81.

[0263] Embodiment 56 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 55, wherein the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2.

[0264] Embodiment 57 includes any one polypeptide or polypeptide complex of Embodiments 1 to 56, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2.

[0265] Embodiment 58 includes any one polypeptide or polypeptide complex of Embodiments 1 to 57, wherein the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2.

[0266] Embodiment 59 includes any one polypeptide or polypeptide complex of Embodiments 1 to 58, wherein the first target antigen includes a tumor cell antigen, and the second target antigen includes an effector cell antigen.

[0267] Embodiment 60 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 59, wherein the tumor cell antigen includes EGFR, HER2, mesothelin, or CEACAM5.

[0268] Embodiment 61 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 60, wherein the effector cell antigen includes CD3.

[0269] Embodiment 62 includes any one polypeptide or polypeptide complex of Embodiments 1 to 61, wherein A1 includes an antibody or an antibody fragment.

[0270] Embodiment 63 includes any one polypeptide or polypeptide complex of Embodiments 1 to 62, wherein A1 includes an antibody or an antibody fragment that is human or humanized.

[0271] Embodiment 64 includes any one polypeptide or polypeptide complex of Embodiments 1 to 63, wherein L1 is bound to the N-terminus of an antibody or antibody fragment.

[0272] Embodiment 65 includes any one polypeptide or polypeptide complex of Embodiments 1 to 64, wherein A2 is bound to the C-terminus of an antibody or antibody fragment.

[0273] Embodiment 66 includes any one polypeptide or polypeptide complex of Embodiments 1 to 65, wherein L1 is bound to the C-terminus of an antibody or antibody fragment.

[0274] Embodiment 67 includes any one polypeptide or polypeptide complex of Embodiments 1 to 66, wherein A2 is bound to the N-terminus of an antibody or antibody fragment.

[0275] Embodiment 68 includes any one polypeptide or polypeptide complex of Embodiments 1 to 67, wherein the antibody or antibody fragment thereof includes a single-chain variable fragment, a single-domain antibody, or a Fab.

[0276] Embodiment 69 includes any one polypeptide or polypeptide complex of Embodiments 1 to 68, wherein the antibody or antibody fragment thereof includes a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain of a single-domain antibody derived from camelids (VHH).

[0277] Embodiment 70 includes any one polypeptide or polypeptide complex of Embodiments 1 to 69, wherein the antibody or antibody fragment thereof is humanized or human.

[0278] Embodiment 71 includes any one polypeptide or polypeptide complex of Embodiments 1 to 70, wherein A1 is a Fab.

[0279] Embodiment 72 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 71, wherein the Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide.

[0280] Embodiment 73 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 72, wherein the antibody or antibody fragment thereof comprises an epidermal growth factor receptor (EGFR) binding domain.

[0281] Embodiment 74 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 73, wherein the antibody or antibody fragment thereof comprises a mesothelin binding domain.

[0282] Embodiment 75 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 74, wherein the antibody or antibody fragment thereof comprises a carcinoembryonic antigen-related cell adhesion molecule CEACAM5 binding domain.

[0283] Embodiment 76 includes a polypeptide polypeptide complex of any one of Embodiments 1 to 75, wherein the tumor cell antigen comprises EGFR and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 56 or SEQ ID NO: 57.

[0284] Embodiment 77 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 76, wherein the tumor cell antigen comprises EGFR and the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 59 or SEQ ID NO: 60.

[0285] Embodiment 78 includes a polypeptide polypeptide complex of any one of Embodiments 1 to 77, wherein the tumor cell antigen comprises HER2 and the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 61.

[0286] Embodiment 79 includes any one polypeptide or polypeptide complex of Embodiments 1-78, wherein the tumor cell antigen includes HER2 and the Fab heavy chain polypeptide includes the amino acid sequence according to SEQ ID NO: 62 or SEQ ID NO: 63.

[0287] Embodiment 80 includes any one polypeptide or polypeptide complex of Embodiments 1-79, wherein A2 includes an antibody or an antibody fragment.

[0288] Embodiment 81 includes any one polypeptide or polypeptide complex of Embodiments 1-80, wherein A2 includes a human or humanized antibody or an antibody fragment.

[0289] Embodiment 82 includes any one polypeptide or polypeptide complex of Embodiments 1-81, wherein the antibody or antibody fragment includes a single-chain variable fragment, a single-domain antibody, or a Fab fragment.

[0290] Embodiment 83 includes any one polypeptide or polypeptide complex of Embodiments 1-82, wherein A2 is a single-chain variable fragment (scFv).

[0291] Embodiment 84 includes any one polypeptide or polypeptide complex of Embodiments 1-83, wherein the scFv includes an scFv heavy chain polypeptide and an scFv light chain polypeptide.

[0292] Embodiment 85 includes any one polypeptide or polypeptide complex of Embodiments 1-84, wherein A2 is a single-domain antibody.

[0293] Embodiment 86 includes any one polypeptide or polypeptide complex of Embodiments 1-85, wherein the single-domain antibody includes a single-chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain of a single-domain antibody derived from camelids (VHH).

