Multispecific binding protein compositions and uses thereof

Abstract

Described herein are single chain polypeptide compositions comprising variable domains linked by a linker, where the variable domains fold into a conformation to form two effector cell antigen interacting domains (E), each effector cell antigen interacting domain comprising two variable domains of a single chain variable fragment (EV and EV), at least one of the effector cell antigen interacting domains binds to one or more target interacting domains. Also described herein are methods of using the single chain polypeptides.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 307,584, filed February 7, 2022, which is incorporated herein by reference in its entirety. Summary of the Invention

[0002] Disclosed herein are single-chain polypeptides comprising variable domains connected by a linker, wherein the variable domains fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains of a single-chain variable fragment (EV and EV), and at least one of the effector cell antigen-interacting domains binds to one or more target-interacting domains. In some embodiments, the target-interacting domain binds to a tumor antigen, a ligand, a protein, a lipid, a nucleic acid, or a polysaccharide. In some embodiments, the tumor antigen is on a cell in the tumor microenvironment. In some embodiments, the target-interacting domain comprises an antibody, an antibody fragment, a ligand, or a cofactor. In some embodiments, the antibody or antibody fragment of the target interacting domain comprises a Fab, Fab', F(ab'), Fv fragment, single chain Fv (scFv), tandem single chain Fv (scFv), dual affinity retargeting antibody, diabody, VHH single domain antibody, TriTac, Adnectin, Anticalin, Avimer, Fynomer, Kunitz domain, knottin, Affibody, or DARPin. In some embodiments, the one or more target interacting domains comprise one or more target cell antigen interacting domains that bind to a target cell antigen. In some embodiments, the target cell antigen interacting domain binds to more than one target cell antigen. In some embodiments, the target cell antigen interacting domain is a bispecific or multispecific antibody or antibody fragment. In some embodiments, the single-chain polypeptide does not comprise a fragment crystallizable (Fc) domain. In some embodiments, the single-chain polypeptide does not comprise a fragment crystallizable (Fc) domain. In some embodiments, the two variable domains of the single-chain variable fragment (EV and EV) comprise a variable heavy chain of the single-chain variable fragment (EVH) and a variable light chain of the single-chain variable fragment (EVL).In some embodiments, at least one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to at least one TVHH, and one EV of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to at least one TVHH. In some embodiments, at least one TVHH that binds to EVL binds to the same target antigen as at least one TVHH that binds to EVH. In some embodiments, at least one TVHH that binds to EVL binds to the same target antigen as at least one TVHH that binds to EVH and has the same complementarity-determining region (CDR) sequence. In some embodiments, at least one TVHH that binds to EVL binds to the same target antigen as at least one TVHH that binds to EVH, but does not share a common complementarity-determining region (CDR) sequence. In some embodiments, at least one TVHH that binds to EVL binds to a different target antigen than at least one TVHH that binds to EVH. In some embodiments, EVL and EVH bind to the same number of TVHH. In some embodiments, the EVLs bind to a different number of TVHHs compared to the number of TVHHs bound to the EVHs. In some embodiments, at least one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to one TVHH, and at least one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to one TVHH. In some embodiments, at least one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to one TVHH, and at least one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to two TVHHs.In some embodiments, at least one EVL of the effector cell antigen interacting domain that binds to at least one or more target cell antigen interacting domains binds to one TVHH, and at least one EVH of the effector cell antigen interacting domain that binds to one or more target cell antigen interacting domains binds to three TVHHs. In some embodiments, at least one EVL of the effector cell antigen interacting domain that binds to one or more target cell antigen interacting domains binds to one TVHH, and at least one EVH of the effector cell antigen interacting domain that binds to one or more target cell antigen interacting domains binds to two TVHHs. In some embodiments, at least one EVH of the effector cell antigen interacting domain that binds to one or more target cell antigen interacting domains binds to one TVHH, and at least one EVL of the effector cell antigen interacting domain that binds to one or more target cell antigen interacting domains binds to three TVHHs. In some embodiments, at least one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to two TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to two TVHHs. In some embodiments, at least one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to three TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to three TVHHs. In some embodiments, at least one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to four TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to four TVHHs.In some embodiments, at least one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to five TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to five TVHHs. In some embodiments, at least one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to six TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to six TVHHs. In some embodiments, at least one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to seven TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to seven TVHHs. In some embodiments, at least one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to eight TVHHs, and at least one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to eight TVHHs. In some embodiments, at least one of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one target cell antigen interacting domain. In some embodiments, one or more target cell antigen interacting domains are C-terminally linked to at least one of the effector cell antigen interacting domains. In some embodiments, one or more target cell antigen interacting domains are N-terminally linked to at least one of the effector cell antigen interacting domains. In some embodiments, at least one of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to more than one target cell antigen interacting domain.In some embodiments, all of the more than one target cell antigen interacting domains bind to the same target antigen. In some embodiments, all of the more than one target cell antigen interacting domains bind to the same target antigen and all have the same CDR sequences. In some embodiments, all of the more than one target cell antigen interacting domains bind to the same target antigen, but at least two of the more than one target cell antigen interacting domains do not share a common CDR sequence. In some embodiments, at least two of the more than one target cell antigen interacting domains bind to different target antigens. In some embodiments, at least one of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to two target cell antigen interacting domains. In some embodiments, the two target cell antigen interacting domains are C-terminally linked to one of the effector cell antigen interacting domains. In some embodiments, the two target cell antigen interacting domains are N-terminally linked to one of the effector cell antigen interacting domains. In some embodiments, at least one of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to three target cell antigen interacting domains. In some embodiments, the three target cell antigen interaction domains are C-terminally linked to one of the effector cell antigen interaction domains. In some embodiments, the three target cell antigen interaction domains are N-terminally linked to one of the effector cell antigen interaction domains. In some embodiments, at least one target of the effector cell antigen interaction domains that binds to one or more target cell antigen interaction domains is linked to four target cell antigen interaction domains. In some embodiments, the four target cell antigen interaction domains are C-terminally linked to one of the effector cell antigen interaction domains. In some embodiments, the four target cell antigen interaction domains are N-terminally linked to one of the effector cell antigen interaction domains.In some embodiments, the variable domain, and the one or more target cell antigen interaction domains are as follows, where (-) is the linker: TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH, TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH, TVHH-EV-EV-EV-EV-TVHH, TVHH-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-EV-EV-EV-EV-TVHH, TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH, TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH, They are ordered according to the following arrangement from N-terminus to C-terminus: TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, or TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, or TVHH-EV-EV-EV-EV-TVHH, where (-) is a linker. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, where (-) is a linker. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-EV-EV-EV-EV-TVHH, where (-) is a linker. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following, where (-) is a linker: TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following, where (-) is a linker: TVHH-EVL-EVH-EVL-EVH, TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH、EVH-EVL-EVH-EVL-TVHH、TVHH-TVHH-EVL-EVH-EVL-EVH、TVHH-TVHH-EVH-EVL-EVH-EVL、EVL-EVL-EVL-EVH-TVHH-TVHH、EVH-EVL-EVH-EVL-TVHH-TVHH、 TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-EVL-EVH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH, TVH They are ordered according to their N- to C-terminal arrangement: H-TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following, where (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH, TVHH-EVL-EVH-EVL-EVH-EVL-EVH-TVHH, or TVHH-EVH-EVL-EVH-EVL-EVH-EVL-TVHH. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following, where (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVH-EVL-EVH-EVL-TVHH-TVHH. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following, where (-) is a linker: TVHH-EVL-EVH-EVL-EVH-TVHH, or TVHH-EVH-EVL-EVH-EVL-TVHH. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following, where (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH.In some embodiments, the variable domain and the one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH, where (-) is a linker. In some embodiments, the variable domain and the one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-EVL-EVH-EVL-EVH-TVHH, where (-) is a linker. The single-chain polypeptide of claim 53, wherein the variable domain and the one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-EVH-EVL-EVH-EVL-TVHH, where (-) is a linker. In some embodiments, the linker connecting the two effector cell antigen interacting domains (E) to each other comprises an internal linker. In some embodiments, the effector cell antigen interacting domain that is not directly linked to the one or more target cell antigen interacting domains comprises a terminal linker connecting the EVL and EVH. In some embodiments, each of the internal linkers is 6 to 8 amino acids in length. In some embodiments, the terminal linker is 15 or 16 amino acids in length. In some embodiments, each of the effector cell antigen interacting domains binds to an effector cell antigen. In some embodiments, the effector cell antigen comprises CD3, CD16a, or death receptor 5 (DR5). In some embodiments, CD3 is CD3 delta, CD3 gamma, or CD3 epsilon. In some embodiments, the effector cell antigen is on an effector cell. In some embodiments, the effector cell comprises a T cell, an NK cell, or a macrophage. In some embodiments, the target cell antigen interacting domain interacts with a target cell (T) that binds to the target cell antigen. In some embodiments, the target cell antigen accumulates in lipid rafts when present on cells associated with a disease state, but does not accumulate in lipid rafts when present on cells not associated with a disease state. In some embodiments, the target cell antigen accumulates when on cells associated with the disease state and does not accumulate when on cells not associated with the disease state.In some embodiments, the target cell antigen is 100 nm or less when on a cell associated with a disease state, but not within 100 nm when on a cell not associated with a disease state. In some embodiments, the target cell antigen is 75 nm or less when on a cell associated with a disease state, but not within 75 nm when on a cell not associated with a disease state. In some embodiments, the target cell antigen is 50 nm or less when on a cell associated with a disease state, but not within 50 nm when on a cell not associated with a disease state. In some embodiments, the target cell antigen is 25 nm or less when on a cell associated with a disease state, but not within 25 nm when on a cell not associated with a disease state. In some embodiments, the target cell antigen is 10 nm or less when on a cell associated with a disease state, but not within 10 nm when on a cell not associated with a disease state. In some embodiments, the target cell antigen is 5 nm or less when on a cell associated with a disease state, but not within 5 nm when on a cell not associated with a disease state. In some embodiments, the target cell antigen is 2 nm or less when on a cell associated with a disease state, but not within 2 nm when on a cell not associated with a disease state. In some embodiments, the target cell antigens are in close proximity when present on cells associated with a disease state and are not in close proximity when present on cells not associated with a disease state. In some embodiments, the target cell antigen is a dimer, trimer, tetramer, or oligomer when present on cells associated with a disease state and a monomer or dimer when present on cells not associated with a disease state. In some embodiments, the target cell antigen is part of a cell signaling complex when present on cells associated with a disease state and is not part of the same cell signaling complex when present on cells not associated with a disease state. In some embodiments, the target cell antigen is part of a cell signaling complex when present on cells associated with a disease state and is not part of a cell signaling complex when present on cells not associated with a disease state. In some embodiments, the target cell antigen comprises CD33, FAP, EGFR, HER2, or EpCAM. In some embodiments, the cells associated with a disease state are myeloid cells, fibroblasts, or cancer cells.

[0003] Disclosed herein is a single-chain polypeptide comprising variable domains joined by a linker, which fold into a conformation to form one or more effector cell antigen-interacting domains (E), wherein the effector cell antigen-interacting domain comprises two variable domains of a single-chain variable fragment (EV and EV), wherein the effector cell antigen-interacting domain directly binds to one or more target cell antigen-interacting domains that interact with target cells, and wherein the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH), and wherein the single-chain polypeptide does not contain an antibody constant domain. Disclosed herein is a single-chain polypeptide comprising variable domains joined by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), wherein each effector cell antigen-interacting domain comprises two variable domains of a single-chain variable fragment (EV and EV), wherein one of the effector cell antigen-interacting domains directly binds to one or more target cell antigen-interacting domains that interact with target cells, and wherein the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH), and wherein the single-chain polypeptide does not contain an antibody constant domain. In some embodiments, the two variable domains of the single-chain variable fragment (EV and EV) comprise a variable heavy chain (EVH) of the single-chain variable fragment and a variable light chain (EVL) of the single-chain variable fragment. In some embodiments, one of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains is bound to one target cell antigen interacting domain. In some embodiments, one target cell antigen interacting domain is bound to one of the effector cell antigen interacting domains at the C-terminus. In some embodiments, one target cell antigen interacting domain is bound to one of the effector cell antigen interacting domains at the N-terminus. In some embodiments, one of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains is bound to two target cell antigen interacting domains. In some embodiments, two target cell antigen interacting domains are bound to one of the effector cell antigen interacting domains at the C-terminus.In some embodiments, two target cell antigen interaction domains are N-terminally linked to one of the effector cell antigen interaction domains. In some embodiments, one of the effector cell antigen interaction domains that bind to one or more target cell antigen interaction domains is linked to three target cell antigen interaction domains. In some embodiments, three target cell antigen interaction domains are C-terminally linked to one of the effector cell antigen interaction domains. In some embodiments, three target cell antigen interaction domains are N-terminally linked to one of the effector cell antigen interaction domains. In some embodiments, one of the effector cell antigen interaction domains that bind to one or more target cell antigen interaction domains is linked to four target cell antigen interaction domains. In some embodiments, four target cell antigen interaction domains are C-terminally linked to one of the effector cell antigen interaction domains. In some embodiments, four target cell antigen interaction domains are N-terminally linked to one of the effector cell antigen interaction domains. In some embodiments, one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to one TVHH, and one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to one TVHH. In some embodiments, one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to one TVHH, and one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to two TVHH. In some embodiments, one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to one TVHH, and one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to three TVHH.In some embodiments, one EVKH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to two TVHHs. In some embodiments, one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to three TVHHs. In some embodiments, one EVKH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to two TVHHs, and one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to two TVHHs. In some embodiments, the variable domain and the one or more target cell antigen interacting domains are selected from the following, where (-) is a linker: TVHH-EV-EV-EV-EV, EV-EV-EV-EV-EV-TVHH, TVHH-TVHH-EV-EV-EV-EV-EV, EV-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EV-EV-EV, EV-EV They are ordered according to their arrangement from N-terminus to C-terminus: V-EV-EV-TVHH-TVHH-TVHH-TVHH, TVHH-EV-EV-EV-EV-TVHH, TVHH-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, TVHH-EV-EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH, or TVHH-TVHH-EV-EV-EV-EV-EV-TVHH-TVHH.In some embodiments, the variable domain and the one or more target cell antigen interacting domains are selected from the following, where (-) is a linker: TVHH-EVL-EVH-EVL-EVH, TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH, EVH-EVL-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH, EVH-EVL-EVH- EVL-TVHH-TVHH, TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH, EVH-EVL-EV H-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVH H-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH-TVHH, TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-EVL-EVH-EVL- EVH-TVHH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-EVL-E The effector cell antigen interacting domains (E) are ordered according to the N- to C-terminal arrangement of VH-EVL-EVH-TVHH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-EVH-EVL-EVH-EVL-TVHH-TVHH. In some embodiments, the linker connecting the two effector cell antigen interacting domains (E) to each other comprises an internal linker.In some embodiments, the effector cell antigen interacting domain that is not directly bound to one or more target cell antigen interacting domains comprises a terminal linker connecting the EVL and the EVH. In some embodiments, each of the internal linkers is 6 to 8 amino acids in length. In some embodiments, the terminal linker is 15 or 16 amino acids in length. In some embodiments, both effector cell antigen interacting domains are directly bound to the target cell antigen interacting domain. In some embodiments, both effector cell antigen interacting domains are bound to the target cell antigen interacting domain at the N-terminus and C-terminus. In some embodiments, both effector cell antigen interacting domains comprise a terminal linker connecting the EVL and the EVH. In some embodiments, the terminal linker is 15 or 16 amino acids in length. In some embodiments, the variable domains and target cell antigen interacting domains are ordered according to the N-terminal to C-terminal configuration of the following: TVHH-EVH-EVL-TVHH-TVHH-EVH-EVL-TVHH, TVHH-EVL-EVH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-EVH-EVL-TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH-EVL-EVH-TVHH, and TVHH-EVL-EVH-TVHH-TVHH-EVL-EVH-TVHH, where (-) is a linker.