[0294] Embodiment 87 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 86, wherein A2 includes an anti-CD3e single-chain variable fragment.

[0295] Embodiment 88 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 87, wherein A2 includes an anti-CD3e single-chain variable fragment that has a K D binding of 1 μM or less to CD3 on CD3-expressing cells.

[0296] Embodiment 89 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 88, wherein A2 includes a variable light chain and a variable heavy chain, each of which can specifically bind to human CD3.

[0297] Embodiment 90 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 89, wherein A2 includes a complementarity-determining region (CDR) selected from the group consisting of muromonab-CD3 (OKT3), otrexup (TRX4), teprotumumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19.

[0298] Embodiment 91 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 90, wherein the polypeptide or polypeptide complex of Formula I binds to effector cells.

[0299] Embodiment 92 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 91, wherein the effector cell is a T cell.

[0300] Embodiment 93 includes any one polypeptide or polypeptide complex of Embodiments 1 to 92, wherein A2 binds to a polypeptide that is part of the TCR-CD3 complex on effector cells.

[0301] Embodiment 94 includes any one polypeptide or polypeptide complex of Embodiments 1 to 93, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3ε.

[0302] Embodiment 95 includes any one polypeptide or polypeptide complex of Embodiments 1 to 94, wherein the effector cell antigen includes CD3, and the scFv includes an amino acid sequence according to SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO: 68.

[0303] Embodiment 96 includes any one polypeptide or polypeptide complex of Embodiments 1 to 95, wherein the Fab light chain polypeptide of A1 is bound to the C-terminus of the single-chain variable fragment (scFv) of A2.

[0304] Embodiment 97 includes any one polypeptide or polypeptide complex of Embodiments 1 to 96, wherein the Fab heavy chain polypeptide of A1 is bound to the C-terminus of the single-chain variable fragment (scFv) of A2.

[0305] Embodiment 98 includes any one polypeptide or polypeptide complex of Embodiments 1 to 97, wherein the Fab light chain polypeptide of A1 is bound to the N-terminus of the single-chain variable fragment (scFv) of A2.

[0306] Embodiment 99 includes any one polypeptide or polypeptide complex of Embodiments 1 to 98, wherein the Fab heavy chain polypeptide of A1 is bound to the N-terminus of the single-chain variable fragment (scFv) of A2.

[0307] Embodiment 100 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 99, wherein the Fab heavy chain polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2, and L1 is bound to the Fab light chain polypeptide of A1.

[0308] Embodiment 101 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 100, wherein the Fab light chain polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2, and L1 is bound to the Fab heavy chain polypeptide of A1.

[0309] Embodiment 102 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 101, wherein the Fab heavy chain polypeptide of A1 is bound to the scFv light chain polypeptide of A2, and L1 is bound to the Fab light chain polypeptide of A1.

[0310] Embodiment 103 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 102, wherein the Fab light chain polypeptide of A1 is bound to the scFv light chain polypeptide of A2, and L1 is bound to the Fab heavy chain polypeptide of A1.

[0311] Embodiment 104 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 103, wherein A2 further includes P2 and L2, where P2 includes a peptide that binds to A2, and L2 includes a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0312] Embodiment 105 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 104, wherein the polypeptide or polypeptide complex is according to Formula Ia. P2-L2-A2-A1-L1-P1-H1 (Formula Ia)

[0313] Embodiment 106 includes any one polypeptide or polypeptide complex of Embodiments 1 to 105, wherein the Fab heavy chain polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2, and L1 is bound to the Fab light chain polypeptide of A1, and L2 is bound to the scFv light chain polypeptide of A2.

[0314] Embodiment 107 includes any one polypeptide or polypeptide complex of Embodiments 1 to 106, wherein the Fab heavy chain polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2, and L1 is bound to the Fab light chain polypeptide of A1, and L2 is bound to the scFv light chain polypeptide of A2, and the polypeptide complex includes the amino acid sequences of SEQ ID NO: 72 and SEQ ID NO: 71.

[0315] Embodiment 108 includes any one polypeptide or polypeptide complex of Embodiments 1 to 107, wherein the Fab light chain polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2, and L1 is bound to the Fab heavy chain polypeptide of A1, and L2 is bound to the scFv light chain polypeptide of A2.

[0316] Embodiment 109 includes any one polypeptide or polypeptide complex of Embodiments 1 to 108, wherein the Fab heavy chain polypeptide of A1 is bound to the scFv light chain polypeptide of A2, and L1 is bound to the Fab light chain polypeptide of A1, and L2 is bound to the scFv heavy chain polypeptide of A2.

[0317] Embodiment 110 includes any one polypeptide or polypeptide complex of Embodiments 1 to 109, wherein the Fab light chain polypeptide of A1 is bound to the scFv light chain polypeptide of A2, and L1 is bound to the Fab heavy chain polypeptide of A1, and L2 is bound to the scFv heavy chain polypeptide of A2.

[0318] Embodiment 111 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 110, which has a weaker binding affinity for a tumor cell antigen than the binding affinity of a polypeptide or polypeptide complex having no P1 or L1 for the tumor cell antigen.

[0319] Embodiment 112 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 111, which has a weaker binding affinity for a tumor cell antigen, and which is at least 10-fold (10X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex having no P1 or L1.

[0320] Embodiment 113 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 112, which has a weaker binding affinity for a tumor cell antigen, and which is at least 100-fold (100X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex having no P1 or L1.

[0321] Form 114 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 113, which has a weaker binding affinity for a tumor cell antigen than the binding affinity of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen.

[0322] Form 115 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 114, which has a weaker binding affinity for a tumor cell antigen, and which is at least 10-fold (10X) higher than the binding affinity for the tumor cell antigen in the form of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease.

[0323] Form 116 comprises a polypeptide or polypeptide complex having a weaker binding affinity for a tumor cell antigen, which is at least 100-fold (100X) higher than the binding affinity of the form of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease for the tumor cell antigen, and is any one of polypeptides or polypeptide complexes of Embodiments 1 to 115.

[0324] Embodiment 117 comprises a polypeptide or polypeptide complex of any one of Embodiments 1 to 116, wherein P1 impairs the binding of A1 to a first target antigen.

[0325] Embodiment 118 comprises a polypeptide or polypeptide complex of any one of Embodiments 1 to 117, wherein P1 binds to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, pi-stacking interactions, and H-bonding interactions, or a combination thereof.

[0326] Embodiment 119 comprises a polypeptide or polypeptide complex of any one of Embodiments 1 to 118, wherein P1 has less than 70% sequence identity to a first target antigen.

[0327] Embodiment 120 comprises a polypeptide or polypeptide complex of any one of Embodiments 1 to 119, wherein P2 impairs the binding of A2 to a second target antigen.

[0328] Embodiment 121 comprises a polypeptide or polypeptide complex of any one of Embodiments 1 to 120, wherein P2 binds to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, pi-stacking interactions, and H-bonding interactions, or a combination thereof.

[0329] Embodiment 122 comprises a polypeptide or polypeptide complex of any one of Embodiments 1 to 121, wherein P2 binds to A2 at or near the antigen-binding site.

[0330] Embodiment 123 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 122, wherein P2 has less than 70% sequence identity to the second target antigen.

[0331] Embodiment 124 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 123, wherein P1 or P2 includes a peptide sequence having a length of at least 10 amino acids.

[0332] Embodiment 125 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 124, wherein P1 or P2 includes a peptide sequence having a length of at least 10 amino acids and a maximum length of 20 amino acids.

[0333] Embodiment 126 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 125, wherein P1 or P2 includes a peptide sequence having a length of at least 16 amino acids.

[0334] Embodiment 127 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 126, wherein P1 or P2 includes a peptide sequence having a maximum length of 40 amino acids.

[0335] Embodiment 128 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 127, wherein P1 or P2 includes at least two cysteine - amino acid residues.

[0336] Embodiment 129 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 128, wherein P1 or P2 includes a cyclic peptide or a linear peptide.

[0337] Embodiment 130 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 129, wherein P1 or P2 includes a cyclic peptide.

[0338] Embodiment 131 includes any one of the polypeptides or polypeptide complexes of Embodiments 1 to 130, wherein P1 or P2 includes a linear peptide.

[0339] Embodiment 132 includes any one of the polypeptides or polypeptide complexes of Embodiments 1 to 131, wherein P1 includes at least two cysteine - amino acid residues.

[0340] Embodiment 133 includes a tumor cell antigen that includes EGFR, and P1 or P2 is

[0341]

Chemical formula

[0342] Embodiment 134 includes a tumor cell antigen that includes HER2, and P1 or P2 is

[0343]

Chemical formula

[0344] Embodiment 135 includes an effector cell antigen that includes CD3, and P1 or P2 is

[0345]

Chemical formula

[0346] Embodiment 136 includes any one of the polypeptides or polypeptide complexes of Embodiments 1 to 135, wherein L1 is bound to the N - terminus of A1.

[0347] Embodiment 137 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 135, wherein L1 is bound to the C-terminus of A1.

[0348] Embodiment 138 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 137, wherein L2 is bound to the N-terminus of A2.

[0349] Embodiment 139 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 138, wherein L2 is bound to the C-terminus of A2.

[0350] Embodiment 140 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 139, wherein L1 or L2 is a peptide sequence having at least 5 to a maximum of 50 amino acids.

[0351] Embodiment 141 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 140, wherein L1 or L2 is a peptide sequence having at least 10 to a maximum of 30 amino acids.

[0352] Embodiment 142 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 141, wherein L1 or L2 is a peptide sequence having at least 10 amino acids.

[0353] Embodiment 143 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 142, wherein L1 or L2 is a peptide sequence having at least 18 amino acids.

[0354] Embodiment 144 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 143, wherein L1 or L2 is a peptide sequence having at least 26 amino acids.

[0355] Embodiment 145, wherein L1 or L2 is (G2S) nhaving a formula including, wherein n is an integer from 1 to 3 (SEQ ID NO: 29), and including any one polypeptide or polypeptide complex of Embodiments 1 to 144.