[0004] In some embodiments, each of the effector cell antigen-interacting domains binds to an effector cell antigen. In some embodiments, the effector cell antigen comprises CD3, CD16a, or death receptor 5 (DR5). In some embodiments, the CD3 is CD3 delta, CD3 gamma, or CD3 epsilon. In some embodiments, the effector cell antigen is on an effector cell. In some embodiments, the effector cell comprises a T cell, an NK cell, or a macrophage. In some embodiments, the target cell antigen-interacting domain that interacts with a target cell (T) binds to the target cell antigen. In some embodiments, the target cell antigen accumulates in lipid rafts when on cells associated with a disease state, and does not accumulate in lipid rafts when on cells not associated with a disease state. In some embodiments, the target cell antigen accumulates when on cells associated with a disease state, and does not accumulate when on cells not associated with a disease state. In some embodiments, the target cell antigen is a dimer, trimer, tetramer, or oligomer when on cells associated with a disease state, and is a monomer or dimer when on cells not associated with a disease state. In some embodiments, the target cell antigen is part of a cell signaling complex when present on a cell associated with a disease state, but is not part of the same cell signaling complex when present on a cell not associated with the disease state. In some embodiments, the target cell antigen is part of a cell signaling complex when present on a cell associated with a disease state, but is not part of the same cell signaling complex when present on a cell not associated with the disease state. In some embodiments, the target cell antigen comprises CD33, FAP, EGFR, HER2, or EpCAM. In some embodiments, the cell associated with a disease state is a myeloid cell, a fibroblast, or a cancer cell.

[0005] Disclosed herein is a pharmaceutical composition comprising (a) a single-chain polypeptide according to any one of the above embodiments, and (b) a pharmaceutically acceptable excipient.

[0006] Disclosed herein is an isolated recombinant nucleic acid encoding a single chain polypeptide according to any one of the above embodiments.

[0007] Disclosed herein are vectors comprising the isolated nucleic acids according to the above-described embodiments.

[0008] Disclosed herein is a host cell comprising an isolated nucleic acid according to the above embodiments, or a vector according to the above embodiments.

[0009] Disclosed herein is a method for treating cancer, comprising administering to a subject who needs cancer treatment the single-chain polypeptide according to any one of above-mentioned embodiments.In some embodiments, cancer is solid tumor cancer.In some embodiments, cancer is blood cancer.

[0010] Disclosed herein is a method of treating an inflammatory disease or condition comprising administering to a subject in need thereof a single chain polypeptide according to any one of the above embodiments.

[0011] Disclosed herein is a method of treating an autoimmune disease or condition comprising administering to a subject in need thereof a single chain polypeptide according to any one of the above embodiments.

[0012] Disclosed herein are methods of treating a cardiovascular disease or condition, comprising administering to a subject in need thereof a single chain polypeptide according to any one of the above embodiments.

[0013] Disclosed herein is a method of treating a fibrotic disease or condition comprising administering to a subject in need thereof a single chain polypeptide according to any one of the above embodiments.

[0014] Disclosed herein is a method of treating a bacterial infection, comprising administering to a subject in need thereof a single chain polypeptide according to any one of the above-described embodiments.

[0015] Disclosed herein is a method of treating a viral infection, comprising administering to a subject in need thereof a single chain polypeptide according to any one of the above-described embodiments.

[0016] Disclosed herein is a single-chain polypeptide comprising two variable domains of a single-chain variable fragment (EV and EV) linked by a linker that fold into a three-dimensional structure to form an effector cell antigen-interacting domain (E), wherein the effector cell antigen-interacting domain is linked by one or more linkers to two or more target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH), and does not contain an antibody constant domain. In some embodiments, the two variable domains of the single-chain variable fragment (EV and EV) comprise a variable heavy chain of the single-chain variable fragment (EVH) and a variable light chain of the single-chain variable fragment (EVL). In some embodiments, the two or more target cell antigen-interacting domains are linked to the effector cell antigen-interacting domain at the C-terminus. In some embodiments, the two or more target cell antigen-interacting domains are linked to the effector cell antigen-interacting domain at the N-terminus. In some embodiments, the two or more target cell antigen-interacting domains are linked to the effector cell antigen-interacting domain at both the N-terminus and the C-terminus. In some embodiments, the effector cell antigen interacting domain binds to two target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to three target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to four target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to five target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to six target cell antigen interacting domains.In some embodiments, the effector cell antigen interacting domain is linked to two target cell antigen interacting domains, and the variable domain and the two target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal configuration: TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH, EVL-EVH-TVHH-TVHH, TVHH-TVHH-EVH-EVL, TVHH-EVL-EVH-TVHH, or TVHH-EVH-EVL-TVHH, where (-) is a linker. In some embodiments, the effector cell antigen interacting domain is linked to three target cell antigen interacting domains, and the variable domain and three target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal configuration: TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EVH-EVL, TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH, TVHH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-TVHH, where (-) is a linker. In some embodiments, the effector cell antigen interacting domain is linked to four target cell antigen interacting domains, and the variable domain and the four target cell antigen interacting domains are arranged in the following order, where (-) is a linker: TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EV They are ordered according to their arrangement from N-terminus to C-terminus: H-EVL, TVHH-TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH-TVHH, TVHH-EVL-EVH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-TVHH-TVHH.In some embodiments, the effector cell antigen interacting domain is linked to five target cell antigen interacting domains, and the variable domain and the five target cell antigen interacting domains are arranged in the following order, where (-) is a linker: TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL, TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH , TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH, TVHH-EVL-EVH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH, TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH, or TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH.In some embodiments, the effector cell antigen interacting domain is linked to six target cell antigen interacting domains, and the variable domain and the six target cell antigen interacting domains are arranged in the following order, where (-) is a linker: TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL, TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH -EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH, TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-EVL-EV In some embodiments, the effector cell antigen interacting domain is linked to six target cell antigen interacting domains, and the variable domain and six target cell antigen interacting domains are ordered according to the N- to C-terminal arrangement of the following: TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH. In some embodiments, the effector cell antigen interacting domain is linked to six target cell antigen interacting domains, and the variable domain and six target cell antigen interacting domains are ordered according to the N- to C-terminal arrangement of the following: TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH, or EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH, where (-) is a linker. In some embodiments, the effector cell antigen interacting domain comprises a terminal linker connecting the EVL and EVH. In some embodiments, the terminal linker is 15 or 16 amino acids in length. In some embodiments, one or more linkers connecting two or more target cell antigen interacting domains comprise an internal linker.In some embodiments, each of the internal linkers is 6 to 8 amino acids in length. In some embodiments, the effector cell antigen interacting domain binds to an effector cell antigen. In some embodiments, the effector cell antigen comprises CD3, CD16a, or death receptor 5 (DR5). In some embodiments, CD3 is CD3 delta, CD3 gamma, or CD3 epsilon. In some embodiments, the effector cell antigen is on an effector cell. In some embodiments, the effector cell comprises a T cell, an NK cell, or a macrophage. In some embodiments, the target cell antigen interacting domain that interacts with a target cell (T) binds to the target cell antigen. In some embodiments, the target cell antigen accumulates in lipid rafts when on cells associated with a disease state, but does not accumulate in lipid rafts when on cells not associated with a disease state. In some embodiments, the target cell antigen accumulates when on cells associated with a disease state, but does not accumulate when on cells not associated with a disease state. In some embodiments, the target cell antigen is a dimer, trimer, tetramer, or oligomer when present on a cell associated with a disease state, and a monomer or dimer when present on a cell not associated with a disease state. In some embodiments, the target cell antigen is part of a cell signaling complex when present on a cell associated with a disease state, and is not part of the same cell signaling complex when present on a cell not associated with a disease state. In some embodiments, the target cell antigen is part of a cell signaling complex when present on a cell associated with a disease state, and is not part of a cell signaling complex when present on a cell not associated with a disease state. In some embodiments, the target cell antigen comprises CD33, FAP, EGFR, HER2, or EpCAM. In some embodiments, the cell associated with a disease state is a myeloid cell, a fibroblast, or a cancer cell. In some embodiments, the single-chain polypeptide has one effector cell antigen-interacting domain.

[0017] Disclosed herein is a pharmaceutical composition comprising (a) a single-chain polypeptide according to any one of the above embodiments, and (b) a pharmaceutically acceptable excipient.

[0018] Disclosed herein is an isolated recombinant nucleic acid encoding a single chain polypeptide according to any one of the above embodiments.

[0019] Disclosed herein are vectors comprising the isolated nucleic acids according to the aforementioned embodiments.

[0020] Disclosed herein is a host cell comprising an isolated nucleic acid according to any one of the preceding embodiments, or a vector according to the preceding embodiments.

[0021] Disclosed herein are methods of treating cancer, comprising administering a single-chain polypeptide according to any one of the preceding embodiments to a subject in need thereof. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer is a hematological cancer. Some embodiments are methods of treating an inflammatory disease or condition, comprising administering a single-chain polypeptide according to any one of the preceding embodiments to a subject in need thereof. Disclosed herein are methods of treating an autoimmune disease or condition, comprising administering a single-chain polypeptide according to any one of the preceding embodiments to a subject in need thereof. Disclosed herein are methods of treating a cardiovascular disease or condition, comprising administering a single-chain polypeptide according to any one of the preceding embodiments to a subject in need thereof. Disclosed herein are methods of treating a fibrotic disease or condition, comprising administering a single-chain polypeptide according to any one of the preceding embodiments to a subject in need thereof. Disclosed herein are methods of treating a bacterial infection, comprising administering a single-chain polypeptide according to any one of the preceding embodiments to a subject in need thereof. Disclosed herein is a method of treating a viral infection, comprising administering to a subject in need thereof a single chain polypeptide according to any one of the preceding embodiments. [Brief explanation of the drawings]

[0022] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Figure 1A]

[0023] Specific non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains of a single-chain variable fragment (EV and EV), one of which binds to two target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: EVL-EVH-EVL-EVH-TVHH-TVHH, where (-) is the linker. [Figure 1B]

[0033] Specific non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains of a single-chain variable fragment (EV and EV), one of which binds to two target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: EVH-EVL-EVH-EVL-TVHH-TVHH, where (-) is the linker. [Figure 2A]

[0033] Specific non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains (EVL and EVH) of a single-chain variable fragment, one of which binds to four target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domains are VHH single-domain antibodies (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH-TVHH, where (-) is the linker. [Figure 2B]

[0023] Specific, non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains (EVL and EVH) of a single-chain variable fragment, one of which binds to four target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH-TVHH. (-) is the linker. [Figure 3A]

[0023] Specific non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains (EVL and EVH) of a single-chain variable fragment, one of which binds to four target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, where (-) is the linker. [Figure 3B]

[0023] Specific non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains (EVL and EVH) of a single-chain variable fragment, one of which binds to four target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH, where (-) is the linker. [Figure 4A]

[0033] Specific non-limiting embodiments of the single-chain polypeptide of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains of a single-chain variable fragment (EV and EV), one of which binds to one target cell antigen-interacting domain that interacts with a target cell, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: EVL-EVH-EVL-EVH-TVHH, where (-) is the linker. [Figure 4B]

[0023] Specific non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains of a single-chain variable fragment (EV and EV), one of which binds to one target cell antigen-interacting domain that interacts with a target cell, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: EVH-EVL-EVH-EVL-TVHH, where (-) is the linker. [Figure 5A]

[0023] Specific non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains (EVL and EVH) of a single-chain variable fragment, one of which binds to three target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH, where (-) is the linker. [Figure 5B]

[0023] Specific non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains (EVL and EVH) of a single-chain variable fragment, one of which binds to three target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH, where (-) is the linker. [Figure 6A]

[0033] Specific non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains (EVL and EVH) of a single-chain variable fragment, one of which binds to two target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: TVHH-EVL-EVH-EVL-EVH-TVHH, where (-) is the linker. [Figure 6B]

[0033] Specific, non-limiting embodiments of single-chain polypeptides of the present disclosure are shown, in which the single-chain polypeptide comprises variable domains connected by a linker, which fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains (EVL and EVH) of a single-chain variable fragment, one of which binds to two target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH). The variable domains and target cell antigen-interacting domains are ordered according to the following N- to C-terminal arrangement: TVHH-EVH-EVL-EVH-EVL-TVHH, where (-) is the linker. [Figure 7A] 1 illustrates T cell killing activity in a cytotoxicity assay of MCF7 cancer cells by exemplary single polypeptide constructs. [Figure 7B] 1 illustrates T cell killing activity in a cytotoxicity assay of MCF7 cancer cells by exemplary single polypeptide constructs. [Figure 8A] 1 illustrates T cell killing activity in a cytotoxicity assay of SKBR3 cancer cells by exemplary single polypeptide constructs. [Figure 8B] 1 illustrates T cell killing activity in a cytotoxicity assay of SKBR3 cancer cells by exemplary single polypeptide constructs. [Figure 9] 1 shows T cell activation of MCF7 cancer cells by exemplary single polypeptide constructs. [Figure 10] 1 shows T cell activation of SKBR3 cancer cells by exemplary single polypeptide constructs. DETAILED DESCRIPTION OF THE INVENTION

[0023] The novel features described herein are set forth with particularity in the appended claims. The features and advantages of the features described herein will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the features described herein are utilized, and the accompanying drawings, in which:

[0024] Disclosed herein are single-chain polypeptides comprising variable domains connected by a linker, wherein the variable domains fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains of a single-chain variable fragment (EV and EV), and at least one of the effector cell antigen-interacting domains binds to one or more target-interacting domains. In some embodiments, the target-interacting domain binds to a tumor antigen, a ligand, a protein, a lipid, a nucleic acid, or a polysaccharide. In some embodiments, the tumor antigen is on a cell in the tumor microenvironment. In some embodiments, the target-interacting domain comprises an antibody, an antibody fragment, a ligand, or a cofactor. In some embodiments, the antibody or antibody fragment of the target interacting domain comprises a Fab, Fab', F(ab'), Fv fragment, single chain Fv (scFv), tandem single chain Fv (scFv), dual affinity retargeting antibody, diabody, VHH single domain antibody, TriTac, Adnectin, Anticalin, Avimer, Fynomer, Kunitz domain, knottin, Affibody, or DARPin.

[0025] In some embodiments, the one or more target interacting domains comprise one or more target cell antigen interacting domains that bind to a target cell antigen. In some embodiments, the target cell antigen interacting domain binds to more than one target cell antigen. In some embodiments, the target cell antigen interacting domain is bispecific or multispecific. In some embodiments, the target cell antigen interacting domain comprises a VHH single domain antibody (TVHH). In some embodiments, the single-chain polypeptide does not comprise a fragment crystallizable (Fc) domain.

[0026] Described herein is a single-chain polypeptide comprising variable domains joined by a linker, which variable domains fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains of a single-chain variable fragment (EV and EV), one of the effector cell antigen-interacting domains directly linked to one or more target cell antigen-interacting domains that interact with target cells, wherein the target cell antigen-interacting domains are VHH single-domain antibodies (TVHH), and the single-chain polypeptide does not contain any antibody constant domains.

[0027] definition In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the provided embodiments may be practiced without these details. Throughout the following specification and claims, the word "comprises" and its variants, such as "comprises" and "comprising," should be interpreted in an open and inclusive sense, i.e., "including, but not limited to," unless the context requires otherwise. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense to include "and / or" unless the content clearly dictates otherwise. Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0028] As used herein, the term "about" refers to an amount that is closer to the recited amount by no more than 10%.

[0029] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of suffering from, or at risk of developing at least one disease that the described compositions and methods are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In certain embodiments, the individual is a human.

[0030] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided polypeptides and antibody chains, as well as other peptides, e.g., linkers and connecting peptides, can contain amino acid residues, including natural and / or unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, a polypeptide can contain modifications relative to its natural or native sequence, so long as the protein maintains the desired activity. These modifications can be deliberate, such as through site-directed mutagenesis, or can be accidental, such as through mutations of the host producing the protein or errors due to PCR amplification.