[0356] Embodiment 146 is such that L1 has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 30), (GGGS) n (SEQ ID NO: 31), (GGGGS) n (SEQ ID NO: 32), or (GSSGGS) n (SEQ ID NO: 33), wherein n is an integer of at least 1, and including any one polypeptide or polypeptide complex of Embodiments 1 to 145.

[0357] Embodiment 147 is such that P1 becomes unbound from A1 when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to the first target antigen, and including any one polypeptide or polypeptide complex of Embodiments 1 to 146.

[0358] Embodiment 148 is such that P2 becomes unbound from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to the second target antigen, and including any one polypeptide or polypeptide complex of Embodiments 1 to 147.

[0359] Embodiment 149 is such that the tumor-specific protease is selected from the group consisting of metalloprotease, serine protease, cysteine protease, threonine protease, and aspartic protease, and including any one polypeptide or polypeptide complex of Embodiments 1 to 148.

[0360] Embodiment 150 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 149, wherein L1 or L2 includes a urokinase-cleavable amino acid sequence, a matriptase-cleavable amino acid sequence, a matrix metalloprotease-cleavable amino acid sequence, or a legumain-cleavable amino acid sequence.

[0361] Embodiment 151 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 150, wherein L1 or L2 includes

[0362]

Chemical Formula

[0363] Embodiment 152 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 151, wherein L1 or L2 includes

[0364]

Chemical Formula

[0365] Embodiment 153 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 152, wherein H1 includes a polymer.

[0366] Embodiment 154 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 153, wherein the polymer is polyethylene glycol (PEG).

[0367] Embodiment 155 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 154, wherein H1 includes albumin.

[0368] Embodiment 156 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 155, wherein H1 includes an Fc domain.

[0369] Embodiment 157 includes any one polypeptide or polypeptide complex of Embodiments 1 to 156, wherein the albumin is serum albumin.

[0370] Embodiment 158 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 157, wherein the albumin is human serum albumin.

[0371] Embodiment 159 includes any one polypeptide or polypeptide complex of Embodiments 1 to 158, wherein H1 includes a polypeptide, a ligand, or a small molecule.

[0372] Embodiment 160 includes any one polypeptide or polypeptide complex of Embodiments 1 to 159, wherein the polypeptide, ligand, or small molecule binds to a serum protein or a fragment thereof, a blood immunoglobulin or a fragment thereof, or CD35 / CR1.

[0373] Embodiment 161 includes any one polypeptide or polypeptide complex of Embodiments 1 to 160, wherein the serum protein includes thyroxine-binding protein, transthyretin, alpha-1-acid glycoprotein, transferrin, transferrin receptor, or a transferrin-binding portion thereof, fibrinogen, or albumin.

[0374] Embodiment 162 includes any one polypeptide or polypeptide complex of Embodiments 1 to 161, wherein the blood immunoglobulin molecule includes IgG1, IgG2, IgG3, IgG4, sIgA, IgM, or IgD.

[0375] Embodiment 163 includes any one polypeptide or polypeptide complex of Embodiments 1 to 162, wherein the serum protein is albumin.

[0376] Embodiment 164 includes any one polypeptide or polypeptide complex of Embodiments 1 to 163, wherein the polypeptide is an antibody.

[0377] Embodiment 165 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 164, wherein the antibody includes a single-domain antibody, a single-chain variable fragment, or a Fab.

[0378] Embodiment 166 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 165, wherein the single-domain antibody binds to albumin and the single-domain antibody is a human antibody or a humanized antibody.

[0379] Embodiment 166 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 165, wherein the single-domain antibody is 645gH1gL1.

[0380] Embodiment 168 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 167, wherein the single-domain antibody is 645dsgH5gL4.

[0381] Embodiment 169 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 168, wherein the single-domain antibody is 23-13-A01-sc02.

[0382] Embodiment 170 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 169, wherein the single-domain antibody is A10m3 or a fragment thereof.

[0383] Embodiment 171 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 170, wherein the single-domain antibody is DOM7r-31.

[0384] Embodiment 172 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 171, wherein the single-domain antibody is DOM7h-11-15.

[0385] Embodiment 173 includes any one polypeptide or polypeptide complex of Embodiments 1 to 172, wherein the single-domain antibody is Alb-1, Alb-8, or Alb-23.

[0386] Embodiment 174 includes any one polypeptide or polypeptide complex of Embodiments 1 to 173, wherein the single-domain antibody is 10G or 10GE.

[0387] Embodiment 175 includes any one polypeptide or polypeptide complex of Embodiments 1 to 174, wherein the single-domain antibody is 10G, and the single-domain antibody includes the following amino acid sequence.

[0388]

Chemical formula

[0389] Embodiment 176 includes any one polypeptide or polypeptide complex of Embodiments 1 to 175, wherein the single-domain antibody is SA21.

[0390] Embodiment 177 includes any one polypeptide or polypeptide complex of Embodiments 1 to 176, wherein the polypeptide or polypeptide complex includes a modified amino acid, or a non-natural amino acid, or a modified non-natural amino acid, or a combination thereof.

[0391] Embodiment 178 includes any one polypeptide or polypeptide complex of Embodiments 1 to 177, wherein the modified amino acid or modified non-natural amino acid includes a post-translational modification.