[0031] The percent sequence identity (%) to a reference polypeptide sequence is the percent of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved by various known methods using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment across the entire length of the sequences being compared. For purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code has been submitted to the U.S. Copyright Office (Washington, DC 20559) along with user documentation, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, CA), or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0032] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively referred to as an amino acid sequence A having or containing a particular % amino acid sequence identity with a given amino acid sequence B) is hereinafter 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0033] In some embodiments, amino acid sequence variants of the polypeptides provided herein are contemplated. Variants typically differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the present invention and evaluating one or more biological activities of the polypeptide as described herein and / or using any of numerous known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0034] In some embodiments, an "antigen-binding domain" or "antigen-interacting domain" refers to an immunoglobulin derivative having antigen-binding properties, i.e., an immunoglobulin polypeptide or a fragment thereof comprising an antigen-binding site. In some cases, the binding domain comprises an antibody variable domain or a fragment thereof. Each antigen-binding domain is formed by an antibody that binds to the same epitope, i.e., an immunoglobulin variable heavy domain (VH) and an antibody variable light domain (VL), where the variable heavy domain (VH) comprises three heavy chain complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3, and the variable light domain (VL) comprises three light chain complementarity-determining regions (CDRs): DR1, CDR2, and CDR3. In some examples, the binding domain according to some embodiments herein lacks an immunoglobulin constant domain. In some examples, the variable light chain domain and variable heavy chain domain that form the antigen-binding site are covalently linked to each other, for example, by a peptide linker, while in other examples, the variable light chain domain and variable heavy chain domain are non-covalently associated with each other to form the antigen-binding site. Binding domains and interaction domains also refer to antibody fragments or antibody derivatives, including, for example, Fab, Fab', F(ab'), Fv fragments, single chain Fv, tandem single chain Fv ((scFv)), bispecific T cell engagers (BiTEs), dual affinity retargeting antibodies (DARTs), diabodies, DuoBody IgG molecules, single domain antibodies (e.g., VHHs), TriTacs, etc. Furthermore, in certain instances, the binding domain is multivalent, i.e., has two, three, or more binding sites for one or more antigens.

[0035] In certain embodiments, the antigen-binding domain or antigen-interacting domain comprises a sequence derived from an antibody's complementarity-determining region. The terms "complementarity-determining region" and "CDR," which are synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3), and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).The exact amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Maryland ("Kabat" numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol. 2003 Jan, 27(1):55-77 ("IMGT" numbering scheme), Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, 2001 Jun 8, 309(3):657-70, ("Aho" numbering scheme), and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modeling antibodies on the WEB", Protein Eng. 2000 Dec, 13(12):819-24 ("AbM" numbering scheme).In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0036] The boundaries of a given CDR or FR may differ depending on the scheme used to identify it. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia numbering schemes are based on the most common antibody region sequence lengths, accommodating insertions with an insertion letter, e.g., "30a," and deletions occurring in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in differential numbering. The contact scheme is based on analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0037] In certain embodiments, the antigen-binding domain or antigen-interacting domain comprises a sequence derived from an antibody's complementarity-determining region. The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. In general, the variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies have similar structures, and each domain contains four conserved framework regions (FR) and three CDRs (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by using a VH or VL domain from an antibody that binds to the antigen to screen a complementary library of VL or VH domains, respectively (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0038] In certain embodiments, the antigen-binding or antigen-interacting domain is "humanized." A "humanized" polypeptide or antibody is one in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has been humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0039] Among the provided polypeptides and antibodies are human antibodies. A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell using a human antibody repertoire or other human antibody coding sequence, such as a human antibody library, or a non-human source. "Human antibody" excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.

[0040] The polypeptides described herein can be encoded by nucleic acids. A nucleic acid is a type of polynucleotide containing two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to introduce a polynucleotide encoding a polypeptide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a genome-integrating vector, or "integrating vector," which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal vector," e.g., a nucleic acid capable of extrachromosomal replication. A vector capable of operably linking to a gene and inducing its expression is referred to herein as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements for controlling transcription, such as promoters, enhancers, and polyadenylation signals, can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene for easy recognition of transformants can also be incorporated. Vectors derived from viruses such as lentivirus, retrovirus, adenovirus, and adeno-associated virus may also be used. Plasmid vectors can be linearized for integration into chromosomal locations. Vectors can contain sequences that direct site-specific integration (e.g., AttP-AttB recombination) into a defined location or a limited set of sites in the genome. Additionally, vectors can contain sequences derived from transposable elements.

[0041] As used herein, "homology," "homology," or "percent homology," when used herein to describe amino acid or nucleic acid sequences, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, as modified in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula has been incorporated into the Basic Local Alignment Search Tool (BLAST) of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent sequence identity can be determined using the most recent version of BLAST as of the filing date of this application.

[0042] Nucleic acids encoding the polypeptides described herein can be used to infect, transfect, transform, or otherwise render appropriate cells transgenic for the nucleic acid, thereby enabling production of the polypeptide for commercial or therapeutic use. Standard cell lines and methods for producing antibodies or polypeptides from large-scale cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production," Mabs. 2010 Sep-Oct, 2(5):466-477. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a cell line useful for producing polypeptides or antibodies, such as a Chinese hamster ovary (CHO) cell, an NS0 mouse myeloma cell, or a PER.C6® cell. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell useful for producing the polypeptide or antibody. In certain embodiments, methods of producing an antibody are described herein, comprising culturing a cell containing a nucleic acid encoding the antibody under in vitro conditions sufficient to allow for the production and secretion of the antibody.

[0043] In certain embodiments, the present invention provides a master cell bank comprising (a) a mammalian cell line comprising a nucleic acid encoding an antibody or polypeptide described herein integrated into a genomic location, and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank is contained in a suitable vial or container that can withstand freezing and storage at liquid nitrogen or an equivalent temperature.

[0044] Also described herein are methods for producing the polypeptides described herein. Such methods include incubating cells or cell lines containing nucleic acids encoding the polypeptides in a cell culture medium under conditions sufficient to allow expression and secretion of the antibody, and further recovering the polypeptide from the cell culture medium. The recovering step may further include one or more purification steps to remove viable cells, cell debris, non-target proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, additional purification steps include centrifugation, ultracentrifugation, purification of Protein A, Protein G, Protein A / G, or Protein L, size exclusion chromatography, hydrophobic interaction chromatography, and / or ion exchange chromatography.

[0045] Single-chain polypeptide compositions Disclosed herein are single-chain polypeptides that fold to form a three-dimensional binding structure. The single-chain polypeptide comprises antigen-binding domains capable of binding to an effector cell antigen and a target cell antigen. In certain embodiments, the single-chain polypeptide is bispecific and bivalent, and in other cases, bispecific and trivalent (e.g., comprising three antigen-binding domains). In certain embodiments, the single-chain polypeptide is trivalent with respect to the effector cell antigen or the target cell antigen. In certain embodiments, the single-chain polypeptide is bispecific and bivalent, and in other cases, bispecific and tetravalent (e.g., comprising four antigen-binding domains). In certain embodiments, the single-chain polypeptide may be tetravalent with respect to the effector cell antigen or the target cell antigen. In certain embodiments, the target cell antigen is a tumor-associated antigen (e.g., CD33, FAP, EGFR, HER2, or EpCAM). In certain embodiments, the effector cell antigen is an effector cell-associated antigen expressed by immune cells (e.g., T cells, NK cells, or NKT cells). Certain non-limiting embodiments of single chain polypeptides are discussed in the following paragraphs.

[0046] In certain embodiments, disclosed herein is a single-chain polypeptide comprising variable domains joined by a linker, which variable domains fold into a conformation to form one or more effector cell antigen-interacting domains (E), wherein the effector cell antigen-interacting domain comprises two variable domains of a single-chain variable fragment (EV and EV), which effector cell antigen-interacting domain is directly linked to one or more target cell antigen-interacting domains that interact with target cells, and wherein the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH), and the single-chain polypeptide does not contain any antibody constant domains.

[0047] In certain embodiments, described herein is a single-chain polypeptide comprising variable domains joined by a linker, which variable domains fold into a conformation to form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains of a single-chain variable fragment (EV and EV), one of the effector cell antigen-interacting domains directly binding to one or more target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domains are VHH single-domain antibodies (TVHH), and the single-chain polypeptide does not contain any antibody constant domains.

[0048] In certain embodiments, the two variable domains of the single-chain variable fragment (EV and EV) comprise the variable heavy chain of the single-chain variable fragment (EVH) and the variable light chain of the single-chain variable fragment (EVL).

[0049] Branched three-dimensional organization of target cell antigen-interacting domains In some embodiments, one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to at least one TVHH, and one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to at least one TVHH. In some embodiments, at least one TVHH that binds to EVL binds to the same target antigen as at least one TVHH that binds to EVH. In some embodiments, at least one TVHH that binds to EVL binds to the same target antigen as at least one TVHH that binds to EVH and has the same complementarity-determining region (CDR) sequence. In some embodiments, at least one TVHH that binds to EVL binds to the same target antigen as at least one TVHH that binds to EVH, but does not share a common complementarity-determining region (CDR) sequence. In some embodiments, at least one TVHH that binds to EVL binds to a different target antigen than at least one TVHH that binds to EVH.

[0050] In some embodiments, EVL and EVH bind to the same number of TVHHs. In some embodiments, EVL binds to a different number of TVHHs compared to the number of TVHHs that bind to EVHs.

[0051] In some embodiments, one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH.

[0052] In some embodiments, one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to two TVHH.

[0053] In some embodiments, one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to three TVHH.

[0054] In some embodiments, one EVKH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to two TVHH.

[0055] In some embodiments, one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to three TVHH.

[0056] In some embodiments, one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH. Figures 6A and 6B illustrate examples of single-chain polypeptides in which one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH.

[0057] In some embodiments, one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to one TVHH, and one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to two TVHHs. Figures 3A and 3B illustrate examples of single-chain polypeptides in which one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to two TVHHs, and one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to two TVHHs.

[0058] In some embodiments, one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to three TVHHs, and one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to three TVHHs.

[0059] In some embodiments, one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to four TVHHs, and one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to four TVHHs.

[0060] In some embodiments, one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to five TVHHs, and one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to five TVHHs.

[0061] In some embodiments, one EVH of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to six TVHHs, and one EVL of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to six TVHHs.

[0062] In some embodiments, one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to seven TVHHs, and one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to seven TVHHs.

[0063] In some embodiments, one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to eight TVHHs, and one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to eight TVHHs.

[0064] Linear three-dimensional structure of the target cell antigen interaction domain In some embodiments, one of the effector cell antigen interaction domains that bind to one or more target cell antigen interaction domains is bound to one target cell antigen interaction domain. In some embodiments, one target cell antigen interaction domain is bound to one of the effector cell antigen interaction domains at the C-terminus. In some embodiments, one target cell antigen interaction domain is bound to one of the effector cell antigen interaction domains at the N-terminus. Figures 4A and 4B illustrate single-chain polypeptides in which one target cell antigen interaction domain is bound to one of the effector cell antigen interaction domains at the C-terminus.

[0065] In some embodiments, at least one of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to more than one target cell antigen interacting domain. In some embodiments, all of the more than one target cell antigen interacting domains bind to the same target antigen. In some embodiments, all of the more than one target cell antigen interacting domains bind to the same target antigen and all have the same CDR sequence. In some embodiments, all of the more than one target cell antigen interacting domains bind to the same target antigen, but at least two of the more than one target cell antigen interacting domains do not share a common CDR sequence. In some embodiments, at least two of the more than one target cell antigen interacting domains bind to different target antigens.

[0066] In some embodiments, one of the effector cell antigen interaction domains that bind to one or more target cell antigen interaction domains binds to two target cell antigen interaction domains. In some embodiments, the two target cell antigen interaction domains are C-terminally linked to one of the effector cell antigen interaction domains. Figures 1A and 1B illustrate single-chain polypeptides in which two target cell antigen interaction domains are C-terminally linked to an effector cell antigen interaction domain.

[0067] In some embodiments, one of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to three target cell antigen interacting domains. In some embodiments, the three target cell antigen interacting domains are C-terminally linked to one of the effector cell antigen interacting domains. In some embodiments, the three target cell antigen interacting domains are N-terminally linked to one of the effector cell antigen interacting domains. Figures 5A and 5B illustrate single-chain polypeptides in which three target cell antigen interacting domains are C-terminally linked to the latter one of the effector cell antigen interacting domains.

[0068] In some embodiments, one of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to four target cell antigen interacting domains. In some embodiments, the four target cell antigen interacting domains are C-terminally linked to one of the effector cell antigen interacting domains. Figures 2A and 2B illustrate single-chain polypeptides in which four target cell antigen interacting domains are C-terminally linked to one of the effector cell antigen interacting domains.

[0069] N- to C-terminal arrangement of a single polypeptide chain Further configurations and specific embodiments regarding the placement of the target cell antigen-interacting domain (T) or effector cell antigen-interacting domain (E) are provided below.

[0070] In certain embodiments, the single-chain polypeptide comprises one or more target cell antigen interacting domains (T) and one or more effector cell antigen interacting domains (E), wherein the effector cell antigen interacting domains and the one or more target cell antigen interacting domains are selected from the following, where (-) is a linker: TVHH-EV-EV-EV-EV, EV-EV-EV-EV-EV-TVHH, TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH-TVHH, T and are ordered according to any one or more of the following N- to C-terminal configurations: VHH-TVHH-TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH, TVHH-EV-EV-EV-EV-EV-TVHH, TVHH-EV-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, TVHH-EV-EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH, or TVHH-TVHH-EV-EV-EV-EV-EV-TVHH-TVHH.

[0071] In some embodiments, the variable domain, and the one or more target cell antigen interaction domains are ordered according to the arrangement from the N-terminus to the C-terminus of the following, where (-) is a linker: TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH, TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH, TVHH-EV-EV-EV-EV-TVHH, TVHH-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-EV-EV-EV-EV-TVHH, TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH, TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, or TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH.

[0072] In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, or TVHH-EV-EV-EV-EV-TVHH, where (-) is a linker. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, where (-) is a linker. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-EV-EV-EV-EV-TVHH, where (-) is a linker.

[0073] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N- to C-terminal configuration: TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, where (-) is a linker.

[0074] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following arrangement from N-terminus to C-terminus: TVHH-EV-EV-EV-EV-TVHH, where (-) is a linker.

[0075] In certain embodiments, the single-chain polypeptide comprises one or more target cell antigen interacting domains (T) and one or more effector cell antigen interacting domains (E), wherein the one or more effector cell antigen interacting domains and the one or more target cell antigen interacting domains are arranged in the following order, where (-) is a linker: TVHH-EVL-EVH-EVL-EVH, TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH, EVH-EVL-EVH-EVL-TVHH, TVHH-TVHH-EVL-EV H-EVL-EVH, TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH, TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-T VHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH, TV HH-TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH-TVHH, TVHH-EVL-EVH-EVL-EV H-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH , TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EVL-EV H-EVL-EVH-TVHH, TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH. The amino acids are ordered according to any one or more of the following arrangements from N-terminus to C-terminus:

[0076] In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following, where (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, TVHH-TVHH-EVH-EVL-EVH-EVL-EVH-TVHH-TVHH, TVHH-EVL-EVH-EVL-EVH-EVL-EVH-TVHH, or TVHH-EVH-EVL-EVH-EVL-EVH-EVL-TVHH. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following, where (-) is a linker: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVH-EVL-EVH-EVL-TVHH-TVHH. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-EVL-EVH-EVL-EVH-TVHH, or TVHH-EVH-EVH-EVL-EVH-EVL-TVHH, where (-) is a linker. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, where (-) is a linker. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N-terminal to C-terminal arrangement of the following: TVHH-TVHH-EVL-EVH-EVL-TVHH-TVHH, where (-) is a linker. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N- to C-terminus arrangement of the following, where (-) is a linker: TVHH-EVL-EVH-EVL-EVH-TVHH. In some embodiments, the variable domain and one or more target cell antigen interacting domains are ordered according to the N- to C-terminus arrangement of the following, where (-) is a linker: TVHH-EVH-EVL-EVH-EVL-TVHH.