[0392] Embodiment 179 includes any one polypeptide or polypeptide complex of Embodiments 1 to 178, wherein H1 includes a linking portion (L3) that connects H1 to P1.

[0393] Embodiment 180 includes any one polypeptide or polypeptide complex of Embodiments 1 to 179, wherein L3 is a peptide sequence having at least 5 to a maximum of 50 amino acids.

[0394] Embodiment 181 includes any one polypeptide or polypeptide complex of Embodiments 1 to 180, wherein L3 is a peptide sequence having at least 10 to a maximum of 30 amino acids.

[0395] Embodiment 182 includes any one polypeptide or polypeptide complex of Embodiments 1 to 181, wherein L3 is a peptide sequence having at least 10 amino acids.

[0396] Embodiment 183 includes any one polypeptide or polypeptide complex of Embodiments 1 to 182, wherein L3 is a peptide sequence having at least 18 amino acids.

[0397] Embodiment 184 includes any one polypeptide or polypeptide complex of Embodiments 1 to 183, wherein L3 is a peptide sequence having at least 26 amino acids.

[0398] Embodiment 185, wherein L3 has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 30), (GGGS) n (SEQ ID NO: 31), (GGGGS) n (SEQ ID NO: 32), or (GSSGGS) n (SEQ ID NO: 33), wherein n is an integer of at least 1, and includes any one polypeptide or polypeptide complex of Embodiments 1 to 184.

[0399] Embodiment 186 includes any one polypeptide or polypeptide complex of Embodiments 1 to 185, wherein L3 includes the amino acid sequence by GGGGSGGGS (SEQ ID NO: 51).

[0400] Embodiment 187 A pharmaceutical composition comprising any one polypeptide or polypeptide complex of Embodiments 1 to 186, and a pharmaceutically acceptable excipient.

[0401] Embodiment 188 comprises an isolated recombinant nucleic acid molecule encoding any one polypeptide or polypeptide complex of Embodiments 1 to 187.

[0402] Embodiment 189 comprises a polypeptide or polypeptide complex according to formula II, L 1a -P 1a -H 1a (Formula II) Wherein L 1a , when not cleaved, comprises a tumor-specific protease-cleavable linker moiety that connects P 1a to an antigen recognition molecule that binds to a target antigen, and P 1a comprises a peptide that binds to the antigen recognition molecule when L 1a is not cleaved, and H 1a comprises a half-life extending molecule.

[0403] Embodiment 190 comprises any one polypeptide or polypeptide complex of Embodiments 1 to 189, wherein P 1a impairs the binding of the antigen recognition molecule to the target antigen when L1 is not cleaved.

[0404] Embodiment 191 comprises any one polypeptide or polypeptide complex of Embodiments 1 to 190, wherein the antigen recognition molecule comprises an antibody or antibody fragment.

[0405] Embodiment 192 comprises any one polypeptide or polypeptide complex of Embodiments 1 to 191, wherein the target antigen is an anti-CD3 effector cell antigen.

[0406] Embodiment 193 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 192, wherein the target antigen is a tumor cell antigen.

[0407] Embodiment 194 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 193, wherein the tumor cell antigen is EGFR, HER2, mesothelin or CEACAM5.

[0408] Embodiment 195 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 194, wherein P 1a has a sequence identity of less than 70% to the second target antigen.

[0409] Embodiment 196 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 195, wherein P 1a includes a peptide sequence having a length of at least 10 amino acids.

[0410] Embodiment 197 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 196, wherein P 1a includes a peptide sequence having a length of at least 10 amino acids and a maximum length of 20 amino acids.

[0411] Embodiment 198 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 197, wherein P 1a includes a peptide sequence having a length of at least 16 amino acids.

[0412] Embodiment 199 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 198, wherein P 1a includes a peptide sequence having a maximum length of 40 amino acids.

[0413] Embodiment 200 includes the polypeptide or polypeptide complex according to any one of Embodiments 1 to 199, wherein P 1a includes at least two cysteine - amino acid residues.

[0414] Embodiment 201 includes any one polypeptide or polypeptide complex of Embodiments 1 to 200, wherein P 1a includes a cyclic peptide or a linear peptide.

[0415] Embodiment 202 includes any one polypeptide or polypeptide complex of Embodiments 1 to 201, wherein P 1a includes a cyclic peptide.

[0416] Embodiment 203 includes any one polypeptide or polypeptide complex of Embodiments 1 to 202, wherein P 1a includes a linear peptide.

[0417] Embodiment 204 includes any one polypeptide or polypeptide complex of Embodiments 1 to 203, wherein the target antigen includes EGFR, and P 1a includes an amino acid sequence selected from the group consisting of

[0418]

Chemical formula

[0419] Embodiment 205 includes any one polypeptide or polypeptide complex of Embodiments 1 to 204, wherein the target includes HER2, and P 1a includes an amino acid sequence selected from the group consisting of

[0420]

Chemical formula

[0421] Embodiment 206 includes any one polypeptide or polypeptide complex of Embodiments 1 to 205, wherein the target includes CD3, and P 1a includes an amino acid sequence selected from the group consisting of

[0422]

Chemical formula

[0423] Embodiment 207 is such that H 1a comprises any one polypeptide or polypeptide complex of Embodiments 1 to 206, which comprises a polymer.