[0077] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N- to C-terminal configuration: TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, where (-) is a linker.

[0078] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N- to C-terminal configuration: TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH, where (-) is a linker.

[0079] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N- to C-terminal configuration: TVHH-EVL-EVH-EVL-EVH-TVHH, where (-) is a linker.

[0080] In some embodiments, the variable domains and one or more target cell antigen interacting domains are ordered according to the following N- to C-terminal configuration: TVHH-EVH-EVL-EVH-EVL-TVHH, where (-) is a linker.

[0081] In some embodiments, the variable domains and target cell antigen interacting domains are ordered according to the N-terminal to C-terminal configuration of the following: TVHH-EVH-EVL-TVHH-TVHH-EVH-EVL-TVHH, TVHH-EVL-EVH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-EVH-EVL-TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH-EVL-EVH-TVHH, and TVHH-EVL-EVH-TVHH-TVHH-EVL-EVH-TVHH, where (-) is a linker.

[0082] In some embodiments, the variable domains and target cell antigen interacting domains are ordered according to the following N- to C-terminal configuration: TVHH-EVH-EVL-TVHH-TVHH-EVH-EVL-TVHH, where (-) is a linker.

[0083] In some embodiments, the variable domains and target cell antigen interacting domains are ordered according to the following N- to C-terminal configuration: TVHH-EVL-EVH-TVHH-TVHH-EVH-EVL-TVHH, where (-) is a linker.

[0084] In some embodiments, the variable domains and target cell antigen interacting domains are ordered according to the following N- to C-terminal configuration: TVHH-EVH-EVL-TVHH-TVHH-EVL-EVH-TVHH, where (-) is a linker.

[0085] In some embodiments, the variable domains and target cell antigen interacting domains are ordered according to the following N- to C-terminal configuration: TVHH-EVL-EVH-TVHH-TVHH-EVL-EVH-TVHH, where (-) is a linker.

[0086] Further configuration Disclosed herein is a single-chain polypeptide comprising two variable domains (EV and EV) of a single-chain variable fragment joined by a linker, which fold into a three-dimensional structure to form an effector cell antigen-interacting domain (E), wherein the effector cell antigen-interacting domain is linked by one or more linkers to two or more target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH), and the single-chain polypeptide does not contain any antibody constant domains.

[0087] In some embodiments, the two variable domains of the single-chain variable fragment (EV and EV) comprise the variable heavy chain of the single-chain variable fragment (EVH) and the variable light chain of the single-chain variable fragment (EVL).

[0088] In some embodiments, two or more target cell antigen interacting domains are linked to the effector cell antigen interacting domain at the C-terminus. Figure 4B illustrates a single-chain polypeptide in which six target cell antigen interacting domains are linked to the effector cell antigen interacting domain at the C-terminus. In some embodiments, two or more target cell antigen interacting domains are linked to the effector cell antigen interacting domain at the N-terminus. In some embodiments, two or more target cell antigen interacting domains are linked to the effector cell antigen interacting domain at the N-terminus and C-terminus. Figure 4A illustrates a single-chain polypeptide in which six target cell antigen interacting domains are linked to the effector cell antigen interacting domain at the N-terminus and C-terminus.

[0089] In some embodiments, the effector cell antigen interacting domain binds to two target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to three target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to four target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to five target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to six target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to seven target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to eight target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to nine target cell antigen interacting domains. In some embodiments, the effector cell antigen interacting domain binds to ten target cell antigen interacting domains.

[0090] N- to C-terminal arrangement of a single polypeptide chain Further configurations and specific embodiments regarding the placement of the target cell antigen-interacting domain (T) or effector cell antigen-interacting domain (E) are provided below.

[0091] In some embodiments, the effector cell antigen interacting domain is linked to two target cell antigen interacting domains, and the variable domain and the two target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal configuration: TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH, EVL-EVH-TVHH-TVHH, TVHH-TVHH-EVH-EVL, TVHH-EVL-EVH-TVHH, or TVHH-EVH-EVL-TVHH, where (-) is a linker.

[0092] In some embodiments, the effector cell antigen interacting domain is linked to three target cell antigen interacting domains, and the variable domain and three target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal configuration: TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EVH-EVL, TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH, TVHH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-TVHH, where (-) is a linker.

[0093] In some embodiments, the effector cell antigen interacting domain is linked to four target cell antigen interacting domains, and the variable domain and the four target cell antigen interacting domains are arranged in the following order, where (-) is a linker: TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EV They are ordered according to their arrangement from N-terminus to C-terminus: H-EVL, TVHH-TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH-TVHH, TVHH-EVL-EVH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-TVHH-TVHH.

[0094] In some embodiments, the effector cell antigen interacting domain is linked to five target cell antigen interacting domains, and the variable domain and the five target cell antigen interacting domains are arranged in the following order, where (-) is a linker: TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL, TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH , TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH, TVHH-EVL-EVH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH, TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH, or TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH.

[0095] In some embodiments, the effector cell antigen interacting domain is linked to six target cell antigen interacting domains, and the variable domain and the six target cell antigen interacting domains are arranged in the following order, where (-) is a linker: TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL, TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH -EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH, TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-EVL-EV They are ordered according to the following arrangement from N-terminus to C-terminus: H-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH, TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH, or TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH.

[0096] In some embodiments, the effector cell antigen interacting domain is linked to six target cell antigen interacting domains, and the variable domain and six target cell antigen interacting domains are ordered according to the following N-terminal to C-terminal configuration: TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH, or EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, where (-) is a linker.

[0097] In some embodiments, the effector cell antigen interacting domain is linked to six target cell antigen interacting domains, and the variable domains and target cell antigen interacting domains are ordered according to the following arrangement from N-terminus to C-terminus: TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH, where (-) is a linker.

[0098] In some embodiments, the effector cell antigen interacting domain is linked to six target cell antigen interacting domains, and the variable domains and target cell antigen interacting domains are ordered according to the following arrangement from N-terminus to C-terminus: EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, where (-) is a linker.

[0099] Linker sequence In certain embodiments, the linker connecting the two effector cell antigen-interacting domains (E) to each other comprises an internal linker.

[0100] In certain embodiments, the effector cell antigen interacting domain that is not directly linked to the one or more target cell antigen interacting domains comprises a terminal linker connecting the EVL and EVH.

[0101] In some embodiments, the effector cell antigen interacting domain comprises a terminal linker connecting the EVL and EVH.

[0102] In some embodiments, the one or more linkers joining two or more target cell antigen interacting domains comprise an internal linker.

[0103] In some embodiments, the length of the internal linker is about 4 amino acids to about 30 amino acids. For example, the length of the internal linker can be about 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 5 to 29, 6 to 29, 7 to 29, 8 to 29, 9 to 29, 10 to 29, 5 to 28, 6 to 28, 7 to 28, 8 to 28, 9 to 28, 10 to 28, 5 to 27, 6 to 27, 7 to 27, 8 to 27, 9-27, 10-27, 5-26, 6-26, 7-26, 8-26, 9-26, 10-26, 5-25, 6-25, 7-25, 8-25, 9-25, 10-25, 5-24, 6-24, 7-24, 8-24, 9-24, 10-24, 5-23, 6-23, 7-23, 8-23, 9-23, 1 0~23, 5~22, 6~22, 7~22, 8~22, 9~22, 10~22, 5~21, 6~21, 7~21, 8~21, 9~21, 10~21, 5~20, 6~20, 7~20, 8~20, 9~20, 10~20, 5~19, 6~19, 7~19, 8~19, 9~19, 10~19, In some embodiments, the internal linkers are about 5, 6, 7, 8, 9, 10, 18, 5, 17, 6, 17, 7, 17, 8, 17, 9, 17, 10, 17, 5, 16, 6, 16, 7, 16, 8, 16, 9, 16, 10, 16, 5, 15, 6, 15, 7, 15, 8, 15, 9, 15, or 10 to 15 amino acids in length. In some embodiments, the internal linkers are about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In certain embodiments, each of the internal linkers is 6 to 8 amino acids in length.

[0104] In some embodiments, the length of the terminal linker is about 10 amino acids to about 30 amino acids. For example, the length of the terminal linker is about 10 to 30, 11 to 30, 12 to 30, 13 to 30, 14 to 30, 15 to 30, 10 to 29, 11 to 29, 12 to 29, 13 to 29, 14 to 29, 15 to 29, 10 to 28, 11 to 28, 12 to 28, 13 to 28, 14 to 28, 15 to 28, 10 to 27, 11 to 27, 12 to 2 7, 13-27, 14-27, 15-27, 10-26, 11-26, 12-26, 13-26, 14-26, 15-26, 10-25, 11-25, 12-25, 13-25, 14-25, 15-25, 10-24, 11-24, 12-24, 13-24, 14-24, 15-24, 10-23, 11-23, 1 2-23, 13-23, 14-23, 15-23, 10-22, 11-22, 12-22, 13-22, 14-22, 15-22, 10-21, 11-21, 12-21, 13-21, 14-21, 15-21, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 10-19, 11- In some embodiments, the terminal linker is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 12-19, 13-19, 14-19, 15-19, 10-18, 11-18, 12-18, 13-18, 14-18, 15-18, 10-17, 11-17, 12-17, 13-17, 14-17, 15-17, 10-16, 11-16, 12-16, 13-16, 14-16, or 15-16. In some embodiments, the terminal linker is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In certain embodiments, the terminal linker is 15 or 16 amino acids in length. In some embodiments, the terminal linker is about 16 amino acids in length.

[0105] In some embodiments, the internal linker is about 25 to about 45 amino acids in length. For example, the internal linker is about 25 to 45, 26 to 44, 27 to 43, 28 to 42, 29 to 41, 30 to 40, 31 to 39, 32 to 38, 33 to 37, or 34 to 36 amino acids in length. In some embodiments, the internal linker is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids in length. In some embodiments, the internal linker is 35 amino acids in length.

[0106] In some embodiments, the terminal linker is about 25 to about 45 amino acids in length. For example, the terminal linker is about 25 to 45, 26 to 44, 27 to 43, 28 to 42, 29 to 41, 30 to 40, 31 to 39, 32 to 38, 33 to 37, or 34 to 36 amino acids in length. In some embodiments, the terminal linker is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids in length. In some embodiments, the terminal linker is 35 amino acids in length.

[0107] In some embodiments, the linker is sized to force pairing of the heavy and light chain variable domains to form two interacting effector cell antigens in a single polypeptide chain, eliminating mismatch pairing, preventing aggregation and multiple species during purification, diffusion, and thus facilitating manufacturing.

[0108] In some embodiments, the linker comprises glycine (G) and / or serine (S). In some embodiments, the linker comprises a sequence as disclosed in Table 1, or a sequence substantially identical to a sequence as disclosed in Table 1 (e.g., a sequence with 0-2 amino acid modifications, substitutions, deletions).

[0109] [Table 1]

[0110] Effector cell antigen-binding domain In some embodiments, the effector cell antigen interaction domain binds to an effector cell antigen. In some embodiments, the CD3 is CD3 delta, CD3 gamma, or CD3 epsilon. In some embodiments, the effector cell antigen is on an effector cell. In some embodiments, the effector cell comprises a T cell, an NK cell, or a macrophage. In some embodiments, the effector cell antigen comprises CD3, CD16a, or death receptor 5 (DR5).

[0111] CD3-binding domain In some embodiments, EVH comprises a variable heavy chain of a single chain variable fragment that binds CD3, and EVL comprises a variable light chain of a single chain variable fragment that binds CD3.

[0112] In some embodiments, the EVH comprises an amino acid sequence set forth in Table 2, or a sequence substantially identical to an amino acid sequence set forth in Table 2 (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the EVH comprises the CDR1 sequence of STYAMN (SEQ ID NO: 13), or a sequence having 0-2 amino acid modifications, substitutions, or deletions. In some embodiments, the EVH comprises the CDR2 sequence of RIRSKYNNYATYYADSVKD (SEQ ID NO: 14), or a sequence having 0-2 amino acid modifications, substitutions, or deletions. In some embodiments, the EVH comprises the CDR3 sequence of HGNFGNSYVSWFAY (SEQ ID NO: 15), or a sequence having 0-2 amino acid modifications, substitutions, or deletions. In some embodiments, the EVH comprises the CDR3 sequence of HGNFGNSYVSYFAY (SEQ ID NO: 16), or a sequence having 0-2 amino acid modifications, substitutions, or deletions.

[0113] In some embodiments, the EVL comprises an amino acid sequence set forth in Table 3, or a sequence substantially identical to an amino acid sequence set forth in Table 3 (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the EVL comprises the CDR1 sequence of RSSTGAVTTSNYAN (SEQ ID NO: 17), or a sequence having 0-2 amino acid modifications, substitutions, or deletions. In some embodiments, the EVL comprises the CDR2 sequence of GTNKRAP (SEQ ID NO: 18), or a sequence having 0-2 amino acid modifications, substitutions, or deletions. In some embodiments, the EVL comprises the CDR3 sequence of ALWYSNL (SEQ ID NO: 19), or a sequence having 0-2 amino acid modifications, substitutions, or deletions.

[0114] [Table 2]

[0115] [Table 3]

[0116] In some examples, the CDR1, CDR2, and CDR3 from EVH or EVL are derived from other CD3 antibodies, such as, for example, UCHT1, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), and visilizumab (Nuvion).

[0117] In some aspects, described herein are anti-CD3 binding molecules that comprise a humanized or fully human, i.e., human-derived, anti-CD3 binding domain. In some embodiments, described herein are anti-CD3 binding molecules that comprise a camel or llama anti-CD3 binding domain.

[0118] In certain instances, the CD3 binding site has moderate or low affinity as a monomer and high affinity and / or specificity as a bivalent molecule due to avidity.

[0119] CD16a-binding domain In some embodiments, EVH comprises a variable heavy chain of a single chain variable fragment that binds to CD16a, and EVL comprises a variable light chain of a single chain variable fragment that binds to CD16a.

[0120] In some embodiments, the EVH comprises the amino acid sequence set forth in SEQ ID NO: 20, or a sequence substantially identical to the amino acid sequence set forth in SEQ ID NO: 20 (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0121] In some embodiments, the EVL comprises the amino acid sequence set forth in SEQ ID NO:21, or a sequence substantially identical to the amino acid sequence set forth in SEQ ID NO:21 (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0122] In some embodiments, described herein are anti-CD16a binding molecules that comprise a humanized or fully human, i.e., human-derived, anti-CD16a binding domain.

[0123] [Table 4]

[0124] Death receptor 5 binding domain In some embodiments, EVH comprises the variable heavy chain of a single chain variable fragment that binds to DR5, and EVL comprises the variable light chain of a single chain variable fragment that binds to DR5.

[0125] In some embodiments, the EVH comprises an amino acid sequence set forth in SEQ ID NOs: 22-26, or a sequence substantially identical to the amino acid sequence set forth in SEQ ID NOs: 22-26 (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0126] In some embodiments, the EVL comprises an amino acid sequence set forth in SEQ ID NOs: 27-31, or a sequence substantially identical (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence set forth in SEQ ID NOs: 227-31.

[0127] In some embodiments, described herein are anti-DR5 binding molecules that comprise a humanized or fully human, i.e., human-derived, anti-DR5 binding domain.

[0128] [Table 5]

[0129] Target cell antigen interaction domain In some embodiments, the target cell antigen-interacting domain that interacts with target cells (T) binds to the target cell antigen. In some embodiments, the target cell antigen accumulates in lipid rafts when present on cells associated with a disease state, but does not accumulate in lipid rafts when present on cells not associated with a disease state. In some embodiments, the target cell antigen accumulates when present on cells associated with a disease state, but does not accumulate when present on cells not associated with a disease state. In some embodiments, the target cell antigen is 100 nm or smaller when present on cells not associated with a disease state, but is not within 100 nm when present on cells not associated with a disease state. In some embodiments, the target cell antigen is 75 nm or smaller when present on cells associated with a disease state, but is not within 75 nm when present on cells not associated with a disease state. In some embodiments, the target cell antigen is 50 nm or smaller when present on cells associated with a disease state, but is not within 50 nm when present on cells not associated with a disease state. In some embodiments, the target cell antigen is 25 nm or smaller when present on cells associated with a disease state, but is not within 25 nm when present on cells not associated with a disease state. In some embodiments, the target cell antigen is 10 nm or less when on a cell associated with a disease state, but not within 10 nm when on a cell not associated with a disease state. In some embodiments, the target cell antigen is 5 nm or less when on a cell associated with a disease state, but not within 5 nm when on a cell not associated with a disease state. In some embodiments, the target cell antigen is 2 nm or less when on a cell associated with a disease state, but not within 2 nm when on a cell not associated with a disease state.

[0130] In some embodiments, the target cell antigens are in close proximity when present on cells associated with a disease state and are not in close proximity when present on cells not associated with a disease state. In some embodiments, the target cell antigen is a dimer, trimer, tetramer, or oligomer when present on cells associated with a disease state and a monomer or dimer when present on cells not associated with a disease state. In some embodiments, the target cell antigen is part of a cell signaling complex when present on cells associated with a disease state and is not part of the same cell signaling complex when present on cells not associated with a disease state. In some embodiments, the target cell antigen is part of a cell signaling complex when present on cells associated with a disease state and is not part of a cell signaling complex when present on cells not associated with a disease state. In some embodiments, the cells associated with a disease state are myeloid cells, fibroblasts, or cancer cells. In some embodiments, the target cell antigen comprises CD33, FAP, EGFR, HER2, or EpCAM.

[0131] CD33-binding domain In some embodiments, the TVHH comprises a VHH single domain antibody that binds to CD33. In some embodiments, the TVHH comprises an amino acid sequence set forth in SEQ ID NOs: 32-43, 72-74, or a sequence substantially identical (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) to an amino acid sequence set forth in SEQ ID NOs: 32-43, 72-74.

[0132] [Table 6]

[0133] HER2-binding domain In some embodiments, the TVHH comprises a VHH single domain antibody that binds to HER2. In some embodiments, the TVHH comprises the amino acid sequence set forth in SEQ ID NOs: 44-46, or a sequence substantially identical to the amino acid sequence set forth in SEQ ID NOs: 44-46 (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0134] [Table 7]

[0135] EGFR-binding domain In some embodiments, the TVHH comprises a VHH single domain antibody that binds to EGFR. In some embodiments, the TVHH comprises the amino acid sequence set forth in SEQ ID NOs: 47-49, or a sequence substantially identical to the amino acid sequence set forth in SEQ ID NOs: 47-49 (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0136] [Table 8]

[0137] FAP-binding domain In some embodiments, the TVHH comprises a VHH single domain antibody that binds to a FAP. In some embodiments, the TVHH comprises the amino acid sequence set forth in SEQ ID NOs: 51, 65-67, or a sequence substantially identical (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence set forth in SEQ ID NOs: 51, 65-67.

[0138] [Table 9]

[0139] EpCAM-binding domain In some embodiments, the TVHH comprises a VHH single domain antibody that binds to EpCAM. In some embodiments, the TVHH comprises the amino acid sequence set forth in SEQ ID NOs: 68-71, or a sequence substantially identical to the amino acid sequence set forth in SEQ ID NOs: 68-71 (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0140] [Table 10]

[0141] Exemplary Single Chain Polypeptide (SCP) Compositions SCP1 In some embodiments, the single-chain polypeptide comprises two target cell antigen-interacting domains. In some embodiments, the variable domain and the target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: EVH-EVL-EVH-EVL-TVHH-TVHH, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 52. A summary of the amino acid sequences of the domains of SCP1 is shown in Table 11.

[0142] [Table 11]

[0143] SCP2 In some embodiments, the single-chain polypeptide comprises two target cell antigen-interacting domains. In some embodiments, the variable domain and the target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: EVL-EVH-EVL-EVH-TVHH-TVHH, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 53. A summary of the amino acid sequences of the domains of SCP2 is shown in Table 12.

[0144] [Table 12-1]

[0145] [Table 12-2]

[0146] SCP3 In some embodiments, the single-chain polypeptide comprises two target cell antigen-interacting domains. In some embodiments, the variable domain and the target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: EVH-EVL-EVH-EVL-TVHH-TVHH, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 54. A summary of the amino acid sequences of the domains of SCP3 is shown in Table 13.

[0147] [Table 13]

[0148] SCP4 In some embodiments, the single-chain polypeptide comprises two target cell antigen-interacting domains. In some embodiments, the variable domain and the target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: EVL-EVH-EVL-EVH-TVHH-TVHH, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 55. A summary of the amino acid sequences of the domains of SCP4 is shown in Table 14.

[0149] [Table 14-1]

[0150] [Table 14-2]

[0151] SCP5 In some embodiments, the single-chain polypeptide comprises two target cell antigen-interacting domains. In some embodiments, the variable domain and the target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: TVHH-TVHH-EVH-EVL-EVH-EVL, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 56. A summary of the amino acid sequences of the domains of SCP5 is shown in Table 15.

[0152] [Table 15-1]

[0153] [Table 15-2]

[0154] SCP6 In some embodiments, the single-chain polypeptide comprises two target cell antigen-interacting domains. In some embodiments, the variable domain and the target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: TVHH-TVHH-EVL-EVH-EVL-EVH, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 57. A summary of the amino acid sequences of the domains of SCP6 is shown in Table 16.

[0155] [Table 16]

[0156] SCP7 In some embodiments, the single-chain polypeptide comprises two target cell antigen-interacting domains. In some embodiments, the variable domain and the target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: TVHH-TVHH-EVH-EVL-EVH-EVL, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 58. A summary of the amino acid sequences of the domains of SCP7 is shown in Table 17.

[0157] [Table 17-1]

[0158] [Table 17-2]

[0159] SCP8 In some embodiments, the single-chain polypeptide comprises two target cell antigen-interacting domains. In some embodiments, the variable domain and the target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: TVHH-TVHH-EVL-EVH-EVL-EVH, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 59. A summary of the amino acid sequences of the domains of SCP8 is shown in Table 18.

[0160] [Table 18-1]

[0161] [Table 18-2]

[0162] SCP9 In some embodiments, the single-chain polypeptide comprises one target cell antigen-interacting domain. In some embodiments, the variable domain and one target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: EVH-EVL-EVH-EVL-TVHH, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 60. A summary of the amino acid sequences of the domains of SCP9 is shown in Table 19.

[0163] [Table 19-1]

[0164] [Table 19-2]

[0165] SCP10 In some embodiments, the single-chain polypeptide comprises one target cell antigen-interacting domain. In some embodiments, the variable domain and one target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: EVL-EVH-EVL-EVH-TVHH, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61. A summary of the amino acid sequences of the domains of SCP10 is shown in Table 20.

[0166] [Table 20-1]

[0167] [Table 20-2]

[0168] SCP11 In some embodiments, the single-chain polypeptide comprises one target cell antigen-interacting domain. In some embodiments, the variable domain and one target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: EVH-EVL-EVH-EVL-TVHH, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 62. A summary of the amino acid sequences of the domains of SCP11 is shown in Table 21.

[0169] [Table 21-1]

[0170] [Table 21-2]

[0171] SCP12 In some embodiments, the single-chain polypeptide comprises one target cell antigen-interacting domain. In some embodiments, the variable domain and one target cell antigen-interacting domain are ordered according to the following N- to C-terminus arrangement: EVL-EVH-EVL-EVH-TVHH, where (-) is a linker. In some embodiments, EVH comprises a variable heavy chain of a single-chain variable fragment that binds to CD3, and EVL comprises a variable light chain of a single-chain variable fragment that binds to CD3. In some embodiments, TVHH comprises a VHH single-domain antibody that binds to CD33. In some embodiments, the single-chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 63. A summary of the amino acid sequences of the domains of SCP12 is shown in Table 22.

[0172] [Table 22-1]

[0173] [Table 22-2]

[0174] CDR sequences and conservative variants of heavy and light chain domains In some embodiments, the binding domain of the binding molecule comprises an immunologically active homolog or variant of the CDR sequences described herein. Thus, in some embodiments, the CDR sequences in the heavy or light chain domain that bind to the target antigen or effector cell are similar, but not identical, to the amino acid sequences set forth in SEQ ID NOs: 7-63. In particular examples, the sequences of the CDR variants have 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity compared to the sequences of SEQ ID NOs: 13-19 and are immunologically active.

[0175] In further examples, the sequence of a DR variant incorporates 1, 2, 3, 4, or 5 conservative amino acid substitutions. Conservative substitutions include those that substitute a given amino acid for another amino acid with similar properties, and further include aliphatic amino acid interchanges of alanine, valine, leucine, and isoleucine, interchanges of the hydroxylated residues serine and threonine, interchanges of the acidic residues aspartate and glutamate, interchanges between the amide residues asparagine and glutamine, interchanges of the basic residues lysine and arginine, and interchanges among the aromatic residues phenylalanine and tyrosine.

[0176] In another further example, the sequences of the CDR variants incorporate substitutions that enhance properties of the CDRs, such as stability, resistance to proteases, and / or improved binding affinity for the target antigen or CD3.

[0177] In other examples, CDR variant sequences are modified to alter non-critical residues or residues in non-critical regions. Non-critical amino acids can be identified by known methods such as affinity maturation, CDR walking, site-directed mutagenesis, crystallization, nuclear magnetic resonance, photoaffinity labeling, alanine scanning mutagenesis, etc.

[0178] In further embodiments, the binding molecules comprise heavy and light chain domains that are immunologically active homologs or variants of the heavy and light chain domain sequences provided herein. Thus, in some embodiments, the binding domains comprise heavy or light chain domain sequences that are similar but not identical to the binding domains provided herein. In certain examples, the variant heavy or light chain domain sequences have 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the sequences herein and are immunologically active.

[0179] In further embodiments, the variant heavy or light chain domain sequences incorporate one, two, three, four, or five conservative amino acid substitutions. Conservative substitutions include those that substitute a given amino acid for another amino acid with similar properties, and further include aliphatic amino acid interchanges of alanine, valine, leucine, and isoleucine, interchanges of the hydroxylated residues serine and threonine, interchanges of the acidic residues aspartate and glutamate, interchanges between the amide residues asparagine and glutamine, interchanges of the basic residues lysine and arginine, and interchanges among the aromatic residues phenylalanine and tyrosine.

[0180] In a further example, the variant heavy or light chain domain sequences incorporate substitutions that enhance properties of the CDRs, such as stability, resistance to proteases, and / or improved binding affinity for the antigen.

[0181] In a further example, the variant heavy or light chain domain sequences are modified to alter non-critical residues or residues in non-critical regions. Non-critical amino acids can be identified by known methods such as affinity maturation, CDR walking, site-directed mutagenesis, crystallization, nuclear magnetic resonance, photoaffinity labeling, alanine scanning mutagenesis, etc.

[0182] The percent sequence identity (%) to a reference polypeptide sequence is the percent of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved by various known methods using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment across the entire length of the sequences being compared. For purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code has been submitted to the U.S. Copyright Office (Washington, DC 20559) along with user documentation, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, CA), or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0183] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively referred to as an amino acid sequence A having or containing a particular % amino acid sequence identity with a given amino acid sequence B) is hereinafter 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0184] Treatment method In certain embodiments, antibodies useful for treating cancer or tumors are disclosed herein. Treatment refers to a method that seeks to improve or ameliorate the condition being treated. With respect to cancer, treatment includes, but is not limited to, reducing tumor volume, reducing tumor volume growth, extending progression-free survival, or extending overall life expectancy. In certain embodiments, treatment affects remission of cancer during treatment. In certain embodiments, treatment encompasses use as a prophylactic or maintenance administration intended to prevent the recurrence or progression of a previously treated cancer or tumor. Treatment of bacterial or viral diseases includes, but is not limited to, alleviating one or more symptoms associated with a viral or bacterial disease, such as fever, nausea, diarrhea, vomiting, sore throat, cough, runny nose, and / or rash. Treatment of bacterial or viral diseases can reduce the overall level of viruses or bacteria in the body, shorten the period an individual is potentially infectious to others, or shorten the duration of illness or recovery. Treatment of autoimmune or inflammatory diseases includes, but is not limited to, reducing total antibody or autoantibody levels, or reducing total immune response or autocellular immune response. Treatment may also be associated with specific symptoms of autoimmune diseases associated with excessive antibody or cellular immune response. Treatment of fibrotic diseases can reduce or delay the appearance of fibrous tissue or collagen deposition in tissue. Treatment of cardiovascular diseases can increase indicators of cardiovascular health, including lowering blood pressure, reducing atherosclerotic lesions, or improving cardiac function as indicated by the ability to pump blood. Those skilled in the art will understand that not all individuals respond equally or at all to administered treatment, but these individuals are nonetheless considered to be treated.

[0185] In certain embodiments, the single chain polypeptide is for use in the treatment of a viral infection. In certain embodiments, the single chain polypeptide is for use in the treatment of a bacterial infection. In certain embodiments, the single chain polypeptide is for use in the treatment of a solid tumor cancer. In certain embodiments, the single chain polypeptide is for use in the treatment of a hematological cancer. In certain embodiments, the single chain polypeptide is for use in the treatment of an inflammatory condition. In certain embodiments, the single chain polypeptide is for use in the treatment of an autoimmune disease. In certain embodiments, the single chain polypeptide is for use in the treatment of a cardiovascular disease. In certain embodiments, the single chain polypeptide is for use in the treatment of a fibrotic disease.

[0186] The single-chain polypeptide molecules described herein are intended for use as pharmaceuticals.Administration can be carried out by different methods, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, intralesional, topical or intradermal administration.In some embodiments, the administration route varies depending on the type of treatment and the type of compound contained in the pharmaceutical composition.The administration regimen is determined by the attending physician and other clinical factors.The dosage per patient varies depending on many factors, including the patient's size, body surface area, age, sex, the specific compound administered, the time and route of administration, the type of treatment, general health condition, and other drugs administered at the same time.

[0187] An "effective dose" refers to an amount of the active ingredient sufficient to affect the course and severity of the disease, leading to the reduction or remission of such pathology. + An "effective dose" useful for treating and / or preventing cancer can be determined using known methods. The maximum tolerated dose (MTD) and maximum response dose (MRD) can be determined through established animal and human experimental protocols, as well as in the examples described herein.

[0188] In some embodiments, the polypeptides described herein are administered at dosage levels determined and planned by a physician.In certain therapeutic applications, the polypeptides are administered to patients already suffering from cancer in an amount sufficient to cure or at least partially prevent the symptoms of cancer.The amount effective for this use will vary depending on the severity and course of the cancer, previous treatments, the patient's health, weight, and response to drugs, and the judgment of the treating physician.The therapeutically effective amount can optionally be determined by methods including, but not limited to, dose escalation clinical trials.

[0189] Pharmaceutical Composition In some embodiments, provided herein are pharmaceutical compositions comprising a single-chain polypeptide molecule, a vector containing a polynucleotide encoding the single-chain polypeptide, or a host cell transformed with this vector, and at least one pharmaceutically acceptable carrier. 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 component 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-in-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 an appropriate dosage. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersing agents. Prevention of microbial attack can be ensured by the inclusion of various antibacterial and antifungal agents. In further embodiments, the pharmaceutical composition comprises a sustained-release excipient, such as PLGA nanoparticles. In further embodiments, the pharmaceutical composition is coated onto a device for insertion into the body for sustained release at a specific site.