[0424] Embodiment 208 is such that the polymer is polyethylene glycol (PEG), and comprises any one polypeptide or polypeptide complex of Embodiments 1 to 207.

[0425] Embodiment 209 is such that H 1a comprises any one polypeptide or polypeptide complex of Embodiments 1 to 208, which comprises albumin.

[0426] Embodiment 210 is such that H 1a comprises any one polypeptide or polypeptide complex of Embodiments 1 to 209, which comprises an Fc domain.

[0427] Embodiment 211 is such that the albumin is serum albumin, and comprises any one polypeptide or polypeptide complex of Embodiments 1 to 210.

[0428] Embodiment 212 is such that the albumin is human serum albumin, and comprises any one polypeptide or polypeptide complex of Embodiments 1 to 211.

[0429] Embodiment 213 is such that H 1a comprises any one polypeptide or polypeptide complex of Embodiments 1 to 212, which comprises a polypeptide, a ligand, or a small molecule.

[0430] Embodiment 214 includes a polypeptide, ligand, or small molecule that binds to a serum protein or a fragment thereof, a blood immunoglobulin or a fragment thereof, or any one polypeptide or polypeptide complex of Embodiments 1 to 213 that binds to CD35 / CR1.

[0431] Embodiment 215 includes any one polypeptide or polypeptide complex of Embodiments 1 to 214, wherein the serum protein includes thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor, or a transferrin-binding portion thereof, fibrinogen, or albumin.

[0432] Embodiment 216 includes any one polypeptide or polypeptide complex of Embodiments 1 to 215, wherein the blood immunoglobulin molecule includes IgGl, IgG2, IgG3, IgG4, slgA, IgM, or IgD.

[0433] Embodiment 217 includes any one polypeptide or polypeptide complex of Embodiments 1 to 216, wherein the serum protein is albumin.

[0434] Embodiment 218 includes any one polypeptide or polypeptide complex of Embodiments 1 to 217, wherein the polypeptide is an antibody.

[0435] Embodiment 219 includes any one polypeptide or polypeptide complex of Embodiments 1 to 218, wherein the antibody includes a single-domain antibody, a single-chain variable fragment, or a Fab.

[0436] Embodiment 220 includes any one polypeptide or polypeptide complex of Embodiments 1 to 219, wherein the antibody includes a single-domain antibody that binds to albumin.

[0437] Embodiment 221 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 220, wherein the antibody is a human antibody or a humanized antibody.

[0438] Embodiment 222 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 221, wherein the single-domain antibody is 645gH1gL1.

[0439] Embodiment 223 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 222, wherein the single-domain antibody is 645dsgH5gL4.

[0440] Embodiment 224 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 223, wherein the single-domain antibody is 23-13-A01-sc02.

[0441] Embodiment 225 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 224, wherein the single-domain antibody is A10m3 or a fragment thereof.

[0442] Embodiment 226 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 225, wherein the single-domain antibody is DOM7r-31.

[0443] Embodiment 227 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 226, wherein the single-domain antibody is DOM7h-11-15.

[0444] Embodiment 228 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 227, wherein the single-domain antibody is Alb-1, Alb-8, or Alb-23.

[0445] Embodiment 229 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 228, wherein the single-domain antibody is 10G or 10GE.

[0446] Embodiment 230 includes a single-domain antibody that is 10G and a polypeptide or polypeptide complex of any one of Embodiments 1 to 229, wherein the single-domain antibody includes an amino acid sequence of

[0447] [Chemical Formula] Embodiment 231 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 230, wherein the single-domain antibody is SA21.

[0448] Embodiment 232 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 231, wherein H

[0449] has a linking moiety (L3) connecting H1 to P1. 1a Embodiment 233 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 232, wherein L

[0450] is a peptide sequence having at least 5 to a maximum of 50 amino acids. 3a Embodiment 234 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 233, wherein L

[0451] is a peptide sequence having at least 10 to a maximum of 30 amino acids. 3a Embodiment 235 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 234, wherein L

[0452] is a peptide sequence having at least 10 to a maximum of amino acids. 3a Embodiment 236 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 235, wherein L

[0453] is a peptide sequence having at least 18 amino acids. 3a Embodiment 236 includes a polypeptide or polypeptide complex of any one of Embodiments 1 to 235, wherein L

[0454] Embodiment 237 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 236, wherein L 3a is a peptide sequence having at least 26 amino acids.

[0455] Embodiment 238 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 237, wherein L 3a has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 30), (GGGS) n (SEQ ID NO: 31), (GGGGS) n (SEQ ID NO: 32), or (GSSGGS) n (SEQ ID NO: 33).

[0456] Embodiment 239 includes a polypeptide or polypeptide complex according to any one of Embodiments 1 to 238, wherein L 3a includes the amino acid sequence of GGGGSGGGS (SEQ ID NO: 51).