[0190] In certain embodiments, the single-chain polypeptide of the present disclosure is contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain embodiments, the antibody of the present disclosure is administered suspended in a sterile solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. Additionally, in certain embodiments, the solution comprises one or more of the following: a buffer, such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, such as glycine or arginine; an antioxidant, such as ascorbic acid, methionine, or a chelating agent, such as EDTA or EGTA.

[0191] In certain embodiments, the single-chain polypeptides of the present disclosure are lyophilized for transport / storage and reconstituted prior to administration. In certain embodiments, the lyophilized antibody formulation includes a bulking agent such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation may be housed in a vial constructed of glass or other suitable non-reactive material. When formulated, the antibody, whether reconstituted or not, may be buffered at a specific pH, generally below 7.0. In certain embodiments, the pH may be 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, or 5.0-6.0.

[0192] Also described herein are kits comprising one or more of the single chain polypeptides described herein in a suitable container and one or more additional components selected from diluents, excipients, carriers, and administration devices.

[0193] In certain embodiments, described herein is a method for preparing a cancer treatment drug, comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an antibody of the present disclosure. In certain embodiments, described herein is a method for preparing a cancer treatment drug for storage or transportation, comprising lyophilizing one or more antibodies of the present disclosure.

[0194] Protein production One aspect provided herein includes a single-chain polypeptide. In certain instances, the polypeptide is an unbranched single-chain fusion protein. For example, the polypeptide contains multiple antigen-binding domains.

[0195] In some embodiments, the polypeptides described herein are produced by expressing a polynucleotide encoding the polypeptide. Thus, another aspect is a polynucleotide, e.g., DNA or RNA, that encodes a polypeptide described herein.

[0196] Polynucleotides are constructed by known methods, such as by combining genes encoding at least two or three binding domains, separated by peptide linkers, or in other embodiments directly linked by peptide bonds, into a single gene construct operably linked to a suitable promoter and, optionally, a suitable transcription terminator, and expressed in bacteria or other suitable expression systems, such as, for example, CHO cells. The polynucleotides encoding the peptide linkers are suitably linked to flexible linkers (e.g., Gly-Ser linkers, where n is 1, 2, 3, 4, 5, or more (Gly4Ser)). n , n is 1, 2, 3, 4, 5, or more (Gly3Ser) n , n is 1, 2, 3, 4, 5, or more (Gly3Ser) nDepending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. The promoter is selected to drive expression of the polynucleotide in the respective host cell.

[0197] In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which represents a further embodiment. The recombinant vector can be constructed according to known methods.

[0198] A variety of expression vector / host systems can be utilized to contain and express a polynucleotide encoding a polypeptide. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells.

[0199] Thus, in some embodiments, the polypeptides described herein are produced by introducing a vector encoding such a polypeptide into a host cell and culturing the host cell under conditions whereby the polypeptide chains can be expressed, isolated, and optionally further purified.

[0200] In some embodiments, the single-chain polypeptides described herein have modifications. Exemplary modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, drug conjugation, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, formation of a GPI anchor, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. In further embodiments, the multivalent antibody is modified with additional amino acids, such as a leader sequence or secretory sequence, or a sequence for purifying the polypeptide.

[0201] In some aspects, the single-chain polypeptide molecules described herein comprise a half-life prolonging domain that extends the half-life of the polypeptide. In some embodiments, the half-life prolonging domain is attached to the N-terminus of the single-chain polypeptide molecule. In some embodiments, the half-life prolonging domain is attached to the C-terminus of the single-chain polypeptide molecule. In some embodiments, the half-life prolonging domain replaces a binding domain, such as a VHH domain, of the molecules described herein. In some embodiments, the half-life prolonging domain replaces a binding domain, such as a VH and VL pair, of the single-chain polypeptide molecules described herein. Such domains are contemplated to include, but are not limited to, HSA binding domains, pegylated, small molecule, and other half-life prolonging domains known in the art. Human serum albumin (HSA) (molecular weight approximately 67 kDa) is the most abundant protein in plasma, present at approximately 50 mg / ml (600 μM), with a half-life in humans of approximately 20 days. HSA maintains plasma pH, contributes to colloidal blood pressure, functions as a carrier of many metabolites and fatty acids, and functions as the main drug transport protein in plasma. Non-covalent binding to albumin extends the elimination half-time of the protein. In some embodiments, the half-life extending domain is a domain that binds to HSA, and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies such as camelid-derived heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH) specific to HSA, peptides, ligands, or small molecule entities.

[0202] List of Embodiments Embodiment 1 comprises a single-chain polypeptide comprising variable domains joined by a linker, which fold into a conformation such that the variable domains form two effector cell antigen-interacting domains (E), each effector cell antigen-interacting domain comprising two variable domains of a single-chain variable fragment (EV and EV), one of the effector cell antigen-interacting domains directly binding to one or more target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domains are VHH single-domain antibodies (TVHH), and the single-chain polypeptide does not comprise an antibody constant domain.

[0203] Embodiment 2 comprises the single chain polypeptide of embodiment 1, wherein the two variable domains of the single chain variable fragment (EV and EV) comprise a variable heavy chain of the single chain variable fragment (EVH) and a variable light chain of the single chain variable fragment (EVL).

[0204] Embodiment 3 comprises the single chain polypeptide of embodiment 1 or 2, wherein one of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains is bound to one target cell antigen interacting domain.

[0205] Embodiment 4 comprises the single-chain polypeptide of embodiment 3, in which one target cell antigen interacting domain is C-terminally linked to one of the effector cell antigen interacting domains.

[0206] Embodiment 5 comprises the single chain polypeptide of embodiment 3, in which one target cell antigen interacting domain is N-terminally linked to one of the effector cell antigen interacting domains.

[0207] Embodiment 6 comprises the single chain polypeptide of embodiment 1 or 2, wherein one of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to two target cell antigen interacting domains.

[0208] Embodiment 7 comprises the single chain polypeptide of embodiment 6, wherein the two target cell antigen interacting domains are linked at their C-terminus to one of the effector cell antigen interacting domains.

[0209] Embodiment 8 comprises the single chain polypeptide of embodiment 6, wherein the two target cell antigen interacting domains are N-terminally linked to one of the effector cell antigen interacting domains.

[0210] Embodiment 9 comprises the single chain polypeptide of embodiment 1 or 2, wherein one of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to three target cell antigen interacting domains.

[0211] Embodiment 10 comprises the single chain polypeptide of embodiment 9, wherein three target cell antigen interacting domains are linked at their C-terminus to one of the effector cell antigen interacting domains.

[0212] Embodiment 11 comprises the single chain polypeptide of embodiment 9, wherein three target cell antigen interacting domains are N-terminally linked to one of the effector cell antigen interacting domains.

[0213] Embodiment 12 comprises the single chain polypeptide of embodiment 1 or 2, wherein one of the effector cell antigen interacting domains that binds to one or more target cell antigen interacting domains binds to four target cell antigen interacting domains.

[0214] Embodiment 13 comprises the single chain polypeptide of embodiment 12, wherein the four target cell antigen interacting domains are linked at their C-terminus to one of the effector cell antigen interacting domains.

[0215] Embodiment 14 comprises the single chain polypeptide of embodiment 12, wherein the four target cell antigen interacting domains are N-terminally linked to one of the effector cell antigen interacting domains.

[0216] Embodiment 15 comprises the single-chain polypeptide of embodiment 2, wherein one EVL of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains is bound to one TVHH, and one EVH of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains is bound to one TVHH.

[0217] Embodiment 16 comprises the single-chain polypeptide of embodiment 2, wherein one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to two TVHH.

[0218] Embodiment 17 comprises the single-chain polypeptide of embodiment 2, wherein one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to one TVHH, and one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to three TVHH.

[0219] Embodiment 18 comprises the single-chain polypeptide of embodiment 2, wherein one EVH of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains binds to one TVHH, and one EVL of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains binds to two TVHH.

[0220] Embodiment 19 comprises the single-chain polypeptide of embodiment 2, wherein one EVH of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains binds to one TVHH, and one EVL of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains binds to three TVHH.

[0221] Embodiment 20 comprises the single-chain polypeptide of embodiment 2, wherein one EVH of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to two TVHHs, and one EVL of the effector cell antigen interacting domains that bind to one or more target cell antigen interacting domains binds to two TVHHs.

[0222] Embodiment 21 is a method for producing a mAb-like antibody comprising the steps of: TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH, TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV, EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH, TVHH-EV-EV-EV-EV-TVHH, TVHH-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-EV-EV-EV-EV-TVHH, TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH, or TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH 21. The single-chain polypeptides of embodiments 2 to 20, which are ordered according to their N-terminal to C-terminal arrangement.

[0223] Embodiment 22 is a method for producing a mAb-like antibody comprising the steps of: TVHH-EVL-EVH-EVL-EVH, TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH, EVH-EVL-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH, TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH-TVHH, TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH 21. The single-chain polypeptides of embodiments 2 to 20, which are ordered according to their N-terminal to C-terminal arrangement.

[0224] Embodiment 23 comprises the single-chain polypeptide of embodiments 1 to 22, wherein the linker connecting the two effector cell antigen-interacting domains (E) to each other comprises an internal linker.

[0225] Embodiment 24 includes the single-chain polypeptide of any of embodiments 2 to 23, wherein the effector cell antigen interacting domain that is not directly linked to the one or more target cell antigen interacting domains comprises a terminal linker connecting the EVL and EVH.

[0226] Embodiment 25 comprises the single-chain polypeptide of embodiment 24, wherein each of the internal linkers is 6 to 8 amino acids in length.

[0227] Embodiment 26 comprises the single-chain polypeptide of embodiment 25, wherein the terminal linker is 15 or 16 amino acids in length.

[0228] Embodiment 27 comprises the single chain polypeptide of embodiment 2, wherein both effector cell antigen interacting domains are directly linked to the target cell antigen interacting domain.

[0229] Embodiment 28 comprises the single chain polypeptide of embodiment 27, wherein both of the effector cell antigen interacting domains are linked at the N-terminus and C-terminus to the target cell antigen interacting domain.

[0230] Embodiment 29 comprises the single-chain polypeptide of embodiments 27-28, wherein both effector cell antigen interacting domains comprise a terminal linker connecting the EVL and EVH.

[0231] Embodiment 30 comprises the single chain polypeptide of embodiment 29, wherein the terminal linker is 15 or 16 amino acids in length.

[0232] Embodiment 31 is a method for producing a target cell antigen interacting domain comprising the following: TVHH-EVH-EVL-TVHH-TVHH-EVH-EVL-TVHH, TVHH-EVL-EVH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-EVH-EVL-TVHH-TVHH-EVL-EVH-TVHH, and TVHH-EVL-EVH-TVHH-TVHH-EVL-EVH-TVHH The single-chain polypeptides of embodiments 27 to 30 are ordered according to their N-terminal to C-terminal arrangement.

[0233] Embodiment 32 comprises the single chain polypeptide of any of embodiments 1-31, wherein each of the effector cell antigen interacting domains binds to an effector cell antigen.

[0234] Embodiment 33 comprises the single chain polypeptide of embodiment 32, wherein the effector cell antigen comprises CD3, CD16a, or death receptor 5 (DR5).

[0235] Embodiment 34 comprises the single chain polypeptide of embodiment 33, wherein the CD3 is CD3 delta, CD3 gamma, or CD3 epsilon.

[0236] Embodiment 35 comprises the single chain polypeptide of embodiment 32, wherein the effector cell antigen is on an effector cell.

[0237] Embodiment 36 comprises the single chain polypeptide of embodiment 35, wherein the effector cell comprises a T cell, an NK cell, or a macrophage.

[0238] Embodiment 37 includes the single-chain polypeptide of any one of embodiments 1 to 36, wherein the target cell antigen-interacting domain that interacts with a target cell (T) binds to a target cell antigen.

[0239] Embodiment 38 comprises a single chain polypeptide of embodiment 37, wherein the target cell antigen accumulates in lipid rafts when on cells associated with a disease state, but does not accumulate in lipid rafts when on cells not associated with a disease state.

[0240] Embodiment 39 comprises the single chain polypeptide of embodiment 38, wherein the target cell antigen accumulates when on cells associated with a disease state and does not accumulate when on cells not associated with the disease state.

[0241] Embodiment 40 comprises the single chain polypeptide of embodiment 38, wherein the target cell antigen is a dimer, trimer, tetramer, or oligomer when on cells associated with a disease state, and is a monomer or dimer when on cells not associated with a disease state.

[0242] Embodiment 41 comprises the single chain polypeptide of embodiment 38, wherein the target cell antigen is part of a cell signaling complex when on a cell associated with a disease state, and is not part of the same cell signaling complex when on a cell not associated with the disease state.

[0243] Embodiment 42 comprises the single chain polypeptide of embodiment 38, wherein the target cell antigen is part of a cell signaling complex when on cells associated with a disease state, and is not part of a cell signaling complex when on cells not associated with a disease state.

[0244] Embodiment 43 comprises the single chain polypeptide of embodiments 37-40, wherein the target cell antigen comprises CD33, FAP, EGFR, HER2, or EpCAM.

[0245] Embodiment 44 includes the single-chain polypeptide of any one of embodiments 38 to 41, wherein the cell associated with the disease state is a myeloid cell, a fibroblast, or a cancer cell.

[0246] Embodiment 45 comprises a single-chain polypeptide comprising variable domains joined by a linker, which fold into a conformation such that the variable domains form one or more effector cell antigen-interacting domains (E), wherein the effector cell antigen-interacting domain comprises two variable domains of a single-chain variable fragment (EV and EV), and the effector cell antigen-interacting domain is directly linked to one or more target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH), and the single-chain polypeptide does not contain an antibody constant domain.

[0247] Embodiment 46 comprises a single-chain polypeptide comprising two variable domains (EV and EV) of a single-chain variable fragment joined by a linker, which fold into a three-dimensional structure to form an effector cell antigen-interacting domain (E), wherein the effector cell antigen-interacting domain is linked by one or more linkers to two or more target cell antigen-interacting domains that interact with target cells, and the target cell antigen-interacting domain is a VHH single-domain antibody (TVHH), and the single-chain polypeptide does not contain any antibody constant domains.

[0248] Embodiment 47 comprises the single chain polypeptide of embodiment 46, wherein the two variable domains (EV and EV) of the single chain variable fragment comprise a variable heavy chain of the single chain variable fragment (EVH) and a variable light chain of the single chain variable fragment (EVL).

[0249] Embodiment 48 comprises the single-chain polypeptide of embodiments 46-47, wherein two or more target cell antigen-interacting domains are linked at the C-terminus to the effector cell antigen-interacting domain.

[0250] Embodiment 49 comprises the single-chain polypeptide of embodiments 46-47, wherein two or more target cell antigen interacting domains are linked at the N-terminus to the effector cell antigen interacting domain.

[0251] Embodiment 50 comprises the single-chain polypeptide of embodiments 46-47, wherein two or more target cell antigen interacting domains are linked at the N-terminus and C-terminus to the effector cell antigen interacting domain.

[0252] Embodiment 51 comprises the single-chain polypeptide of any of embodiments 46 to 50, wherein the effector cell antigen interacting domain is linked to two target cell antigen interacting domains.

[0253] Embodiment 52 comprises the single-chain polypeptide of any of embodiments 46 to 50, wherein the effector cell antigen interacting domain is linked to three target cell antigen interacting domains.

[0254] Embodiment 53 comprises the single chain polypeptide of any of embodiments 46 to 50, wherein the effector cell antigen interacting domain is linked to four target cell antigen interacting domains.

[0255] Embodiment 54 comprises the single chain polypeptide of any of embodiments 46 to 50, wherein the effector cell antigen interacting domain is linked to five target cell antigen interacting domains.

[0256] Embodiment 55 comprises the single chain polypeptide of any of embodiments 46-50, wherein the effector cell antigen interacting domain is linked to six target cell antigen interacting domains.

[0257] Embodiment 56 is a polypeptide comprising an effector cell antigen interacting domain linked to two target cell antigen interacting domains, and a variable domain and two target cell antigen interacting domains, each of which has the following structure: TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH, EVL-EVH-TVHH-TVHH, TVHH-TVHH-EVH-EVL, TVHH-EVL-EVH-TVHH, or TVHH-EVH-EVL-TVHH The single-chain polypeptides of embodiments 46 to 50 are ordered according to their N-terminal to C-terminal arrangement.

[0258] Embodiment 57 is a polypeptide comprising an effector cell antigen interacting domain linked to three target cell antigen interacting domains, and a variable domain and three target cell antigen interacting domains, each of which has the following structure: TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EVH-EVL, TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH, TVHH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-TVHH The single-chain polypeptides of embodiments 46 to 50 are ordered according to their N-terminal to C-terminal arrangement.

[0259] Embodiment 58 is a polypeptide comprising an effector cell antigen interacting domain linked to four target cell antigen interacting domains, and the variable domain and the four target cell antigen interacting domains are linked to the following, wherein (-) is a linker: TVHH-TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVH-EVL, TVHH-TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH-TVHH, TVHH-EVL-EVH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-TVHH-TVHH The single-chain polypeptides of embodiments 46 to 50 are ordered according to their N-terminal to C-terminal arrangement.

[0260] Embodiment 59 is a polypeptide comprising an effector cell antigen interacting domain linked to five target cell antigen interacting domains, and the variable domain and the five target cell antigen interacting domains are linked to the following, wherein (-) is a linker: TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL, TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH, TVHH-EVL-EVH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH, TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH, or TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH The single-chain polypeptides of embodiments 46 to 50 are ordered according to their N-terminal to C-terminal arrangement.

[0261] Embodiment 60 is a polypeptide comprising an effector cell antigen interacting domain linked to six target cell antigen interacting domains, and the variable domain and the six target cell antigen interacting domains are linked to the following, wherein (-) is a linker: TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH, EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL, TVHH-TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH, TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-EVL-EVH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH, TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH, TVHH-TVHH-TVHH-EVL-EVH-TVHH-TVHH-TVHH, or TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH The single-chain polypeptides of embodiments 46 to 50 are ordered according to their N-terminal to C-terminal arrangement.

[0262] Embodiment 61 is an antibody fragment comprising an effector cell antigen interacting domain linked to six target cell antigen interacting domains, and the variable domain and the six target cell antigen interacting domains are linked to the following, wherein (-) is a linker: TVHH-TVHH-TVHH-EVH-EVL-TVHH-TVHH-TVHH, or EVH-EVL-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH The single-chain polypeptides of embodiments 46 to 50 are ordered according to their N-terminal to C-terminal arrangement.

[0263] Embodiment 62 comprises the single-chain polypeptide of any of embodiments 47 to 61, wherein the effector cell antigen interacting domain comprises a terminal linker connecting the EVL and EVH.

[0264] Embodiment 63 comprises the single chain polypeptide of embodiment 62, wherein the terminal linker is 15 or 16 amino acids in length.

[0265] Embodiment 64 comprises the single-chain polypeptide of embodiments 46 to 63, wherein the one or more linkers connecting the two or more target cell antigen interacting domains comprise an internal linker.

[0266] Embodiment 65 comprises the single-chain polypeptide of embodiment 64, wherein each of the internal linkers is 6 to 8 amino acids in length.

[0267] Embodiment 66 comprises the single chain polypeptide of any of embodiments 46-65, wherein the effector cell antigen interacting domain binds to an effector cell antigen.

[0268] Embodiment 67 comprises the single chain polypeptide of embodiment 66, wherein the effector cell antigen comprises CD3, CD16a, or death receptor 5 (DR5).

[0269] Embodiment 68 comprises the single chain polypeptide of embodiment 67, wherein the CD3 is CD3 delta, CD3 gamma, or CD3 epsilon.

[0270] Embodiment 69 comprises the single chain polypeptide of embodiment 68, wherein the effector cell antigen is on an effector cell.

[0271] Embodiment 70 comprises the single chain polypeptide of embodiment 69, wherein the effector cell comprises a T cell, an NK cell, or a macrophage.

[0272] Embodiment 71 comprises a single-chain polypeptide according to any one of embodiments 46 to 70, wherein the target cell antigen-interacting domain that interacts with a target cell (T) binds to a target cell antigen.

[0273] Embodiment 72 comprises a single chain polypeptide of embodiment 71, wherein the target cell antigen accumulates in lipid rafts when on cells associated with a disease state, but does not accumulate in lipid rafts when on cells not associated with a disease state.

[0274] Embodiment 73 comprises the single chain polypeptide of embodiment 71, wherein the target cell antigen accumulates when on cells associated with a disease state and does not accumulate when on cells not associated with the disease state.

[0275] Embodiment 74 comprises the single chain polypeptide of embodiment 71, wherein the target cell antigen is a dimer, trimer, tetramer, or oligomer when on cells associated with a disease state, and is a monomer or dimer when on cells not associated with a disease state.

[0276] Embodiment 75 comprises the single chain polypeptide of embodiment 71, wherein the target cell antigen is part of a cell signaling complex when on a cell associated with a disease state, and is not part of the same cell signaling complex when on a cell not associated with the disease state.

[0277] Embodiment 76 comprises the single chain polypeptide of embodiment 71, wherein the target cell antigen is part of a cell signaling complex when on cells associated with a disease state, and is not part of a cell signaling complex when on cells not associated with a disease state.

[0278] Embodiment 77 comprises the single chain polypeptide of embodiments 71-76, wherein the target cell antigen comprises CD33, FAP, EGFR, HER2, or EpCAM.

[0279] Embodiment 78 includes the single chain polypeptide of any of embodiments 71-77, wherein the cell associated with the disease state is a myeloid cell, a fibroblast, or a cancer cell.

[0280] Embodiment 79 comprises a single-chain polypeptide according to any one of embodiments 46 to 78, which has one effector cell antigen-interacting domain.

[0281] Embodiment 80 comprises a tablet pharmaceutical composition comprising (a) the single-chain polypeptide of any one of embodiments 1 to 79, and (b) a pharmaceutically acceptable excipient.

[0282] Embodiment 81 comprises an isolated recombinant nucleic acid encoding the single chain polypeptide of any one of embodiments 1 to 78.

[0283] Embodiment 82 includes a vector comprising the isolated nucleic acid of embodiment 81.

[0284] Embodiment 83 includes a host cell comprising the isolated nucleic acid of embodiment 81 or the vector of embodiment 81.

[0285] Embodiment 84 includes a method of treating cancer comprising administering to a subject in need thereof a single chain polypeptide according to any one of embodiments 1-79.

[0286] Embodiment 85 includes the method of embodiment 84, wherein the cancer is a solid tumor cancer.

[0287] Embodiment 86 includes the method of embodiment 84, wherein the cancer is a blood cancer.

[0288] Embodiment 87 includes a method of treating an inflammatory disease or condition comprising administering to a subject in need thereof a single chain polypeptide according to any one of embodiments 1-78.

[0289] Embodiment 88 includes a method of treating an autoimmune disease or condition comprising administering to a subject in need thereof a single chain polypeptide according to any one of embodiments 1-79.

[0290] Embodiment 89 includes a method of treating a cardiovascular disease or condition comprising administering to a subject in need thereof a single chain polypeptide according to any one of embodiments 1-79.

[0291] Embodiment 90 includes a method of treating a fibrotic disease or condition comprising administering to a subject in need thereof a single chain polypeptide according to any one of embodiments 1-79.

[0292] Embodiment 91 includes a method of treating a bacterial infection comprising administering to a subject in need thereof a single chain polypeptide according to any one of embodiments 1-79.

[0293] Embodiment 92 includes a method of treating a viral infection comprising administering to a subject in need thereof a single chain polypeptide according to any one of embodiments 1-79. [Example]

[0294] The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0295] Example 1 Cloning of DNA expression constructs encoding single-chain polypeptides specific for CD3 and HER2. A coding sequence for a single polypeptide chain specific for CD3 and HER2 is generated.

[0296] To express the single polypeptide chains in CHO cells, each coding sequence was cloned into an animal expression vector system, and the expression constructs were designed to contain a coding sequence for a C-terminal hexahistidine (6xHis) tag to facilitate antibody secretion and purification.

[0297] Expression of single polypeptide chains in stably transfected CHO cells The CHO cell expression system (Flp-In®, Life Technologies) is used, which is a derivative of CHO-K1 Chinese hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad Sci USA 1968, 60(4):1275-81). Adherent cells are subcultured according to the standard cell culture protocol provided by Life Technologies.

[0298] To adapt to growth in suspension, cells are removed from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium containing 10% DMSO.

[0299] Recombinant CHO cell lines stably expressing the single-chain polypeptide are generated by transfecting suspension-adapted cells. During selection with the antibiotic hygromycin B, viable cell density is determined twice a week, and cells are centrifuged and harvested at 0.1 x 10 6 Resuspend in fresh selection medium at a maximum density of viable cells / mL. Cell pools stably expressing the single-chain polypeptide are recovered after 2–3 weeks of selection, at which point the cells are transfected in standard medium in shake flasks. Expression of the recombinant secreted protein is confirmed by protein gel electrophoresis or flow cytometry. Stable cell pools are cryopreserved in DMSO-containing medium.

[0300] Single-chain polypeptides are produced in a 10-day fed-batch culture of a stably transfected CHO cell line by secretion into the cell culture supernatant. Cell culture supernatant is harvested after 10 days, when culture viability typically exceeds 75%. Samples are collected from the production culture every other day to assess cell density and viability. On the day of harvest, the cell culture supernatant is removed by centrifugation and vacuum filtration before further use.

[0301] Protein expression titers and product integrity in cell culture supernatants are analyzed by SDS-PAGE.

[0302] Purification of single-chain polypeptides Single-chain polypeptides are purified from CHO cell culture supernatants in a two-step procedure. In the first step, His6-tagged constructs are applied to Ni-NTA Superflow chromatography, followed by preparative size-exclusion chromatography (SEC) on a Superdex 200 in the second step. The eluted single-chain polypeptides are characterized for size and pooled. The pooled sample is buffer-exchanged and concentrated by ultrafiltration to a concentration typically exceeding 1 mg / mL. The purity and homogeneity of the final sample are assessed by SDS-PAGE under reducing and non-reducing conditions, followed by immunoblotting with an anti-His-Tag antibody and analytical SEC, respectively. The purified protein is stored in aliquots at -80°C until use.

[0303] [Table 23-1]

[0304] [Table 23-2]

[0305] [Table 23-3]

[0306] [Table 23-4]

[0307] [Table 23-5]

[0308] Example 2 Measurement of SCP-CD3 / HER2 affinity for huHER2 The purpose of this example was to measure the affinity of SCP-CD3 / HER2 construct samples for huHER2-His protein using SPR in multi-cycle kinetics (MCK) using a Biacore 8K+ instrument. The construct was injected so that it bound to the immobilized huHER2-His protein in solution. The instrument used was a Biacore 8K+ instrument, CFJB614 CM5 Series S sensor chip (Cytivia, Cat. nr. 29104992), with amine-coupled huHER2-His (AcroBiosystems, Cat. nr. HE2-H5225) immobilized at low density (≈1200 RU) in flow channels 1–4 and at high density (≈5000 RU) in flow channels 5–8. Construct samples (construct numbers 1, 2, 3, 5, and 6) engaging huHER2 were injected at 1, 10, and 100 nM at 30 μl / min for 2 min into flow cells 1 and 2. A control mAb, human anti-HER2 (trastuzumab) (R&D Systems, Cat. nr. MAB9589), was injected at 5 nM at 30 μl / min for 2 min into flow cells 1 and 2. An irrelevant construct engaging huCD33 was injected at 1, 10, and 100 nM at 30 μl / min for 2 min into flow cells 1 and 2. Off-rate measurements were performed for 300 s, and baseline levels were restored with regeneration solution. The buffer used was 1x HBS-EP + pH 7.4. The data were analyzed using the multi-cycle kinetics predefined evaluation method in Biacore Insight Evaluation Software. Kinetic parameters were calculated using a 1:1 binding fitting model. The results are shown in Table 24.

[0309] [Table 24] Example 3 Characterization of single polypeptide chains Single chain polypeptide constructs according to Table 25 and Table 26 were made and purified according to the protocol outlined in Example 1.

[0310] [Table 25-1]

[0311] [Table 25-2]

[0312] [Table 25-3]

[0313] [Table 25-4]

[0314] [Table 26-1]

[0315] [Table 26-2]

[0316] [Table 26-3]

[0317] [Table 26-4]

[0318] [Table 26-5]

[0319] [Table 26-6]

[0320]

Table 26-7

[0321]

Table 26-8

[0322]

Table 26-9

[0323]

Table 26-10

[0324]

Table 26-11

[0325]

Table 26-12

[0326]

Table 26-13

[0327]

Table 26-14

[0328]

Table 26-15

[0329]

Table 26-16

[0330] [Table 26-17]

[0331] SCP-CD3 / HER2 T cell cytotoxicity assay The T cell killing activity of the single-chain polypeptides was evaluated in MCF-7 and SKBR3 cancer cell lines. T cells were negatively selected from PBMCs (StemCells, Cat. nr. 70025.1, Lot nr. 220370304C, Donor ID RG2512 (Donor 6)). The number of cells used was adjusted to achieve a T cell to cancer cell (effector to target, E:T) ratio of 10:1 (1.0E+05:1.0E+04). T cells were stained with CellTrace Violet (Invitrogen, Cat. nr. C34557), added to a flat-bottom 96-well plate, and incubated for 48 hours in a 5% CO2, 37°C humidified incubator. The single-chain polypeptides were diluted in complete cell culture medium in eleven 10-fold serial dilutions, starting at 30 nM. Negative controls included PBMC, SKBR3, or MCF-7 cell lines alone at 20 nM. Additional controls included unstained cancer cells and T cells, and single cancer cells and co-cultured cells incubated with the highest concentration of construct. SKBR3 and MCF-7 cells were harvested and used in the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Cat. nr. G7571). After 48 hours of incubation, SKBR3 or MCF7 cells were transferred to opaque plates and incubated with a mixture of complete cell culture medium and CellTiter-Glo® Reagent (Promega, Cat. nr. G7571) for 10 minutes at ambient temperature. Cell viability was assessed by luminescence. Controls included unstained cells and CellTiter-Glo® Reagent alone. Luminescence was analyzed using a Pherastar instrument. T cells were recovered and used for cell activation assays (CD25 and CD69 staining). The supernatant containing T cell conditioned medium was stored frozen for further cytotoxicity / activation assays. Buffers used included:Complete cell culture medium: RPMI-1640 (Gibco, Cat. nr. A10491-01) supplemented with 10% FBS (Gibco, Cat. nr. 10270-106) and 1x penicillin-streptomycin (Gibco, Cat. nr. 15140122), and FACS buffer: 1x PBS / 0.5% HI-FBS (heat-inactivated fetal bovine serum) / 1:1000 EDTA.

[0332] SCP-CD3 / HER2 T cell activation assay The T cell activation of the single-chain polypeptide was evaluated in MCF-7 and SKBR3 cancer cell lines. T cell activation was measured 48 hours later by assessing the upregulation of T cell activation markers (CD25 and CD69) by FACS. After 48 hours of incubation, T cells were harvested and labeled with CD25 and CD69 markers. A 1:1 detection mix of mouse antibody huCD25-PE (BD Biosciences, Cat. nr. 567214) at a final concentration of 2 μg / ml and mouse anti-huCD69-APC (BD Biosciences, Cat. nr. 560711) was diluted 1:100 in FACS buffer and incubated on ice for 30 minutes protected from light. Controls used included unstained cells, PE detection only, APC detection only, and dual detection controls. Analysis was performed using the IntelliCyt® iQue Screener PLUS. CD25 and CD69 expression was read in the BL2 (PE) and RL1 (APC) channels of the single cell population, respectively. The percentage (%) of double-positive (CD25+CD69+) cells was measured within the single cell gate. The assay buffer included FACS buffer: 1x PBS / 0.5% HI-FBS (heat-inactivated fetal bovine serum) / 1:1000 EDTA.