[0457] Embodiment 240 includes a polypeptide or polypeptide complex, and the polypeptide or polypeptide complex includes a structural arrangement according to Configuration 1,

[0458] [Chemical Formula] Here, the polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, wherein the scFv is linked to a peptide (P1) that impairs the binding of the scFv to effector cell antigens, and P1 is linked to the N-terminus of the light-chain variable domain of the scFv by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extending molecule and comprises a Fab that binds to a tumor cell antigen, where the Fab comprises a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, where the Fab heavy-chain polypeptide is linked to the C-terminus of the heavy-chain variable domain of the scFv, and where the Fab is linked to P2 and L2, where P2 comprises a peptide that impairs the binding of the Fab to the tumor cell antigen, and L2 comprises a linking moiety that connects the Fab light-chain polypeptide to P2 and is a substrate for a tumor-specific protease.

[0459] Embodiment 241 comprises a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a structural arrangement according to Configuration 2.

[0460]

Chemical formula

[0461] Embodiment 242 includes a polypeptide or a polypeptide complex, and the polypeptide or the polypeptide complex includes a structural arrangement according to Configuration 3.

[0462]

Chemical formula

[0463] Embodiment 243 includes a polypeptide or a polypeptide complex, and the polypeptide or the polypeptide complex includes a structural arrangement according to Configuration 4.

[0464]

Chemical formula

Claims

1. A polypeptide complex, wherein the polypeptide complex comprises a structural arrangement with the following constitution: 【Chemical 1】 Here, the polypeptide complex includes a single-chain variable fragment (scFv) containing a light-chain variable domain and a heavy-chain variable domain, where the scFv is, at the N-terminus of the scFv, linked to a peptide (P １ ), by a linking moiety (L １ ) that is a substrate for a tumor-specific protease, where P １ impairs the binding of the scFv to an effector-cell antigen, and P １ is further linked to a half-life extending molecule. The polypeptide complex includes an antigen recognition molecule that binds to a tumor cell antigen, wherein the antigen recognition molecule includes a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the antigen recognition molecule is linked to the scFv, and wherein the antigen recognition molecule is further linked to P ２ and L ２ and further linked to, wherein P ２ includes a peptide that impairs the binding of the antigen recognition molecule to the tumor cell antigen, and L ２ connects the antigen recognition molecule to P ２ and includes a linking moiety that is a substrate for a tumor-specific protease, a polypeptide complex.

2. The polypeptide complex according to claim 1, wherein the antigen recognition molecule is Fab or Fab'.

3. The heavy chain variable domain is linked to the N-terminus of the Fab heavy chain polypeptide, and L ２ is connected to the N-terminus of the Fab light chain polypeptide, the polypeptide complex according to claim 1.

4. The heavy chain variable domain is linked to the N-terminus of the Fab light chain polypeptide, and L ２ is connected to the N-terminus of the Fab heavy chain polypeptide, the polypeptide complex according to claim 1.

5. The light chain variable domain is linked to the N-terminus of the Fab heavy chain polypeptide, and L ２ is connected to the N-terminus of the Fab light chain polypeptide, the polypeptide complex according to claim 1.

6. The light chain variable domain is linked to the N-terminus of the Fab light chain polypeptide and L ２ is connected to the N-terminus of the Fab heavy chain polypeptide, the polypeptide complex according to claim 1.

7. The polypeptide complex according to claim 1, wherein the polypeptide complex has a molecular weight of less than about 110 kDa.

8. The polypeptide complex according to claim 1, wherein the heavy chain variable domain, the light chain variable domain, the Fab heavy chain polypeptide, the Fab light chain polypeptide, and the half-life extension molecule have a total molecular weight of less than about 100 kDa.

9. The polypeptide complex according to claim 1, wherein the tumor cell antigen comprises epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), or mesothelin.

10. The polypeptide complex according to claim 1, wherein the effector cell antigen comprises surface antigen classification 3 (CD3).

11. The polypeptide complex according to claim 1, wherein the scFv comprises a complementarity-determining region (CDR) selected from the group consisting of muromonab-CD3 (OKT3), otrexup mab (TRX4), teprotumumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19.

12. The polypeptide complex according to claim 1, wherein the scFv comprises the complementarity-determining region (CDR) of SP34.

13. The polypeptide complex according to claim 1, wherein the scFv comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 66, SEQ ID NO: 67, or SEQ ID NO:

68.

14. P １ The polypeptide complex according to claim 1, which binds to the scFv through ionic interaction, electrostatic interaction, hydrophobic interaction, π-stacking interaction, and H-bonding interaction, thereby impairing the binding of the scFv to the effector cell antigen.

15. P １ The polypeptide complex according to claim 1, which binds to the scFv at or near the antigen-binding site, thereby impairing the binding of the scFv to the effector-cell antigen.

16. P １ is the polypeptide complex according to claim 1, comprising an amino acid sequence having a length of at least 10 amino acids and a maximum length of 20 amino acids.

17. P １ The polypeptide complex according to claim 1, which has a sequence identity of less than 70% with respect to the amino acid sequence of the effector cell antigen.

18. P １ The polypeptide complex according to claim 10, which has a sequence identity of less than 70% with respect to the amino acid sequence of CD3.

19. P １ is the polypeptide complex according to claim 1, comprising an amino acid sequence according to SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

20. L １ is the polypeptide complex according to claim 1, comprising a urokinase-cleavable amino acid sequence, a matriptase-cleavable amino acid sequence, a matrix metalloprotease-cleavable amino acid sequence, or a legumain-cleavable amino acid sequence.

21. L １ (G ２ S) ｎ , (GS) ｎ , (GSGGS) ｎ (SEQ ID NO: 30), (GGGS) ｎ (SEQ ID NO: 31), (GGGGS) ｎ (SEQ ID NO: 32), or (GSSGGS) ｎ 2. The polypeptide complex of claim 1, having a formula comprising: (SEQ ID NO: 33), wherein n is an integer of at least 1.

22. L １ The polypeptide complex according to claim 1, comprising an amino acid sequence according to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 55.

23. L １ is the polypeptide complex according to claim 22, comprising the amino acid sequence according to SEQ ID NO:

42.

24. The half-life extending molecule is a linking moiety (L １ ), which connects the half-life extending molecule to P ３ ), the polypeptide complex according to claim 1.

25. L ３ (G ２ S) ｎ , (GS) ｎ , (GSGGS) ｎ (SEQ ID NO: 30), (GGGS) ｎ (SEQ ID NO: 31), (GGGGS) ｎ (SEQ ID NO: 32), and (GSSGGS) ｎ 25. The polypeptide complex of claim 24, having a formula selected from the group consisting of: (SEQ ID NO: 33), wherein n is an integer of at least 1.

26. L ３ is the polypeptide complex according to claim 24, comprising the amino acid sequence according to SEQ ID NO:

51.

27. The half-life extended molecule is the polypeptide complex according to claim 1, comprising an antibody.

28. The polypeptide complex according to claim 27, wherein the antibody comprises a single domain antibody, a single-chain variable fragment, or a Fab.

29. The polypeptide complex according to claim 28, wherein the single domain antibody binds to albumin.

30. The polypeptide complex according to claim 28, wherein the single domain antibody comprises 10G or 10GE.

31. The polypeptide complex according to claim 30, wherein the single domain antibody comprises 10G, and the single domain antibody comprises an amino acid sequence according to SEQ ID NO:

52.

32. P ２ The polypeptide complex according to claim 1, which binds to the antigen recognition molecule through ionic interaction, electrostatic interaction, hydrophobic interaction, π-stacking interaction, and H-bonding interaction, thereby impairing the binding of the antigen recognition molecule to the tumor cell antigen.

33. P ２ The polypeptide complex according to claim 1, which binds to the antigen recognition molecule at or near the antigen binding site, thereby impairing the binding of the antigen recognition molecule to the tumor cell antigen.

34. P ２ The polypeptide complex according to claim 1, comprising an amino acid sequence having a length of at least 10 amino acids and a maximum length of 20 amino acids.

35. P ２ The polypeptide complex according to claim 1, which has a sequence identity of less than 70% with respect to the amino acid sequence of the tumor cell antigen.

36. The polypeptide complex according to claim 1, wherein the tumor cell antigen comprises epidermal growth factor receptor (EGFR).

37. P ２ comprises an amino acid sequence according to SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 The polypeptide complex according to claim 36.

38. The polypeptide complex according to claim 36, wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 56 or SEQ ID NO:

57.

39. The polypeptide complex according to claim 36, wherein the Fab heavy chain polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO:

60.

40. The polypeptide complex according to claim 1, wherein the tumor cell antigen comprises human epidermal growth factor receptor 2 (HER2).

41. P ２ is the polypeptide complex according to claim 40, comprising an amino acid sequence according to SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17.

42. The polypeptide complex according to claim 40, wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:

61.

43. The polypeptide complex according to claim 40, wherein the Fab heavy chain polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 62 or SEQ ID NO:

63.

44. L ２ is the polypeptide complex according to claim 1, comprising a urokinase-cleavable amino acid sequence, a matriptase-cleavable amino acid sequence, a matrix metalloprotease-cleavable amino acid sequence, or a legumain-cleavable amino acid sequence.

45. L ２ is, (G ２ S) ｎ , (GS) ｎ , (GSGGS) ｎ (SEQ ID NO: 30), (GGGS) ｎ (SEQ ID NO: 31), (GGGGGS) ｎ (SEQ ID NO: 32), or (GSSGGS) ｎ (SEQ ID NO: 33), and has a formula including n which is an integer of at least 1, the polypeptide complex according to claim 1.

46. L ２ is the polypeptide complex according to claim 1, comprising an amino acid sequence according to SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 55.

47. L ２ is the polypeptide complex according to claim 46, comprising the amino acid sequence according to SEQ ID NO:

42.

48. A pharmaceutical composition comprising: (i) the polypeptide complex according to any one of claims 1-47; and (ii) a pharmaceutically acceptable excipient.

49. An isolated recombinant nucleic acid molecule encoding the polypeptide or polypeptide complex according to any one of claims 1-47.

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