[0333] T cell proliferation assay T cell proliferation was assessed by Cell Trace Violet dye staining. Cell proliferation was assessed by FACS 48 hours after co-culture with SKBR3 and MCF7 cancer cell lines. T cells were incubated with 1 μM Cell Trace Violet staining solution diluted in 1x PBS for 20 minutes in a 37°C incubator. Cell Trace Violet staining was assessed by FACS at 0 and 48 hours after incubation with cancer cell lines and HER2 engagers. Plates were placed in a humidified incubator at 37°C with 5% CO2 and co-cultured with SKBR3 and MCF7 cancer cell lines for 48 hours. Unstained T cells served as a control. Cells were acquired using the Intellicyt® iQue Screener PLUS and analyzed using Intellicyt Forecyt Software. Cell Trace Violet expression was measured in the VL1 (Pacific Blue) channel in single-cell populations. Sequence of H. pylori BabA_Nb14 / CD3 binder (SEQ ID NO: 85): QVQLQESGGGLVQPGGSLRLSCAASGSIYSLIAMGWYRQAPGKEHELVATISSGSTTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAMYYCAAYSDRLTDCSNCEADYWGQGTQVTVSH

[0334] The results of T cell killing in the MCF7 cancer cell line are shown in Figures 7A and 7B. Table 27 summarizes the IC50s from the plots shown in Figures 7A and 7B. Log(inhibitor) vs. response - % cancer cell kill vs. log(concentration) using a nonlinear regression (curve fitting) with variable slope (four parameters). IC50 values ​​were calculated from the same analysis.

[0335] [Table 27]

[0336] The results of T cell killing in the SKBR3 cancer cell line are shown in Figures 8A and 8B. Table 28 summarizes the IC50s from the plots shown in Figures 8A and 8B. Log(inhibitor) vs. response - % cancer cell kill vs. log(concentration) using a nonlinear regression (curve fitting) with variable slope (four parameters). IC50 values ​​were calculated from the same analysis.

[0337] [Table 28]

[0338] The results of the T cell activation assay in the MCF7 cancer cell line are shown in Figure 9. Table 29 summarizes the EC50 values ​​from the plot shown in Figure 9. The plot illustrates percent cancer cell kill versus log(concentration) plotted using GraphPad Prism 7, applying a nonlinear regression (curve fitting) of log(agonist) versus response-variable slope (four parameters). EC50 values ​​were calculated from the same analysis.

[0339] [Table 29]

[0340] The results of the T cell activation assay in the SKBR3 cancer cell line are shown in Figure 10. Table 30 summarizes the EC50 values ​​from the plot shown in Figure 10. This plot illustrates percent cancer cell kill versus log(concentration) plotted using GraphPad Prism 7, applying a nonlinear regression (curve fitting) of log(agonist) versus response-variable slope (four parameters). EC50 values ​​were calculated from the same analysis.

[0341] [Table 30]

[0342] Example 4 Measurement of SCP-CD3 / HER2 affinity The purpose of this example was to measure the affinity and avidity of single-chain polypeptide constructs for bio-huCD3, huHER2, and cyHER2 proteins in multi-cycle kinetics (MCK) using SPR with a Biacore 8k+. The constructs were injected so that they bound to the immobilized proteins in solution. The affinity measurements were performed on a Biacore 8K+ instrument using a CFJB755 CM5 Series S sensor chip (Cytivia, Cat. nr. 29104992) immobilized by amine coupling to biotinylated huCD3ε / δ (AcroBiosystems, Cat. nr. CDD-H82W0), huHER2-His (AcroBiosystems, Cat. nr. HE2-H5225-100µg), and cyHER2-His (AcroBiosystems, Cat. nr. HE2-C52Hb-100µg). Flow channels 1, 3, and 5 were immobilized at low density (1000–3000RU) and flow channels 2, 4, and 6 at high density (6200–7000RU). Samples of single-chain polypeptide constructs were injected at 0.3, 0.6, 1.3, 2.5, and 5 nM at 30 μL / min for 2 minutes into flow cells 1 and 2. A control monoclonal antibody was injected at 10 nM at 30 μL / min for 2 minutes into flow cells 1 and 2. Mouse anti-huCD3 (R&D, Cat. nr. MAB100-100) was used in flow channels 1 and 2. Human anti-HER2 (R&D, Cat. nr. MAB9589-100) was used in flow channels 3 and 6. Dissociation rate measurements were performed over 300 seconds. Regeneration was performed using regeneration solution to restore baseline levels. The buffer used was 1x HBS-EP + pH 7.4. Data were analyzed using the multi-cycle kinetics predefined evaluation method in Biacore Insight Evaluation Software. Kinetic parameters were calculated using a 1:1 binding fitting model.

[0343] Tables 31A and 31B summarize the results for huCD3. Tables 32A and 32B summarize the results for huHER2. Tables 33A and 33B summarize the results for cyHER2. Table 34 shows that the KD (dissociation constant) for CD3 binding is more than 100-fold higher, and in some cases more than 10,000-fold higher, than the KD of 3.8 nM reported for the HER2 control (construct #47) and anti-CD3 2B2 (SEQ ID NO:75 for VH and SEQ ID NO:76 for VL). Furthermore, the CD3 configurations of the constructs tested retain binding to cynomolgus monkey CD3. A branched construct with four VHHs (construct #18) binds to huHER2 with a 100-fold lower KD. Both HER2 and EGFR constructs with 6-, 7-, and 8-residue linkers between the CD3 domains are active.

[0344] [Table 31]

[0345] [Table 32]

[0346] [Table 33]

[0347] [Table 34-1]

[0348] [Table 34-2]

[0349] [Table 35]

[0350] [Table 36]

[0351] [Table 37-1]

[0352] [Table 37-2]

[0353] Example 5 Efficacy of single-chain polypeptides in breast cancer PDX models in NSG mice 2x10 7 Mice were inoculated subcutaneously (SC) with BT-474 tumor cells, transplanted with 1x10 huPBMCs on day 8, and treated 2 days later with two equimolar doses of antibody (e.g., 5 nmol / kg and 10 nmol / kg daily for 3 weeks) at metabolic tumor volume (MTV). Mice were weighed weekly and then sacrificed on day 38, and organs were harvested for analysis by flow cytometry.

[0354] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0355] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference to the same extent as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference. Definitions contained in texts incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

Claims

1. A single-chain polypeptide comprising variable domains connected by a linker, said variable domains folding into a three-dimensional structure to form two effector cell antigen-interacting domains (E), wherein each effector cell antigen-interacting domain (E) comprises two variable domains (EV and EV) of a single-chain variable fragment, and at least one of said effector cell antigen-interacting domains is bound to one or more target cell antigen-interacting domains.

2. The single-chain polypeptide of claim 1 , wherein the target cell antigen interacting domain binds to a tumor antigen, a ligand, a protein, a lipid, a nucleic acid, or a polysaccharide.

3. 2. The single-chain polypeptide of claim 1, wherein the target cell antigen interacting domain comprises an antibody, an antibody fragment, a ligand, or a cofactor, and the antibody or the antibody fragment of the target cell antigen interacting domain comprises Fab, Fab', F(ab')2, an Fv fragment, a single-chain Fv (scFv), a tandem single-chain Fv (scFv)2, a dual affinity retargeting antibody, a diabody, a VHH single-domain antibody, TriTac, an Adnectin, an Anticalin, an Avimer, a Fynomer, a Kunitz domain, a Knottin, an Affibody, or a DARPin.

4. The single-chain polypeptide described in claim 3, wherein the target cell antigen interacting domain comprises a VHH single domain antibody (TVHH).

5. The single-chain polypeptide of claim 1, which does not contain a fragment crystallizable (Fc) domain.

6. The single-chain polypeptide of claim 1, wherein the two variable domains (EV and EV) of the single-chain variable fragment comprise a variable heavy chain (EVH) of the single-chain variable fragment and a variable light chain (EVL) of the single-chain variable fragment, and wherein the EVL of at least one of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to at least one TVHH, and / or the EVH of one of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to at least one TVHH.

7. (i) at least one of the EVLs of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains binds to one TVHH, and at least one of the EVHs of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains binds to two or three TVHHs, or (ii) at least one of the EVHs of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains binds to one TVHH, and at least one of the EVLs of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains binds to two or three TVHHs; (iii) at least one EVH of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to two TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to two TVHHs; or (iv) at least one EVH of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to three TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to three TVHHs; or (v) at least one EVH of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to four TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to four TVHHs; or (vi) at least one EVH of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to five TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to five TVHHs; or (vii) at least one EVH of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to six TVHHs, and at least one EVL of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to six TVHHs; or (viii) the EVH of at least one of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to seven TVHHs, and the EVL of at least one of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to seven TVHHs; or (ix) the EVH of at least one of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to eight TVHHs, and the EVL of at least one of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to eight TVHHs. The single-chain polypeptide of claim 6. (i) at least one of the effector cell antigen interacting domains that bind to the one or more target cell antigen interacting domains binds to one target cell antigen interacting domain; (a) the one or more target cell antigen interacting domains are C-terminally linked to at least one of the effector cell antigen interacting domains; (b) the one or more target cell antigen interacting domains are N-terminally linked to at least one of the effector cell antigen interacting domains; (ii) at least one of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to two target cell antigen interacting domains; (a) the two target cell antigen interacting domains are C-terminally linked to one of the effector cell antigen interacting domains; (b) the two target cell antigen interacting domains are N-terminally linked to one of the effector cell antigen interacting domains; (iii) at least one target of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to three target cell antigen interacting domains; (a) the three target cell antigen-interacting domains are C-terminally linked to one of the effector cell antigen-interacting domains; (b) the three target cell antigen interacting domains are N-terminally linked to one of the effector cell antigen interacting domains; or (iv) at least one target of the effector cell antigen interacting domains that binds to the one or more target cell antigen interacting domains binds to four target cell antigen interacting domains; (a) the four target cell antigen-interacting domains are C-terminally linked to one of the effector cell antigen-interacting domains; (b) the four target cell antigen interacting domains are N-terminally linked to one of the effector cell antigen interacting domains.

9. The variable domain, and the one or more target cell antigen interacting domains, comprising the following: TVHH-EV-EV-EV-EV-TVHH, TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH, TVHH-EV-EV-EV-EV-TVHH-TVHH, TVHH-TVHH-EV-EV-EV-EV-TVHH, TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH, - - - - - - - - - - TVH-EV-EV-EV-EV、 EV-EV+EV-EV-TVHH、 TVHHTVHHEV-EV-EV-EV EV-EV-EV-EV-TVH-TVHH、 - EV-EV-EV-EV-TVH-TVH-TVHH、 - EV-EV-EV-EV-TVHHOTVH-TVH-TVHH、 - EV-EV-EV-EV-TVH-TVH-TVH-TVH-TVH、 - EV-EV-EV-EV-TVH-TVH--TVH-OTVH-TVHH、 - - - - - TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH, or TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH-TVHH The single-chain polypeptide of claim 4, which is ordered according to the arrangement from the N-terminus to the C-terminus of:

10. The variable domain, and the one or more target cell antigen interacting domains, comprising the following: TVHH-EV-EV-EV-EV-TVHH, or TVHH-TVHH-EV-EV-EV-EV-TVHH-TVHH The single-chain polypeptide of claim 9, ordered according to the arrangement from the N-terminus to the C-terminus of:

11. The variable domain, and the one or more target cell antigen interacting domains, selected from the group consisting of: TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH, TVHH-EVL-EVH-EVL-EVH, TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH, EVH-EVL-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH, TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH, TVHH-TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL, EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH-TVHH, EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH-TVHH, TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH-TVHH, TVHH-TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH, or TVHH-TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH, The single-chain polypeptide of claim 6, which is ordered according to the arrangement from the N-terminus to the C-terminus of:

12. The variable domain, and the one or more target cell antigen interacting domains, comprising the following: TVHH-EVL-EVH-EVL-EVH-TVHH, TVHH-EVH-EVL-EVH-EVL-TVHH, TVHH-TVHH-EVL-EVH-EVL-EVH-TVHH-TVHH, or TVHH-TVHH-EVH-EVL-EVH-EVL-TVHH-TVHH The single-chain polypeptide of claim 11, ordered according to the arrangement from N-terminus to C-terminus of:

13. The single-chain polypeptide of claim 1, wherein the linker that connects the two effector cell antigen-interacting domains (E) to each other comprises an internal linker.

14. The single-chain polypeptide of claim 6, wherein the effector cell antigen interacting domain that is not directly bound to the one or more target cell antigen interacting domains comprises a terminal linker that connects EVL and EVH, and the terminal linker is 15 or 16 amino acids in length.

15. The single-chain polypeptide of claim 1, wherein each of the effector cell antigen interacting domains binds to an effector cell antigen, the effector cell antigen comprising CD3, CD16a, or death receptor 5 (DR5), and the CD3 is CD3 delta, CD3 gamma, or CD3 epsilon.

16. The single-chain polypeptide of claim 1, wherein the effector cell comprises a T cell, a NK cell, or a macrophage. (a) a target cell antigen accumulates in lipid rafts when present on cells associated with a disease state, but does not accumulate in lipid rafts when present on cells not associated with a disease state; (b) the target cell antigen accumulates when present on cells associated with a disease state, but does not accumulate when present on cells not associated with a disease state; (c) the target cell antigen is 100 nm or less when on a cell associated with a disease state and is not greater than 100 nm when on a cell not associated with a disease state; (d) the target cell antigen is 75 nm or less when on a cell associated with a disease state and is not greater than 75 nm when on a cell not associated with a disease state; (e) the target cell antigen is 50 nm or less when on a cell associated with a disease state and is not greater than 50 nm when on a cell not associated with a disease state; (f) the target cell antigen is 25 nm or less when on a cell associated with a disease state and is within 25 nm when on a cell not associated with a disease state; (g) the target cell antigen is 10 nm or less when on a cell associated with a disease state and is not greater than 10 nm when on a cell not associated with a disease state; (h) the target cell antigen is 5 nm or less when on a cell associated with a disease state and is not greater than 5 nm when on a cell not associated with a disease state; (i) the target cell antigen is 2 nm or less when on a cell associated with a disease state and is not within 2 nm when on a cell not associated with a disease state; or (j) the target cell antigen is in close proximity when on a cell associated with a disease state and is not in close proximity when on a cell not associated with a disease state.

18. The single-chain polypeptide of claim 17, wherein the target cell antigen (a) is a dimer, trimer, tetramer, or oligomer when on a cell associated with a disease state and is a monomer or dimer when on a cell not associated with the disease state; (b) is part of a cell signaling complex when on a cell associated with a disease state and is not part of the same cell signaling complex when on a cell not associated with the disease state; or (c) is part of a cell signaling complex when on a cell associated with a disease state and is not part of a cell signaling complex when on a cell not associated with the disease state.

19. The single-chain polypeptide comprising two or more target cell antigen-interacting domains; (i) each of the two or more target cell antigen interacting domains binds to a different target cell antigen; or (ii) each of the two or more target cell antigen interacting domains binds to the same target cell antigen. (a) a single-chain polypeptide according to any one of claims 1 to 19; (b) a pharmaceutically acceptable excipient; and A pharmaceutical composition comprising:

21. An isolated recombinant nucleic acid encoding the single-chain polypeptide of any one of claims 1 to 19.

22. A vector comprising the isolated recombinant nucleic acid of claim 21.

23. A host cell comprising the isolated recombinant nucleic acid of claim 21.

24. Use of a single-chain polypeptide according to any one of claims 1 to 19 in the manufacture of a medicament for treating a disease or disorder.

25. The use of claim 24, wherein the disease or condition is cancer, an inflammatory disease or condition, an autoimmune disease or condition, a cardiovascular disease or condition, a fibrotic disease or condition, a bacterial infection, or a viral infection.

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